-------
620
workers in the departments which process arsenic display completely
different and higher values. However, the women from Burea must be
considered a highly exposed group if we take into account non-
industrial exposure. When the winds are from the north, Burea can
be highly exposed. However, the arsenic levels in the urine samples
from all groups were within acceptable limits.
The low values for the women in Skelleftehamn, who live so close
to the source of emissions, appear confusing at first, but they can
probably be explained by the fact that easterly winds are relatively
uncommon, and by the fact that the stack on the RSnnskar Works is so
high that when the wind is from the east, Skelleftehamn lies, so to
speak, below the line of fire.
As early as 1918, Bang determined the normal value for arsenic
in urine. He also showed how the level of arsenic in the urine
varies considerably from day to day and pointed out the importance
of diet. G. Westb'b and M, Rydalv have reported instances of a
substantial elevation cf the urine arsenic level after the con-
sumption of seafood (plaice) containing arsenic. (See the handbooks
by Koelsch and Patty for further information regarding the
evaluation of the level of arsenic in urine.)
The resulta indicated that there was no difference in the level
of cadmium in the urine from the four groups included in the study.
Nor was any tubular protein found.
In summary, it may be said that the procedures used in the
investigation failed to indicate any health hazards caused by the
substances studied in the area surrounding the smelting plant.
3. Lead in bloojt and ALA in urine
Lead tests on blood samples taken from men in the surrounding
area have been performed since 1950 at the RSnnska'r Works in
connection with the hiring of new employees. The material resulting
from these studies was reported at the occupational medicine
conference in Vienna in -1966 (Holmqvist), The studies have continued
since then, and ALA determinations in the urine were begun in 1968.
The lead level values from the blood of 801 men living at
various distances from the smelting' plant are shown in Pig. 1.
These distances have been divided into regions of <5, 5-15, 15-30,
30-50 and >50 km from the source of emissions. In addition, a large
number of men from Umea and from outside VMsterbotten County have
-------
621
Fig 1
NEW EMPLOYEES
LEAD in blood jug/100ml
Byske
Bolide
N.355 N 104
x 12.49 \S-12.25
urtrask
Lovanger
Over 50 km
N 20
* 1195 .Bygdsiljum
Umea (off the map) An3S6t
N 50 I
x 10.66 J
Outside Vasterbotten County
N 68
x 11.76
-------
622
also been studied.
The highest value, 13-57 jug/100 ml blood, was found in men
residing at a distance of less than 5 km from the Ronnskar Works.
It is clear from the statistical treatment that this value is
significantly higher than all the other values. The mean values
within the region 5-50 km are significantly higher than those more
than 50 km away - outside Vasterbotten County. The differences
between the three groups within the region 5-50 km are not significant.
The corresponding ALA values are reported in Pig. 2. There are
no significant differences,
In the results of the Vienna report, the students had lower
levels of lead in their blood than the working men. The material has
now been divided up into students and working men, and it can be
seen from table 2 that working men have a higher mean lead level in
their blood than students.
Table 2
Lead in blood. Qug/100 ml)t from students and working men
All
Students
Working men
n
801
369
432
Range
4-62
4-40
4-62
X
12.49
11.26
13.50
However, there is no such difference in the ALA values
(Table 3).
Table 3
ALA levelsin urine (mg/100 ml) from students and working men
n Range X
All 790 0.05-2.08 0.3030
Students 361 0.05-2.08 0.3099
Working men 429 0.05-0.87 0.2972
Pig. 3 reports the average lead levels in the blood of
students living at various distances from the source of emissions.
The mean value for students living within 0-50 km was significantly
higher than for those living more than 50 km away - outside Vaster-
botten County. "Umea" is not significantly lower than "50 km" -
"Outside VSsterbotten County".
The students in all the regions had lower levels of lead in
their blood than working men.
No difference in mean ALA levels could be detected between
students and working men at various distances from the smelting
-------
623
Fig 2
NEW EMPLOYEES
ALA in urine mg/100ml
x 0.3044 x 0.3099
Bygdsiljum
Over 50 km •
N = 19
1-0.2826
Umea (off the map):
N 50
x 0.2930
Outside Vasterbotten County:
N 68
x 0.3079
-------
624
Fig 3
NEW EMPLOYEES
LEAD in blood jug/100ml
Students 1968-1972
N 369
x 11.26
Byske
.Boliden
Kage
Skelleftec
5 km
Skelleftehamn
N 143 N-34\
x = 11.53 x 11.08
rtrask
Lovanger
ygdsiljum
Over 50km:
N 6
x-8.00
Umea (off the map):
N=46
x 10.26
Outside Vasterbotten County •
N=32
x=10.31
-------
625
Fig 4
NEW EMPLOYEES
ALA in urine mg/100ml
Students 1968-1972
N 361
x= 0.3099
Lovanger
ygdsiljum
Over 50 km =
N=5
x = 0.3340
Umea (off the map):
N=46
x-0.2965
Outside Vasterbotten County:
N-32
x-0.3000
-------
626
plant. (Pig. 4).
Table 4
Lead level dug/100 ml) in the blood of workers belonging to
different profeasions
Profession
Students
Drivers
Electricians
Metal workers
Lightweight cement workers,
Siporex
Wood and pulp workers
Other professions
n
369
33
7
70
20
39
263
Range
4-40
6-32
6-23
8-36
7-45
4-36
4-62
X
11.26
13.18
16.14
15.94
14.60
11.94
13.05
SD
5.2
4.8
-
6.3
9.1
5.9
5.9
It can be seen from Table 4 that the professional group
which had the highest level was electricians. But this was a small
group (7 persons) and thus not significant. The metal workers had
the highest significant mean value, 15.34/ig/100 ml. Metal workers
living less than 5 km from the source of emissions exhibited higher
values (average 19.06) than metal workers in the other regions.
The greatest difference between professions was between electricians
and students - 4.88 ^ig/100 ml. If we disregard students and
electricians, the biggest difference between working men was that
between metal workers and wood and pulp workers, which amounted
to 4.10 jig/100 ml. Thus, profession is more important for the level
of lead in the blood than distance of abode from the smelting plant.
The largest difference was between metal workers living within
5 km of the plant and students in Umea - 8.80 ug/100 ml.
There was no difference in the ALA values between these two
groups. In fact, there was no difference in the ALA values between
any of the professional groups. Thus, we can conclude that there
were no differences in metabolic response, even though the lead
values were somewhat different (Table 5)*
-------
627
Table 5
ALA in the urine (mg/100 ml) of workers belonging to different
prof easi ana
Profession
Students
Drivers
Electricians
Metal workers
Lightweight cement workers,
Siporex
Wood and pulp workers
Other professions
n
361
33
7
69
19
39
262
Range
0.05-2.08
0.09-0.80
0.24-0.57
0.07-0.64
0.09-0.50
0.09-0.87
0.05-0.73
X
0.3099
0.2948
0.3557
0.3024
0.2778
0.3064
0.2946
As further proof that occupation has some effect on lead level
in the blood, it has been shown that trade school students, who have
some practical experience in their trades during their study years,
exhibit significantly higher lead levels in their blood than do
other students.
Age does not seem to have any influence on lead level in the
blood of lead workers. Exposure is the decisive factor. This
opinion is shared by. a number of researchers in the field,
including Lehnert, who surveyed the problem. The lower lead levels
in the blood which are sometimes obtained from older age groups may
result from the fact that older persons seek work which involves
less physical strain, resulting in less inhalation of lead.
The levels of airborne lead are also of interest in this
connection. The measurements which have been made at a distance of
<:5 km from the smelting plant in Skelleftehamn have given results
which vary in the actual residential area from 0.3 to 0.5^»g/m air.
Other values measured within the region <5 km from the source of
emissions varied between 0.2 and 3.8^ig/m , with a mean of 0.85
ug/m . These data may be compared with the values which were
reported at a heavy metals conference in February of 1973 in
Dilsseldorf. It was for example reported that a lead level in the
air of 3 iig/m could result in a lead level in the blood of 30
jug/100 ml. At a meeting which was held after the conference,
a committee of experts decided that an air level of 2 lig/m could
be recommended as acceptable.
Finally, some experiments which shed light upon the relation-
ship between inhaled lead and blood lead levels deserve mention here.
-------
628
Exposure trials with airborne, inhaled lead, which have been
conducted on prisoners in the USA, have shown that when the
prisoners were allowed to reside in an atmosphere containing
3,2 ug of lead per m for 125 days, the mean level of lead in the
blood rose from about 18 to 27 jug/100 ml. The mean lead level of
a group of subjects who were exposed to air containing 10.9 ug lead
per m for 125 days rose from about 20 to about 38 jig/100 ml. The
control group exhibited no rise. One of the graphs of these
experiments presented by Knelson at the heavy metals conference in
Diisseldorf is reproduced in Pig. 5. From these experiments, we can
conclude that the mean value of 14 ug/100 ml we obtained for all
the subjects in our study and 19 jig/100 ml for the metal workers
living less than 5 km from the source of emissions indicate a low
mean exposure to airborne, inhaleable lead, in any case less than
•z
3.2 ug/m and probably much lower.
The lead levels in the Skelleftehamn air are low, probably
much lower than the guideline value of 2 ug/m" offered in CUsseldorf,
This claim is supported by measurements of the lead content of the
air, the value found for the mean level of lead in the blood of
men in Skelleftehamn and finally the American lead exposure
experiments on prisoners discussed above.
In summary, we can conclude that the lead emissions from the
Rttnnskar Works have not proved to constitute a health hazard for
people living in the surrounding area.
-------
629
DISCUSSION
BERLIN (C.E.C.)
1. Could the author indicate the analytical method used for
ALA determinations and how the quality was carried out?
2. Has any assessment been made of the emissions from the
smelter and of environmental concentrations/ besides air?
HOLMQVIST (Sweden)
1. The ALA determinations were made according to a method
elaborated by Mauzerall and Granick (1956). The quality control
was carried out by several investigations. The recovery from
normal human urine was satisfactory. Comparison with other
laboratories gave comparable results.
2. The emissions from the smelter have continuously been re-
duced during the last decades. The measures for improvements of
the environment have been intensified during the last years,
among other things by new electrofliters and by building a plant
for production of liquid sulphur dioxide from the smelter gases.
-------
631
STUDY ON CADMIUM PROTEINURIA. GLOMERULAR
DYSFUNCTION: AN EARLY SIGN OF RENAL IMPAIRMENT
H, ROELS, R, LAUWERYS, D, MATERNE AND J, P, BUCHET
UnitS de ToxicoLogie Industrielle et M6dicale, UniversitS Catho-
lique de Louvain, Brussels, Belgium
ABSTRACT
The eleatrophoretic patterns of urinary proteins from wor~
kera with prolonged exposure to cadmium, reveal the existence of
two distinct proteinurias, i.e., glomerular and mixed. The for-
mer probably reflects only an enhanced permeability of the glom-
eruli to high molecular weight plasma proteins, whereas in the
latter also a decreased protein reabeorption by the tubuli is
involved. Glomerular patterns were found in workers with less
and more than 20 years exposure; mixed patterns only in workers
with more than 20 years exposure. These observations were con-
firmed by measurements of renal clearance of albumin, lysozyme,
and ribonualease. The results indicate that in workers with a
glomerular pattern only an increased excretion of high molecular
weight plasma proteins occurs, but that in those with a mixed
pattern the excretion of both high and low molecular weight plas-
ma proteins is increased. These results together with a few
recent reports on animals exposed to Cd and on electron microsco-
pic studies in patients suffering from Balkan endemic nephropathy,
reinforce our suggestion that a glomerular dysfunction may pre-
cede the "classical" tubulopathy observed after long-term expo-
sure to cadmium.
-------
632
Introduction
Although the complex handling of plasma proteins in the
kidneys is presently not fully understood, the types and
relative amount of plasma proteins excreted in urine are
useful biological parameters reflecting renal function. The
predominant part of plasma proteins in urine appears to arise
from a process of glomerular filtration followed by reabsorp-
tion and ca^bolism in the proximal tubuli {Schultze and
Heremans (1) .
Cadmium is a systemic cumulative poison frequently
inducing increased proteinuria in workers with long ^xnosure
to cadmium compounds as first described by Friberc (;>; t
This binding has been amply confirmed by results of epidem-
iological surveys among workers chronically exposed to cadmium
oxide dust or fume and by studies with experimentally poisoned
animals, revealing that the injurious action of Cd occurs
especially in the proximal tubuli with a concomitantly elevated
excretion of low molecular weight proteins (for reviews see:
Flick et al. (3) ; Friberg et_al. (4) among them the
plasma protein, /^-microglobulin (Piscator (5) ; Bergg&rd
and Beam (6) ,
Up to now, most reports on cadmium proteinuria have dealt
with workers excreting large amounts of proteins (.5g or more
a day) and because of the obvious tubular lesion, slight
changes in glomerular permeability could have been overlooked.
Recent investigation in our laboratory, however, prompted
us to suggest that glomerular dysfunction may be an early
phase in cadmium-induced renal impairment (Lauwerys et a1.
(7 ). Indeed, electrophoresis on agarose of the urinary
proteins of clinically healthy workers, exposed to an average
airborne concentration of Cd-dust below the current American
3
TLV (0.2 mg/m of air), enabled us to classify the proteinuria
in two categories, i.e., glomerular and mixed (glomerular +
tubular) proteinuria. For both types of proteinuria the
electrophoretic patterns reflect an enhanced excretion of
high molecular weight proteins (i.e., albumin,^-Zn-
-------
633
glycoprotein, transferring), and in cases of mixed proteinuria
also an enhanced excretion of low molecular weight proteins
(i.e..a^-and/^-microglobulin, and in some cases post-P- protein).
The glomerular type was observed in workers with less or more
than 20 years' occupational exposure, whereas the mixed type
only in workers with more than 20 years' exposure.
The present paper reports results of investigations
carried out to test our hypothesis that early glomerular
dysfunction may be induced by cadmium. We have estimated
the relative importance of glomerular and tubular proteinuria
by measuring the renal clearances of albumin, lysozyme, and
ribonuclease (expressed as % of the (creatinine clearance) in
both distinct "electfophoretic groups" of Cd-exposed workers.
Materials and Methods
The total study population consisted of 218 male workers
(110 non-exposed and 108 exposed) from two different cadmium-
producing plants and a Ni-Cd storage battery factory in Bel-
gium. In each factory the selected group of non-exposed
workers matches the group of exposed workers according to age,
weight, height, smoking habit, duration of employment in the
same factory, and socio-economic status. Air sampling for
determination of total airborne and respirable (aerodynamic
diameter<5yu) Cd-dust concentration at different sites of the
factories was carried out as described previously (7) . Cadmium
was measured by atomic absorption spectrophotometry.
The following biological analyses were performed: Cd
concentration in blood and urine (Vens and Lauwerys 8), total
proteinuria (7), totala-amino-aciduria (Hall et al. 9* and
electrophoresis on agarose of urinary proteins (Johansson 10 ;
see also 7 ). Furthermore, the concentration of lysozyme and
ribonuclease (Harrison et al. 11), albumin (Ritchie et al. 12),
and creatinine (Jaffe-method, see Henry 13 )were measured on the
urine and blood specimen (collected at the same time as the
urine samples of 6 normal subjects (laboratory staff),18 Cd-exposed
workers with pathological electrophoretic pattern and 8 patients
with various renal diseases.
-------
634
Results
The workers were exposed to an average respirable Cd-
dust concentration of 0.020 to 0.030 mg/m3 of air (average
total airborne concentration ranged from 0.074 to 0.212 mg/m
of air) and appeared to be in good clinical condition.
Table I shows the biological parameters measured in the
total study population; the results obtained for the exposed
workers are classified according to the electrophoretic
pattern of the urinary proteins, i.e., normal, glomerular
and mixed. All the non-exposed workers showed normal electrop-
horetic pattern, while 18 out of 108 exposed workers exhibited
a pathological one.
As reported previously (7)the glomerular pattern was
found in workers with less (4 cases) or more (5 cases) than
20 years' exposure, whereas the mixed (9 cases) only in those
with more than 20 years' exposure. The mean Cd concentration
in urine (Cd-U) of non-exposed workers is about five times
that (0.38 + 0.10 ^t/g/g creatinine) found in 20 normal male
subjects (students and laboratory staff; non-smokers). This
discrepancy probably reflects a different degree of environ-
mental contamination by Cd between the living areas of the
normal subjects and those of the non-exposed workers which
are likely to be more contaminated because of the proximity
of the Cd plants.
Ninety exposed workers showed apparently normal kidney
function at least on the basis of the electrophoretic pattern
of urinary proteins, total proteinuria, and total a-amino-
aciduria (Table I). For 9 exposed workers a glomerular pattern
was found on the urinary protein electrophoresis. They also
exhibited a significant increased total protei,nuria and a
slight increase in total #-amino-aciduria. It is interesting
to notice that the average Cd-U level of these workers (19.0
^Um/g creatinine) was not different from that of the exposed
group (IQ.l flg/g creatinine) with normal electrophoretic
urinary protein pattern. The 9 other exposed workers showed
a more impaired renal function as evidenced by the finding
-------
Table I Biological parameters in workers non-exposed to cadmium and in
exposed workers classified according to the electrophoretic
pattern of their urinary proteins (mean values + SEM)
Workers
Non-exposed
Exposed
Electrophoretic
pattern
Normal
Normal
Glomerular
Mixed
It
110
90
9
9
Cd in urine
(jig/g creatinine)
1.79 + O.l6-i
18.7 + 2.3 -
-
19.0 + 3-7 -J
-
52.8 + 8.3
*
-
-1
Proteinuria
(mg/g creatinine)
152 + 5.0 -
166 + '\k
296 + kj J
-
700 +131 J
-
=
ct-Aminoaciduria
(mg/g creatinine)
159 + 6.1 -,
158 + 5-9
175 i 10
232 + 25 -
-
-
Cd in blood
(jig *)
0.58 + 0.02-,-]-,
2.18 + 0.19-'
1.87 ± 0.38 -!
2.72 + 0.55 -
Ul
Ul
""significant different mean values {P at least < 0.05 ; Student's t-test).
-------
Table II Renal clearance of three plasma proteins in normal subjects, in
Cd-exposed workers with abnormal electrophoretic pattern (glomerular
and mixed), and in patients with renal diseases (mean and range)
Subjects
Normal subjects (n = 6)
Cd-exposed workers
Glomerular type (n = 9)
Mixed type (n = 9)
Patients with renal diseases
Suspected lithiasis
Uric acid nephrolithiasis
Focal glomerulonephritis
Interstitial nephritis (analgesic)
Interstitial nephritis (analgesic)
Acute interstitial nephritis
Diabetic nephropathy
Chronic pyelonephritis
Renal cleai
% of endog«
Albumin
(icf*)
0.35
0.11-0.70
U.05
0. 76-16. U
8.81
0.89-23.3
0.58
1.13
0.83
5.68
281
18.9
1*2.1*
263
•ance of plasms
>nous creatinir
Lysozyme
0.010
0.006-0.019
O.Oll*
0.005-0.025
2.1*5
0.13-20.7
O.OOU
0.001
0.001
0.027
0.08U
0.037
0.063
0.836
i proteins (as
le clearance)
Ribomiclease
5-31
2.6U-9.06
5-59
1.18-18.5
8.73
U. 19-31. 5
0.62
0.71*
0.70
0.71
5.26
1.81*
1.30
17.8
Proteinuria
(mg/g creatinine)
161
109-228
296
11*1-1*87
700
267-1171
Creatinine
in serum
(mg/100 ml)
1.18
0.97-1.1+1
1.20
0.95-1.81*
1.28
0.89-2.03
0.97
0.99
0.83
1.61
2.01
2.21*
2.63
8.69
-------
637
of a mixed electrophoretic pattern of urinary proteins, a
greater increase in total proteinuria and in total tt-amino-
aciduria. In the last group Cd-U is significantly greater
than in the two preceding groups which is a usual observation
when the proximal tubuli are damaged by cadmium (7); Adams
et__al. (14); Kazantzis et al. (15).
Table II shows the results of renal clearance measure-
ments in 6 normal subjects, in the Cd-exposed workers with
pathological electrophoretic pattern of urinary proteins
(9 glomerular and 9 mixed), and in a group of patients with
various renal diseases. For the normal subjects the clearances
of albumin (mol. wt. 69 000), lysozyme (mol.wt. 14 000), and
ribonuclease {mol. wt. 13 000) expressed as % of the endogenous
creatinine clearance^ are comparable with literature data
(Harrison et al. (11); Peterson et al. (16).
The clearance results obtained for the 18 Cd-exposed
workers are in agreement with our classification of the type
of proteinuria based on the electrophoretic pattern. Indeed,
the moderate increase in proteinuria observed for the 9 workers
with glomerular type electrophoretic pattern can only be ascribed
to an increased clearance of albumin and of other high molecular
weight proteins, since no increased lysozyme and ribonuclease
clearances were observed. On the other hand, in the 9 workers
with mixed type electrophoretic pattern, not only albumin
clearance was increased but also that of low molecular weight
proteins as shown by the results of lysozyme and ribonuclease
and clearance measurements. The group of patients with renal
diseases has been included in this study in order to check the
validity of the methods used for measuring the clearances.
Discussion
Results of the renal clearance of high and low molecular
weight plasma proteins tend to confirm our previous suggestion
(7), that the early kidney lesion in workers developing cadmium-
induced proteinuria could be a glomerular dysfunction, which
precedes the tubulopathy predominantly found in workers with
prolonged exposure to cadmium and excreting large amounts of
-------
638
proteins (14;15) Piscator (17) ). Two recent reports dealing
with investigation of experimentally Cd-poisoned animals
reinforce this suggestion: 1) stop-flow analysis performed
on rabbits at the end of successive Cd administration revealed
a glomerular dysfunction and a normal or slia^tly altered
proximal tubular function (Nomiyama et al.(18) and (2) long-
term (120 days) administration of Ca-adequate and Ca-deficient
diets together with water containing 50 ppm Cd provoqued
extensive glomerular as well as tubular lesions in male rats
(Itokawa et al. (19) ) . Recently, it has been demonstrated by
means of electron microscopy, that in patients with suspected
Balkan endemic nephropathy (20) early glomerular lesions are
involved first in this renal disease that eventually gives
rise to a proteinuria that resembles (Hall et al. (21) ).
the "classical" tubular proteinuria observed in workers with
prolonged exposure to cadmium.
Heinemann et al. (22) (see also Flynn and Platt (23)
proposed two basic mechanisms which can cause pathological
excretion of high molecular weight proteins: 1) increased
glomerular permeability to high molecular weight plasma proteins
and no change in tubular reabsorption of protein (only rise
of high molecular weight proteins in urine), 2) normal glomerular
filtration of high molecular weight proteins with impaired
tubular reabsorption which is usually associated with an
i
increased excretion of both high and low molecular weight protein^
According to this schema, two hypotheses remain to
explain the glomerular type electrophoretic pattern associated
with an increased albumin clearance found in 9 workers exposed
to cadmium: 1) an impaired glomerular permeability with a
normal tubular function or 2) a selective impairment of the
mechanism of high molecular weight protein reabsorption by
the tubuli with a normal glomerular function. In this last
event, the mixed type electrophoretic pattern would then
result from an aggravation of the tubular lesion involving
also the mechanism of low molecular weight protein reabsorption.
-------
639
However, the morphologic studies (19, 20) mentioned above
tend to support the first hypothesis, i.e., that an alteration
of glomerular filtration is responsible for the glomerular
type electrophoretic pattern, and that the mixed pattern
results from lesion at both sites (glomeruli and tubuli)
usually occurring after a much longer exposure to cadmium
than the glomerular lesion.
Measurement of /l-microglobulin clearance will be carried
out to characterize further the two types of proteinuria.
This investigation was supported by the Commission des
CcmmunautSs Europeennes, projet No. 6244-00/2/028.
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-------
640
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Clin. Lab. Invest. 29 (suppl. 124), 7-19 (1972).
11. HARRISON, J. F., LUNT, G. S., SCOTT, P., and BLAINEY, J. D.,
Urinary lysozyme, ribonuclease and low-molecular-weight
protein in renal disease. The Lancet i, 371-374 (1968) .
12. RITCHIE, R. F., ALPER, C. A., GRAVES, J., PEARSON, N., and
LARSON, C., Automated quantitation of proteins in serum
and other biological fluids. Amer. J. Clin. Path. 59,
151-159 (1973).
13. HENRY, R. J., Clinical chemistry. Principles and Technics.
p. 287. Hoeber Medical Division; Harper and Row, ''
Publishers, New Zork (1965).
14. ADAMS, R. G., HARRISON, J., and SCOTT, P., The development
of cadmium-induced proteinuria, impaired renal function,
and osteomalacia in alkaline battery workers. Quart. J.
Med. 38, 425-443 (1969).
15. KAZANTZIS, G., FLYNN, F. V., SPOWAGE, J. S. and TROTT,
D. G., Renal rubular malfunction and pulmonary emphysema
in cadmium pigmet workers. Quart. J. Med. 32, 165-192
(1963). "
16. PETERSON, P. A., EVRIN, P. E., and BERGGARD, I., Different-
iation of glomerular, tubular, and normal proteinuria:
Determinations of urinary excretion of /5--microglobulin,
albumin, and total protein. J. Clin. Invest. 48, 1189-1198
(1969).
17. PISCATOR, M., Proteinuria in chronic cadmium poisoning.
1. An electrophoretic and chemical study of urinary and
serum proteins from workers with chronic cadmium poisoning
Arch. Environ. Health. 4, 607-621 (1962).
18. NOMIYAMA, K., SATO, C., and YAMAMOTO, A., Early signs of
cadmium intoxication in rabbits. Toxicol. Appl. Pharmaco]
24, 625-635 (1973). " *
19. ITOKAWA, Y., ABE, T., TABEI, R., and TANAKA, S., Renal and
skeletal lesions in experimental cadmium poisoning. Hist-
ological and biochemical approaches. Arch. Environ. Health
28, 149-154 (1974). —^
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641
20. The Lancet i, 472 (1973).
21. HALL, P. W., PISCATOR, M., VASILJEVIC, and POPOVIC, N.f
Renal function studies in individuals with the tubular
proteinuria of endemic Balkan nephropathy. Quart. J. Med,
41, 385-393 (1972) .
22. HEINEMANN, H. O., MAACK, T. M., and SHERMAN,'R. L., Prot-
einuria. Am. J. Med. 56, 71-82 (1974).
23. FLYNN, F. V. and PLATT, H. S., The origin of proteins
excreted in tubular proteinuria. Clin. Chem. Acta 21,
377-399 (1968).
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643
UNTERSUCHUNG DER WIRKUNGEN AUF DEN MENSCHEN
HUMAN EFFECTS STUDIES
ETUDES'DES EFFETS SUR L*HOMME
STUDI DE6LI EFFETTI SULL'UOMO
ONDERZOEKINGEN NAAR EFFECTEN BIJ DE MENS
(Continued)
Vova-itzender - Chairman - President - Presidente - Voorzitter
G. JJEAN (Ireland)
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645
INCREASED RISK OF ACUTE AND CHRONIC RESPIRATORY
DISORDERS FOLLOWING LONG-TERM AIR POLLUTION EXPOSURES
R, CHAPMAN/ J, FRENCH, J, FINKLEA, C, HAYES AND G, LOVE
Environmental Protection Agency, Research Triangle Park, NC, USA
ABSTRACT
In 1970 and 1971, the U.S. Environmental Protection Agency
conducted several epidemiologic surveys which related long-term
air pollution exposures to the prevalence of chronic respiratory
disease and the incidence of acute respiratory disease. Chronic
respiratory disease (CRD) was studied in about 29,000 young adults
in New York, Utah and the Idaho-Montana area, and in about 42,000
military inductees in Chicago. Acute respiratory disease (ARD)
was studied in about 5,500 children and adults in the cities of
Chicago and New York.
In all four CRD studies, chronic bronchitis prevalence was
elevated in neighbourhoods of elevated pollution exposure. The
effect of pollution on chronic bronchitis prevalence generally
ranged from about one-tenth to about one-third of the effect of
cigarette smoking. Among fathers in New York, the pollution ef-
fect on CRD was observed to be stronger than the smoking effect.
The latter finding emphasized the considerable effect of air pol-
lution on CRD prevalence.
The findings of the Chicago and flew York ARD studies sugges-
ted that reduced-rates of ARD would result from improvements in
existing air quality. In Chicago, it was estimated that reduc-
tion of annual average sulfur dioxide (S0n) levels from about
3 3
100 ,ug/m to about 57 .ug/m , and of suspended sulfate (SS) levels
-------
646
3 3
from about 18 ,ug/m to about 14 ,ug/m t would promote reductions
•in total ARD incidence. Such reductions in APD could be expec-
ted even in the face of continued exposure to total suspended
particulate levels of about 110 to ISO ,ug/m . In New York,
exposure for one to three years to annual average SO0 levels of
3 . 3
about 250 ,ug/m } accompanied by SS levels of about 22 .ug/m and
3 '
TSP levels of about 94-117 .ug/m , was associated with a signifi-
cant increase in the incidence of acute lower respiratory illness.
-------
647
1. hvtrpdiiction
The U.S. Environmental Protection Agency (EPA) has devoted
considerable effort to characterizing the health effects of long-
term past exposures to ambient air pollution. The purpose of this
report is to summarize four EPA determinations of pollution effects
on the prevalence of chronic respiratory disease (CRD) * ' ' ' '
and two determinations of pollution effects on the incidence of acute
fi 7 ft
respiratory disease (ARD). ' The CRD studies focused on young
adults, and the ARD studies focused on both young adults and their
children. For all six studies, the pollutants of major concern were ,.
oxides of sulfur and suspended particulate matter (TSP). One important
pollutant, suspended sulfates, fits into both of these categories.
2. Methods
2.1 Determinations of CRD Prevalence
In 1969 and 1970, the prevalence of CRD was determined in four
large geographic areas of the United States, the Salt Lake Basin
in Utah, the two-state Idaho-Mo'ntana area, the Chicago, Illinois
area, and the New York City Metropolitan area. The first two areas
are in the western United States, and the bulk of their sulfur oxide
and particulate pollution is emitted by metal smelters. The pollution
in Utah and Idaho-Montana generally contains substantial levels of
sulfur oxides, including suspended sulfates, but moderate levels
of TSP. In Chicago and New York, substantial amounts of both sulfur
oxides and TSP are present.
In each of these four major areas, at least one community or zone of
high pollution exposure, and at least one community or zone of low exposure
were selected for study. Thus, four independent comparisons of CRD
rates in the presence of high and low pollution levels could be made.
The results of each individual CRD study therefore offered a test
of the results of the other three.
In Utah, Idaho-Montana, and New York, CRD prevalence rates were
determined by the distribution of self-administered symptom questionnaires
to about 1400 families of elementary school children in each community.
(In Utah, questionnaires were also distributed to families of high
school students.) Questionnaires were patterned after the 1966 British
Medical Research Council's Questionnaire on Research Into Chronic
Bronchitis. In each community, over 1000 completed questionnaires
-------
648
were returned for data processing. Response rates ranged between
85 to 95 %. In Chicago, similar eelf-administered questionnaires
were distributed to 38,800 white and 3200 black male military
inductees between 18 and 2^ years of age. Study participants who
reported coughing and phlegm from the chest for at least three months
per year were considered to have chronic bronchitis.
2.2 Determinations of ARD Incidence
Determinations of incidence rates of ARD were made in Chicago
from December 1969 through November 1970, and in New York from September
1970 until May 1971. As with CRD, determinations of ARD rates were
made across pollution gradients in both areas. In each area, partici-
pating families were enrolled in a biweekly telephone survey, in which
trained interviewers inquired about the presence of acute upper and
lower respiratory complaints in all family members during the previous
two weeks. In both areas, incidence rates were computed for total
ARD, for lower respiratory disease, and for upper respiratory disease.
For both Chicago and New York, ARD rates were computed for
four family segments, mothers, fathers, elementary school children
(aged 5-12 years), and preschool children (aged less than 5 years).
In Chicago, about 625 families, representing about 2700 individuals,
participated in the ARD survey. In New York, about 650 families,
representing about 3000 individuals, participated. All ARD study
participants in Chicago and New York lived within 1 1/2 miles of
a municipal air monitoring station.
2.3 Exposures to Air Pollution
For all communities or zones in the four study areas of Utah,
Idaho-Montana, Chicago, and New York, estimates of past air pollution
were made for each year of the 20 to 30 years preceding the CRD
survey. Three major tools were used to construct these estimates;
actual aerometric measurements made by local, state, or federal
organizations; emissions data from smelters and other industrial
complexes; and meteorologic models of pollution dispersion. Aerometric
data for the periods of the ARD survey were provided by the cities
of Chicago and New York.
-------
6A9
3. Results
3.1 CRD Prevalence Surveys
In each of the four study areas, levels of sulfur oxide and
particulate pollution tended to decline over the 20 years preceding
the CRD prevalence survey 1n that area (Table 1). The pollutant
showing the most dramatic decreases was S(L. Declines In TSP, though
quite steady, were not proportionally as large as those 1n S02.
Levels of SS were generally estimated to have undergone the smallest
proportional decrements. (An exception to these trends occurred
in Idaho-Montana, where the estimates of maximum levels of SCL
and SS actually Increased slightly between 1950-59 and 1968-70).
In all four study areas, communities were divided Into high-exposure
and low-exposure categories on the basis of estimated exposures
during the 20 to 30 years preceding the CRD surveys. Within each
of the high-exposure and low-exposure categories, chronic bronchitis
prevalence rates were computed separately for mothers and fathers.
(In Chicago, rates were computed separately for black and white inductees.)
Within these categories, rates were computed separately for lifetime
non-smokers, ex-smokers, and current cigarette smokers.
With the exception of black military Inductees from low-pollution
areas around Chicago, all groups reported higher chronic bronchitis
prevalence among smokers than among non-smokers (Table 2). Prevalence
rates among ex-smokers were generally Intermediate between non-smokers'
and current smokers' rates. In both non-smokers and current smokers,
prevalence rates were consistently higher in areas of elevated air
pollution than in those of low pollution. In all four areas, the
highest prevalence rates were found in smokers living In communities
or zones of elevated pollution.
An effort was made to determine the effect of air pollution
relative to cigarette smoking on chronic bronchitis prevalence rates.
The first step in this determination was to calculate the excess prevalences
due to smoking alone and to pollution alone* In each area-apeoifie
and sex-specific group, the excess prevalence attributable to smoking
is equal to the difference between the prevalence among emokera in the
low-pollution communities and the prevalence among non-smokers in the
low-pollution communities. The excess prevalence attributable to
pollution 1s equal to the difference between the prevalence among
-------
TABLE 1
Range of Exposure to Air Pollutants (in
In Four United States Areas, During 1950-1959
and During 1968-1970
Time Period
.
1950-59
1968-70
Q -"
Range of Exposure (in wg/m )
so2
Minimum (Area)*
142 (U)
92 (U)
Maximum (Area)
419 (NY)
210 (NY)
.
TSP
Minimum (Area)
139 (I-M)
88 (U)
- -
Maximum (Area)
214 (C)
155 (C)
•
SS
Minimum (Area)
15 (I-M)
15 (U)
Maximum (Area)
27 (NY)
20 (NY)
*Area abbreviations are as follows:
U = Utah
I-M - Idaho-Montana
C = Chicago
NY = New York
-------
TABLE 2
Chronic Bronchitis Prevalence Rates, Distributed
by Smoking Status and Pollution Exposure: United
States Studies, 1969-1970
Community
Exposure &
Smoking Status
Low Pollution
Nonsmokers
Smokers
High Pollution
Nonsmokers
Smokers
Chronic Bronchitis Prevalence (in %)
Utah
Mothers
4.16
15.80
5.20
22.25
Fathers
3.00
19.60
6.81
26.80
Idaho-Montana .
Mothers
1.08
11.78
2.54
12.88
Fathers
1.25
17.05
3.48
18.36
New York
Mothers
2.00
13.90
6.04
18.08
Fathers
4.60
13.89
15.87
21.71
Chicago
Blacks
9.20
8,74
9.84
13.52
Whites
4.00
15.20
5.32
18.08
-------
652
non-smokers in the high-pollution communities and the prevalence among
non-smokers in the low-pollution communities. (For black inductees
in Chicago, the excess prevalence due to smoking was calculated to be a
negative number. As yet* we have no convincing explanation for this
unusual finding.)
The second step in this determination was to divide the excess
prevalence attributable to pollution by the excess prevalence attributable
to smoking. For mothers, the pollution effect on chronic bronchitis
prevalence rates was 9% to 34% as strong as the smoking effect (Table 3).
For fathers, the pollution effect was 14% to 121% as strong as the smoking
effect. For both mothers and fathers, the effect of pollution relative
to smoking was strongest in New York. In white military inductees,
the pollution effect was 18% as strong as the smoking effect.
2. ARD Incidence Surveys
2.1 Chicago Study
In Chicago, ARD study participants were divided into a high and
a low air pollution exposure group. Mean pollution exposures for
both groups during the study period are presented in Table 4. In
all family segments in Chicago, the incidence- rates of upper and lower
tract ARD combined were higher In the high-exposure group than In
the low-exposure group (Table 5). Increases in total ARD between
exposure groups ranged from 2% in children aged 3 to 5 years, to 37%
In children aged less than three years. Increases in rates between
exposure groups were particularly apparent in families who had lived
at the same address for at least three years. In Chicago, differences
between exposure groups in rates of both upper and lower tract ARD
were statistically significant at a = 0.05.
2.2 New York Study
In New York, ARD incidence rates In the community of lowest air
pollution exposure were compared to the combined rates in the two
communities of higher exposure. Mean pollution exposures for the
high- and low-exposure groups during the study period are presented
In Table 4. Incidence rates of total ARD In New York, as in Chicago,
were generally higher in the high-exposure communities than in the
low-exposure communities. Elevations In rates between exposure areas
were statistically significant for lower, but not for upper respiratory
-------
TABLE 3
Excess Chronic Bronchitis Prevalence Attributable
to Cigarette Smoking and to Air Pollution, United States Studies,
1969-1970
Source of Risk
Pollution
'Smoking
jPolluti on/Smoking
Excess Chronic Bronchitis Prevalence (in %)
Utah
Mothers
1.04
11.64
0.09
Fathers
3.81
16.60
0.23
Idaho-Montana
Mothers
1.46
10.70
0.14
Fathers
2.23
15.80
0.14
New York
Mothers
4.04
11.90
0.34
Fathers
11.27
9.29
1.21
Chicago
Blacks
0.64
-0.46
-1.39
Whites
1.32
11.20
0.18
Oi
Ul
-------
654
TABLE 4
Mean Pollution Exposures, in pg/m , During
Acute Respiratory Disease Surveys in Chicago (1969 - 1970) and
New York (1970-71)
Area
Chicago
New York
Mean Pollution Level (in pg/m3)
so2
.ow
Exposure
57
23
High
Exposure
106
63
TSP
Low
Exposure
111
34
High
Exposure
151
104
SS
Low
Exposure
14.5
10.2
High
Exposure
16.0
14.3
TABLE 5
CHICAGO: RELATIVE RISK OF TOTAL ACUTE
RESPIRATORY DISEASE
FAMILY
SEGMENT
FATHERS
MOTHERS
SCHOOL
CHILDREN
CHILDREN
3-5 .
CHILDREN
0-2
COMMUNITY AIR POLLUTION EXPOSURE
INTERMEDIATE
1.00
1.00
1.00
1.00
LOO
(2.80)*
(4.76)
(7.04)
(9.35)
(9.41)
HIGHEST
1.33
1.25
1.18
1.02
1.37
• (BASELINE ARD RATES, PER 100 PERSON-WEEKS,
ARE IN PARENTHESES.)
-------
655
disease. Elevations in rates were more apparent in people who had
not changed address in the past three years than in those who had
(Figure 1).
4. Discussion
The CRD prevalence surveys consistently demonstrated higher chronic
bronchitis rates in polluted communities than in relatively clean ones
The effect of pollution relative to smoking, which ranged from 9%
to over 100%, was generally stronger than the investigators had expected.
From the CRD survey results, we would not argue that air pollution
is as strong a determinant of chronic bronchitis as is heavy smoking.
We would suggest, however, that long-term exposure to sulfur oxide
ana particulate pollution combined may rival moderate smoking in
promoting the development of that disease.
In the ARD incidence surveys, illness rates were consistently
highest in residentially stable families in high-pollution neighborhoods.
This result suggested that several years of continuous exposure to
elevated pollution levels might be required to develop increased suscepti-
bility to ARD. The ARD survey results also suggested that improvements
in air quality, such as have occurred in many American cities since 1970,
might be accompanied by decreased MKU rates, in the investigators'
best judgment, reductions in annual average S09 levels from about
33
110 ug/m to about 57 vg/m , accompanied by reductions of SS from
about 18 to about 14 yg/m , might well promote reductions in the incidence
of acute respiratory disease.
-------
FIGURE 1
2.0
. 1.5
RELATIVE
RISK
OF 1.0
ACUTE
ILLNESS
0.5
0
CHILDREN AGED
0-5 YEARS
MOTHERS
1.25
1.00
(8.18:
0.98
1.15
1.18
1.30
1.00
(4.31
1.20
DID NOT
MOVE
MOVED
WITH IN 3 YEARS
D I D NOT
MOVE
MOVED
WITH IN 3 YEARS
LOW EXPOSURE
HIGHER EXPOSURE
* (BASELINE ARD RATES, PER 100 PERSON-WEEKS, ARE IN PARENTHESES.)
FIGURE 1: NEW YORK: EFFECT OF MOBILITY ON TOTAL ACUTE RESP/RATORY DISEASE, 1970-1971
-------
657
REFERENCES
1. House, D.E., et al. Prevalence of Chronic Respiratory Disease
Symptoms in Adults: 1970 Survey of Salt Lake Basin Communities.
Health Consequences of Sulfur Oxides: A Report From CHESS,
1970-71. In press at the U.S. Government Printing Office.
2. Hayes, C.6., et al. Prevalence of Chronic Respiratory Disease
Symptoms in Adults: 1970 Survey of Five Rocky Mountain
Communities. Health Consequences of Sulfur Oxides: A Report
From CHESS, 1970-71. In press at the U.S. Government Printing
Office.
3. Finklea, J.F., et al. Prevalence of Chronic Respiratory Disease
Symptoms in Military Recruits, Chicago Induction Center, 1969-
1970. Health Consequences of Sulfur Oxides: A Report from CHESS,
1970-71. In press at the U.S. Government Printing Office.
4. Goldberg, H.E., et al. Prevalence of Chronic Respiratory Disease
Symptoms 1n Adults: 1970 Survey of New York Communities. Health
Consequences of Sulfur Oxides: A Report From CHESS, 1970-71.
In press at the U.S. Government Printing Office.
5. Chapman, R.S., et al. Chronic Respiratory Disease. Arch. Environ,
Health. Vol. 27-3:138-142, September 1973.
6. Love, G.J., et al. Prospective Surveys of Acute Respiratory
Disease in Volunteer Families, 1970-71. Health Consequences
of Sulfur Oxides: A Report From CHESS, 1970-71. In press
at the U.S. Government Printing Office.
7. Finklea, J.F., et al. Prospective Surveys of Acute Respiratory
Disease in Volunteer Families: Chicago Nursery School Study.
Health Consequences of Sulfur Oxides: A Report From CHESS,
1970-71. In press at the U.S. Government Printing Office.
8. French, J.G., et al. The Effect of Sulfur Dioxide and Suspended
Sulfates on Acute Respiratory Disease. Arch. Environ. Health
27-3:129-133, September 1973.
DISCUSSION
SULAIMAN (Nigeria)
In Nigeria where 1O-15% of people carry sickle cell trait
or sickle cell anaemia, what part has carbon monoxide to play
in causing adverse human effects?
-------
658
CHAPMAN (U.S.A.)
To ray knowledge, very little research is available on the
effects of carbon monoxide on patients with sickle cell anaemia.
In people without that disease, levels of about 50 ppm carbon
monoxide have been well demonstrated to shorten the time re-
quired for exercise to produce angina pain in cardiac patients.
FREEMAN (U.S.A.)
Do you happen to have equivalent data with regard to either
NO- or 0_ or both?
CHAPMAN (U.S.A.)
Within the next six months, EPA reports will be written on
the effects of nitrogen oxides and oxidants on chronic respiratory
disease and other indicators.
HICKEY (Ireland)
What is your definition of a smoker? Have you distinguished
between smoking habits of the mothers and the fathers?
CHAPMAN (U.S.A.)
In this report, I have defined a smoker as anyone who cur-
rently smokes cigarettes. Generally, we have observed that
smoking rates are higher in fathers than in mothers, and that
chronic bronchitis rates are highest in fathers who smoke.
However, we have also observed that chronic bronchitis are
higher in non-smoking fathers than in non-smoking mothers. This
last observation was particularly striking in polluted communities
in New York, where non-smoking mothers had a chronic bronchitis
rate of 6.04%, and non-smoking fathers had a rate of 15.87%.
-------
659
BRONCHITE CHRONIQUE, SYMPTOMES RESPIRATOIRES
ET POLLUTION ATMOSPHERIQUE
P, REY, D, RAMACIOTTI, B, VOINIER ET R, LANG
Institut de m^decine sociale et preventive, UniversitS de GenSve,
Suisse
RESUME
Dana cette &tudet on a d'une part applique le questionnaire
sur la bronahite ahronique du British Medical Council, a 2882
•
personnes appartenant a la population active du Canton de Geneve
et d'autre part analyst lea donnees de pollution atmoapherique
enregistrees a 10 pastes situes en ville ou & la aampagne, Les
2
teneurs en SO, vena on trees (teneur moyenne: 30 ,ug/m en ville)
6 /
dependent essentiellement des ohauffages domestiqueet la pollu-
tion induatrielle etant pratiquement negligeable. Pour definir
le niveau de pollution moyen durant la peviode de I'enquete, on
a tout d'abord elimine, d. I'aide d'un programme de dSeaisonnali-
sationf lee variations 8aisonniereet puis aherche^ a I''aide d'une
regression multiple par past une relation entre les niveaux moy-
ens aux 10 pastes et 2 parametres exprimant I'abondance dee sour-
ces de pollution par le SO„, eoit la densite de population et le
&
chiffrtt d'affaires theorique dee ramoneurs. Une carte par com-
mune des niveaux de pollution a ainsi pu itre etablie ofi. I'on a
plac6 lea prevalences de la bronchite chronique ou dea symptdmes
reapiratoirea non bronohitiquea striotu senso. On a enfin intro-
duit un niveau personnel de pollution par individu en tenant
compte du lieu d'habitation.
D'apree lea resultats obtenus juequ'ici et en utiliaant le
SO,, comme indioateur, la prevalence de la bronchite chronique
&
tend d. augmenter avec I'augmentation de la pollution provenant
-------
660
des chauffages. Cette relation devient hautement significative
pour autant que I'on puisse recourir d. plusieurs indices de la
pollution. Il n'est pas exalu que la pollution atmosph&rique
telle qu'elle exists d. Gen&ve contribue aussi d. aggraver les symp-
tomes respiratoires qu'ils soient spe"cifiques ou non.
ABSTRACT
In this study the British Medical Council questionnaire on
chronic bronchitis was used to obtain information on 2882 people
belonging to the working population of the Canton of Geneva;
secondly, atmospheric pollution data recorded at ten points in
urban and rural areas were analysed. The SO,, concentrations
3
encountered (average concentration 30 ,ug/m in town) originated
principally from domestic heating, pollution of industrial ori-
gin being practically negligible. To define the average pollu-
tion level during the survey period we first of all eliminated
seasonal variations by a "deseasonalisation" process and then,
by applying multiple step-by-step regression, sought a relation-
ship between the average levels at the ten control points and two
parameters expressing the frequency of SO- pollution sources, or
lu
the -population density and the theoretical volume of business of
chimney-sweeps. Maps showing pollution levels could thus be
drawn for each commune where chronic bronchitis or respiratory
symptoms not strictly speaking bronchitia were shown to be pre-
valent. Finally, a personal pollution level per individual wae
included, according to the place of residence.
From the results obtained to date, and using SO_ as an in-
dicator, it appears that the prevalence of chronic bronchitis
tends to increase with the increase of pollution from central
heating systems. This connection becomes highly significant
as long as it is possible to refer to several pollution indices.
The possibility is not excluded that atmospheric pollution, as
existing in Geneva, contributes to the aggravation of respiratory
symptoms whether specific or not.
-------
661
INTRODUCTION
La mise en relation de la pollution atmosphe'rique avec ses effets sur la
bronchite chronique a ddja fait 1'objet de nombreux travaux dont quelques
uns ont &t& revus dans un article precedent (1). Actuellement encore, de
larges enquetes sont menses sur ce sujet, attestant par la qu'il est loin
d'etre e"puise". II pr^sente, en effet, cette difficult^ que la pollution at-
mosphe'rique entre en concurrence avec d'autres facteurs exogenes comma la
fume"e du tabac ou 1'exposition aux polluants d'origine professionnelle. De
maniere a distinguer 1*action propre de la pollution atmosphe'rique, on re-
court le plus souvent a la selection de groupes comparables sur plusieurs
points habitant par exemple, soit la ville, soit la campagne. On interroge
communSment les habitants d'un secteur proche d'un point de mesure du pol-
luant mesure. Ce type d'enquete reclame une population assez dense pour qu'
on puisse y trouver les groupes appropri£s et une region g€ographique as-
sez etendue pour que les niveaux de pollution soient tres contrasted, Dans
notre enquete, la situation de depart est diff«Srente i le territoire consi-
dere" est exigu (284 km2 pour 332.000 habitants) et le gradient de pollution
dans 1'espace, faible au depart, a d£cru ces dix dernieres ann£es. C'est
pourquoi nous nous sommes orientes vers une autre me'thodologie qui, sans
nous permettre des conclusions definitives, peut orienter nos recherches
futures et Sventuellement inspirer d'autres enquStes menses dans de aembla-
bles conditions. Cette m^thode est une tentative de roettre en oeuvre des
analyses math€matiques ou d'une part des phfinomenes connus et repertories
peuvent itre utilises a la place des dosages chimiques de la pollution
(qu'on ne saurait multiplier a 1'infini), et oQ d'autre part les facteura
de pollution, introduits dans une se*rie d'autres variables, manifestent
leur pouvoir discriminant vis-a-vis des bronchitiques ou des non-bronchiti-
ques du collectif examine.
METHODOLOGIE ET RESULTATS
1. Pollution atmosphgrique ; les donn^es
II s'agit des niveaux relatifs de SO determines, en une dizaine de
points du Canton de Geneve, a 1'aide d'appareils Leclerc, par les soins du
Laboratoire de toxicologie et d'analyse de 1'air (Dir, P. Desbaumes) de 1*
Institut d'hygiene. Si les appareils Leclerc prfisentent 1'inconvenient de
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662
ne pas fournir de concentration, ils ont 1'avantage d'etre peu couteux et
d'un maniement aise", ce qui a permis le relieve" systdmatique de valeurs du-
rant plus de 10 ans,
2. Conditions atmosphe'riques ! les donnges
II s'agit de releve"s faits a l'ae*roport de Cointrin, portant sur la tem-
pgrature de 1'air, son humidite", le point de rose"e, la visibility, la pres-
sion atmosphe'rique, etc. Etant donn£ 1'exiguite" du canton, on peut admettre
que ces re leva's fournissent une bonne approximation des conditions me'te'o-
rologiques qui rdgnent sur I1ensemble du territoire. La situation gSogra-
phique tres particulidre du canton de Geneve explique en grande partie com-
ment les conditions metSorologiques e"voluent dans l'anne"e et comment elles
influencent la repartition, sur tout le territoire, des polluants atmosphe'-
riques qui proviennent en majority de 1'agglomeration urbaine. Le canton de
Geneva est en effet enferme" entre les barri&res naturelles du Jura et des
Alpes. Les vents sont ainsi canalises dans deux directions privile°gi€es et
sont associSs a des climats trSs opposes. Quand le vent du Nord-Est ("bis^')
souffle, elle entralne les polluants a tr§s grande vitesse et le ciel est
"nettoye"". Les vents du Sud et de 1'Quest apportent surtout la pluie et la
basse pression et leur vitesse est inf£rieure aux vents du Nord. La situa-
tion d'inversion se rencontre en automne et en hiver, et occasionnellement,
au printemps. Les couches de stratus situe"es entre 100 et 300 m au-dessus
du sol envahissent 1'ensemble du territoire. En hiver, oO 1'inversion noc-
turne se prolonge le jour, elle peut durer plusieurs jours conse"cutifs. c1
est a cette occasion qu'on peut voir la pollution atmosphe"rique atteindre
ses niveaux les plus Sieve's.
3• Variations de la^ pollution atmosphe'rique par le SO,.
Vu ce qui precede, on ne s'Stonnera pas de constater que la pollution
par le.SO est caracte'rise'e, en plus de tr£s fortes variations saisonnie-
res, par des fluctuations acycliques, en particulier durant la mauvaise sai-
son. De maniSre a choisir une valeur qui soit representative de la pdriode
de 1'enquete et qui puisse servir de base & la mise au point d'une carte,
nous avons appliquS aux relev^s provenant des dix postes de mesure, une
thode dite de "dgsaisonnalisation" (2). Elle permet de mettre en Evidence
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663
un "trend", une composante saisonniere et une composante alSatoire, A par-
tir de ces calculs, on a obtenu une valeur annuelle pour la pe"riode de 1'en
quete aux different? points d1observation.
4. Indicateurs indirects de la pollution par le SO
Re'sume'e dans I1 introduction (cf supra), la methods appliqufie sera dficri-
te da~s une autre publication (3). A 1'occasion d'une regression par pas, 2
parametres se sont montr6s en tres forte correlation avec les valeurs de
SO calcule'es selon la me'thode de"crite ci-dessus (outre les paramStres me'-
tfiorologiques) : 1'un, Evident, est la densitfi de la population, 1'autre»inat-
tendu/est le chiffre d'affaires th^orique des ramoneurs, qui est proportion-
nel 3 la puissance des installations de chauffage. A partir de ces chiffres
I,
d'affaires th£oriques par hectare, obtenus dans les diffe"rentes communes du
Canton, nous avons pu dresser une premiere carte ou des zones d'"isopollu-
tion" de niveaux croissants recouvrent soit 1'agglomeration urbaine, soit
les zones sub-urbaines, soit les regions rurales, ou semi-rurales.
5. Prevalence de la bronchite chronique et des sympt6mes respiratoires
1681 homines et 1201 femmes appartenant aux secteurs de I1Industrie, du
commerce et de 1'administration ont rempli le questionnaire du British Me-
dical Council, traduit en francais par la CECA. L'enquSte s'est prolonged
durant plusieurs mois. Partant de la definition de la bronchite chronique
donnee par le Ciba Guest Symposium, nous avons extrait du collectif les
bronchitiques sur la base de quatre questions touchant la toux et 1'expec-
toration chronique. Puis nous avons sorti du collectif restant les sujets
dits indemnes de symptdmes respiratoires. Entre deux se trouvent les sujets
pre'sentant des symptomes respiratoires, sans que ceux-ci satisfassent la d6-
finition de la bronchite chronique : ils constituent le groupe dit interme'-
diaire (1). D'apres 1'analyse que nous avons appliqu^e au questionnaire (4)
les questions se'lectionne'es par nous se sont re've'le'es particuli§rement ap-
tes a discriminer les bronchitiques vrais des non bronchitiques; mais tel
n'a pas £t£ le cas pour les questions se rapportant d 1'exposition aux in-
temp€ries ou a 1'exposition aux polluants atmosph^riques. II serait trop
t6t pour en conclure que la pollution ou les intempe'ries n'interviennent
pas dans la discrimination entre bronrhitiques et non bronchitiques, car on
peut aussi mettre en doute la validity des questions qui concernent ces der-
niers facteurs.
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664
6. Mise en relation des prevalences avec lea Equivalents de la pollution
Aprds avoir regroup^ les communes en classes selon la densite de la po-
pulation et le chiffre d'affaires des ramoneurs, on a place* en regard, le
nombre de personnes examinees N_r le nombre de bronchitiques B^, et calculi
le taux (en %) de la prevalence p « 100 x B . On a fait de m€me pour la po-
N
pulation intermediaire. En vue de d€celer une eventuelle relation entre la
prevalence p_ et la densite x_ d'une part, entre la prevalence p_ et le chif-
fre d'affaires des ramoneurs v_ d'autre part, on a transforms' ces prevalen-
ces p_ en probits, puis ajust^ & ces probits une regression lin£aire dont la
variable explicative (independante) est soit x_, soit y_, selon une technique
statistigue d£crite par example par Linder (5). A ces ajustements, sont as-
sociees deux analyses de variance avec leurs tests de signification ( base's
sur la repartition du x2 )> elles permettent les observations suivantes :
a. Dans le cas de la density de la population, le coefficient de la regres-
sion ne diffSre pas significativement de zero (seull : 5 %)j la variabi-
lite residuelle est un peu trop grande, mais ne doit pas itre considered
cependant comroe significative.
b. Dans le cas du chiffre d'affaire, le coefficient de regression ne diffe-
re pas significativement de zero (seuil : 5 %) et la variabilite residu-
elle n'est pas significative (seuil : 5 %).
c. Quand on combine differents parametres, representatifs de la
pollution, dont la densite de population et le chiffre d'affaires
des ramoneurs, le coefficient de regression devient hautement
significatif ( p< 0,001).
D'apres ces resultats, on peut pretendre qu'il existe a Geneve
une relation entre la prevalence de la bronchite chronique et la
pollution atmospherique pour autant qu'on puisse recourir a plusieurs
indices de cette pollution. Avoir mis en evidence cette relation
parait d'autant plus interessant que la prevalence de la bronchite
chronique a Geneve est* basse et que les niveaux de pollution sont
faiblee.
7. Introduction des donnSes de pollution dans 1'analyse typologique
On peut se demander, entre autres choses,si la pollution atmospherique
favorise le developpement de la bronchite chronique en tant que maladie spe-
cif ique ou si elle agit sur les sympt&nes respiratoires meme banalsi si, com-
me cela a ete avance, son effet est augmente quand elle se combine a d'au-
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665
tres facteurs exog£nes comme la fum€e de cigarette. Suffit-il d'etre expose
& la pollution pour etre atteint ? suffit-il de ne pas etre expose* pour 6-
chapper A la bronchite chronique ? ou faut-il posse"der un terrain predispo-
se" ? sans permettre de r£pondre a toutes ces questions, 1'analyse typologi-
que offre la possibility d'en explorer quelques-unes. L'analyse typologique
precede H des regroupements successifs d'individus en des types dont ils
sont le plus proche suivant une ne'trique appliqu€e a des variables-indica-
teurs. Dans un cas comme le notre, chaque rfiponse au questionnaire est con-
sid£re"e comme une variable, mais aussi les donn^es raesur^es comme le de"bit
expiratoire dc pointe. Le processus de constitution des types passent par
des phases successives. Dans la premiere, A partir de plusieurs Schantil-
lons extraits de fa une Evaluation de la variance expliqu^e s'arrStant &
la liraite que 1'utilisateur fixe lui-mfiroe selon le degrfi d'homog«n«it6 qu'
il recherche j le programme attribue de plus, d chaque individu, un num^ro
de type qui permet de le faire entrer dans une nouvelle catSgorie > c'est
ainsi que nous avons pu comparer notre classification • priori, inspired de
la definition du Ciba Guest Symposium, A celle donn6e par le programme. A
cette analyse s'ajoute une €tude topographique des variables ou des types,
dans la representation de I'espace factoriel.
L'analyse typologique a montre" que la pollution atmosphfirique, telle que
nous I1avons dfifinie, 6tait 1'un des parametres qui prfisentait un fort pou-
voir discriminant vis-a-vis de la population e'tudie'e (I1 ensemble du collec-
tif sus-mentionne). En d'autres termes, nous avons examin6 des sujets aussi
bien dans les regions de haute que de basse pollution. Parmi les sujets d«-
finis comme bronchitiques chroniques, certains sont exposes & des hauts ni-
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veaux de pollution, d'autres pas. Cependant, c'est le groupe qui presents
les signes de complication les plus rrpvac «i'l »«+• «munH R anx niveaux les
plus Sieves. L'association entre forte consomniation de tabac et forte pollu-
tion ne s'est pas re've'le'e plus active que chacun des deux parametres pris
isole"ment.
En bref, 1'analyse typologique permet de conclure que, dans notre collectif,
la teneur en SO ne serable pas avoir joue" le role d'un facteur dSclenchant
de la bronchite chronique, mais qu'elle pourrait favoriser 1'aggravation
des symptomes respiratoires, au moins chez les bronchitiques.
References :
1. Rufener-Press, C. , Rey, P. et Press, P. : Une e"tude ^pid^miologique de
la bronchite chronique a Geneve. Respiration, 30, 458-516 (1973).
2. Ramaciotti, D. et Imhoff, C. : Essai de d£saisonnalisation des donn^es
de pollution atmosphe'rique par 1'anhydride sulfureux, relev£s a Geneve
entre 1959 et 1971, Rev. Med. Prey., 1£, 6, 379-384, (1973).
3. Ramaciotti, D. et Imhoff, C. : Recherche d'indicateurs indirects de la
pollution atroosph^rique (5 paraltre).
4. Rey, P. et Voinier, B. : Computational validation of the BMC question-
naire on respiratory symptoms (a paraltre).
5. Linder, A. : Statistische Methoden, Birkhauser Verlag, Basel und Stutt-
gart, 1964.
Reroerciements ; Ce travail a pu etre realise* gr&ce a 1'aide du Fonds natio-
nation suisse de la Recherche scientifique, du Laboratoire de toxicologie
et d'analyse de 1'air, du service de me'te'orologie de I'afiroport de Cointrin,
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DISCUSSION
RYLANDER (Suisse)
Est-ce qu'il y a une influence des classes sociales dans
votre materiel? On sait bien qu'il y a une influence importante
sur la frequence de la bronchite chronique par les classes sociales,
KEY (Suisse)
Toutes les classes sociales ne sont pas represented dans
notre echantillon de la population active mais parmi celles qui
s'y trouvent, nous n'avons pas observe1 de difference dans la
prevalence de la bronchite chronique.
BRILLE (France)
Les valeurs de SO_ prises en compte dans la relation entre
bronchite chronique et pollution sont-elles des moyennes annuel-
les, une moyenne ponderee ou la valeur d'un mois?
REY (Suisse)
II s'agit de raoyennes annuelles calculees sur la base de
relevSs hebdomadaires exprimes, pour permettre la comparaison
entre les differents postes, en mg de S0_ par 30 jours, deposes
sur le papier impregnfi (methode Leclerc).
SCHLIPKOETER (Republique Federale d'Allemagne)
Comme vous avez pu dficeler, en depit de valeurs SO, tres
faibles, une augmentation du syndrome de la bronchite 3. mesure
que les concentrations de S0_ s'elevent - ce qui est etonnant *•
11 y aurait lieu de verifier si les differences ne sont pas
imputables aux differences de densitS demographique. La bronchite
peut resulter d'une ou de plusieurs infections qui sont plus
frequentes lorsque-la densit€ demographique est plus forte. A-
t-il 6tS tenu compte de la densitg demographique dans les calculs
de cofrelation?
REY (Suisse)
Nous avons etudifi la relation entre la prevalence de la
bronchite chronique et la densit£ de la population sans trouver
de resultats significatifs comme cela a et£ indiqufi dans le texte.
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669
EPIDEMIOLOGICAL STUDIES OF LUNG DISEASE IN
URBAN AND RURAL COMMUNITIES
A, BOUHUYS, C, A, MITCHELL/ H, R, HOSEIN/
J, B, SCHOENBERG, R, E, BINDER, R, S, F, SCHILLING
Yale University Lung Research Center, New Haven, Connecticut, USA
ABSTRACT
We have studied over 7000 residents of one urban and two
rural communities in them United States, aged 7 years and older,
residing in geographically determined areas in which a private
census was obtained. The 7000 persons seen comprise about 75%
of the defined populations; additional door-to-door surveys
provided information about those not seen for validation of the
results in terms of the total community populations. A mobile
laboratory with an on-line computerized data collection system
was used to record answers to a questionnaire on respiratory
symptoms, residential and family history, smoking habits and
other environmental exposures, and to record maximum expiratory
flow-volume curves. Outdoor air quality is being determined
over a period of more than one year in each community, using
standard methods for particulates, SO,., NO., ozone, sulfate and
Z Z
nitrate. In addition, studies with portable air samplers for
respirable particulates, SO g and NO-, were carried out by selected
subjects, monitoring their personal environment over 24-hour
periods. The results in the urban and in one rural population,
both in Connecticut, indicate: (1) there is no significant ex-
cess of respiratory symptoms among lifetime urban dwellers, liv-
ing in an area where particulate pollution exceeds the primary
AQS in 39% of 102 samples; (2) sensitive lung function testa do
not show differences attributable to an increased prevalence of
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airway obstruction among urban adults when compared to rural
dwellers; (3) indoor particulate pollution may add considerably
to the total respirable particulate load; (4) occupational or
domestic, toxic and allergenic inhalants., including tobacco
smoke, are important etiological factors in many persons with
airway obstruction; outdoor air pollution may only play a minor
role.
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1. Introduction
Epidemiological studies on chronic bronchitis and other lung diseases
in communities have generally used population samples. Such samples often
contain too small numbers of persons in defined, homogeneous subgroups to
allow study of more than a few variables. Computerized methods now allow
us to broaden the scope of such surveys to total community populations, to
a wider range of questions concerning lung disease, and to more sophisti-
cated lung function tests. We have developed a mobile computerized labora-
tory, in which standardized, computer-prompted interviews on respiratory
symptoms and environmental exposures (for text of questionnaire, see Bou-
huys [1]) as well as lung function testing are conducted simultaneously on
2 terminals connected to a PDP-8E computer, using a time-sharing system.
With this facility, we have recorded symptoms and lung function data in
about 2500 residents of a rural town (Lebanon) and 1500 residents of an
urban area (Ansonla), both in Connecticut, U.S.A. Similar data have re-
cently been obtained in about 4000 residents of a rural town (Winnsboro)
in South Carolina (Feb.-May 1974). In each community, a total population
census as well as door-to-door surveys in selected areas provide informa-
tion on persons not seen in the mobile laboratory, thus enabling us to es-
tablish the validity of our findings in terms of the total population. An
air sampling network with 3-5 stations is used to characterize outdoor air
quality, using standard methods recommended by the Environmental Protection
Agency. 24-hour samples are analyzed for suspended particulates, sulfate
and nitrate, as well as for SO., NO. and ozone. In the two Connecticut
towns, samples were obtained at least weekly; in Winnsboro, S.C., daily
samples are obtained during four 3-week periods in the fall, winter, spring
and summer. This schedule of sampling defines the outdoor pollution load
over a year.
This paper describes initial results of the surveys in Lebanon and An-
sonia, Conn., and of air quality measurements in these communities. In ad-
dition, preliminary data on 24-hour personal environmental sampling to com-
pare individual pollutant exposure and outdoor air quality are included.
2. Lung function tests. We have selected the maximum expiratory flow-
volume (MEFV) curve as a simple, objective test of ventilatory function
which provides: (a) standard spirometric values, (b) measurements of flow
rates at low lung volumes, which are sensitive Indices of airway obstruc-
tion, and (c) visual recognition of obstructive and restrictive function
loss (see review in Bouhuys [!])•
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The mobile laboratory Is equipped for simultaneous recording of MEFV
curves on two terminals, using Fleisch pneumotachographs to measure expira-
tory flow rates. Flow signals are digitized and stored by the computer,
which also integrates flow to volume, presents each MEFV curve on the
graphic terminal, and calculates forced vital capacity (FVC), forced ex-
piratory volumes (FEV. and FEVJ and peak expiratory flow rate (PEF) from
the 2 blows with the highest FEV. « in a series of 5 blows. Average maxi-
J. * U
mum expiratory flow rates at 50% and at 25% of the FVC (i.e., total lung
capacity minus 50 or 75% of the FVC) were measured from the same two blows.
In contrast to PEF, these flow rates (MEF50% and MEF25%) are largely inde-
pendent of muscular effort; while PEF reflects tracheal size and effort,
MEF50% and MEF25% depend primarily on the caliber of small airways.
Quality-control features of the software include rejection of too short
blows, and calibration and zero drift correction subroutines. Precision
flowmeters, as well as a newly designed motor-driven 5-liter syringe which
delivers air at varying rates simulating an MEFV curve, are used to cali-
brate the volume and flow measurements and to check the computation sub-
routines. For a more detailed description of the system, see Bouhuys [1].
Output format. After the questionnaire and MEFV curves have been re-
corded, data output is obtained (a) in a condensed hard copy format for
immediate review and checking, (b) on punched paper tape, for editing and
transfer to magnetic tape on a home-based PDP-11 computer which classifies
the results in tabular form and performs statistical analyses.
3. Personal environment sampling. To obtain air quality data that may more
adequately reflect an individual's changing environment, we have developed
a portable light-weight and low-noise personal environment sampler (PES)
that allows individuals to monitor their environment during 24 hours
(Hoseln et al. [2]). The design is a modification of an instrument devel-
oped by Burgess et al. [3]. The unit is housed in a suitcase, which con-
tains a blower drawing in air at 250 llters/min. From the mainstream, in-
dividual sampling pumps draw air through collection vessels for SO. and NO.,
and through a membrane filter for respirable particulates (approx. <7pm),
using a cyclone.
4. Results. Symptoms and lung function. Analysis of data obtained in
door-to-door surveys in selected areas of each town has shown similar symp-
tom prevalences among those seen in the mobile laboratory and those not
seen, if men and women of similar age are compared. These results suggest
that our results can be extrapolated to the total community population.
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673
There were no significant differences between the prevalences of per-
sistent cough and phlegm, frequent wheezing and dyspnea on exertion among
rural and urban males (age 7-80 yrs); their smoking habits were similar.
The prevalence of persistent cough and phlegm was similar among rural
and urban nonsmoking men and women (25-6A yrs}, except that older urban
women (45-64 yrs) more frequently reported persistent phlegm (x2 * 5.26,
p <0.05). The prevalence of these symptoms was higher among current
smokers (men and women, 25-64 yrs), without significant.differences be-
tween rural and urban dwellers.
The lung function data showed similar differences. For instance,
FEV- - and MEF50Z were lower in smokers than in nonsmokers but there were
no significant differences between nonsmoking or smoking rural and urban
men or women, 25-64 yrs. In this analysis, stature was taken Into account
by including only data from 150-169 cm tall women and 160-179 cm tall men.
The interpretation of lung function in younger persons (7-24 yrs) requires
a more complex analysis of the simultaneous effects of growth, smoking and
urban or rural residence; this.has not yet been completed. Thus, we have
found no evidence of Increased airway obstruction among urban adult men
and women when compared to rural residents, using a sensitive lung function
test (MEF50Z) which, e.g., detects significant airway obstruction in teen-
aged smokers (Seely et al. [4]).
Outdoor environment sampling. In Lebanon (rural), weekly S0« and ozone
levels were always less than about 50% of the primary air quality standard
(AQS), while total particulatea and NO- exceeded the AQS In, respectively,
2 and 1QZ of all samples taken over a 42-week period. In Ansonia (urban),
ozone was always less than 50Z of the AQs, while particulatea, N0_ and SO-
exceeded the AQS in, respectively, 39, 35 and 1Z of all samples taken over
a 42-week period. Ansonia, situated in the heavily industrialized Nauga-
tuck valley, Is generally considered a polluted town, and our measurements
confirm that partlculate and NO. levels frequently exceed those in rural
Connecticut.
Personal environment sampling. The highest outdoor total particulate
level measured in Ansonia was 144 ug/m . The personal environment samplers
(PES) frequently Indicated higher respirable partlculate levels (up to 303
o
pg/m ) when they were carried about by selected residents for 24 hours,
covering their normal dally indoor and outdoor activities, within 2 miles
from the outdoor samplers. In general, SO. and NO- levels with the PES
were less than those In outdoor air. Thus, indoor environments may add
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674
considerably to the total respirable particulate load. In two instances,
T
homes with very high particulate levels (up to 450 pg/m ) were identified
through the clinical data of their inhabitants, which suggested airway
obstruction unexplained by smoking.
5. Conclusions. This is an interim report on a still ongoing study. In
the data analyzed thus far we have not found evidence to warrant the con-
clusion that urban outdoor air pollution in a typical industrialized U.S.
town is a significant causal factor in the pathogenesis of lung disease,
as evidenced by respiratory symptoms and obstructive lung function loss.
Maximal expiratory flow rates at low lung volumes, which are highly sensi-
tive indicators of airway obstruction in smokers, textile workers, hair-
spray users and others exposed to airway constrictor inhalants, are if
anything higher rather than lower among groups of urban dwellers than
among their rural counterparts, smokers or nonsmokers. There are indica-
tions that indoor air pollution may be a more significant factor in the
pathogenesis of airway obstruction than outdoor air pollution. A complete
analysis of smoking habits, occupational and domestic exposures, as well
as of histories of allergy, in relation to respiratory symptoms and ob-
jective, sensitive and quality-controlled lung function test data, is
required before a causal role of outdoor air pollution in the pathogenesis
of obstructive airway disease can be established.
References
[1] Bouhuys, A., Breathing—Physiology, Environment and Lung Disease,
Grune & Stratton, New York, N.Y., 1974.
[2] Hosein, H.R., J.A. Virgulto, C.A. Mitchell and A. Bouhuys, Personal
environment sampler for participates, sulfur dioxide and nitrogen
dioxide. In preparation.
[3] Burgess, W.A., L. DiBerardinis and F. Speizer, Exposure to automobile
exhaust. III. An environmental assessment. Arch. Environ. Health,
26:325-329, 1973.
[4] Seely, J.E., E. Zuskin and A. Bouhuys, Cigarette smoking: Objective
evidence for lung damage in teen-agers. Science, 172:741-743, 1971.
Supported by USPHS grant HL-14179 (SCOR Program) and Contract
73-2904-R, both from the National Heart and Lung Institute, NIH.
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DISCUSSION
GOLDSMITH (U.S.A.)
The manuscript reports two interesting findings which suggest
that community pollution and indoor pollution may produce impair-
ment of lung function. The first is the low level of FEV, Q in
urban non-smoking women. The second in the "clinical data' and
impairment of lung function in persons with high indoor partic-
ulate matter. Could you give any further information on these
points?
BOUHUYS (U.S.A.)
1, The FEV^ Q values of urban and rural non-smoking women did
not differ significantly. There is no evidence for impairment
of lung function by community air pollution in our study.
2. Details on our studies on indoor air pollution could not
be included in the paper for lack of space. They will be pub-
lished elsewhere.
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USES OF MORTALITY AS A MEASURE OF THE HEALTH
EFFECTS OF AIR POLLUTION
S, C, MORRIS+ AND M, A, SHAPIRO*"*"
+ Brookhaven National Laboratory, Upton, New York, USA
++ Graduate School of Public Health, University of Pittsburgh,
Pennsylvania, USA
ABSTRACT
The data obtained in- a 13 year follow-up of mortality in
two communities subjected to widely different air pollution lev-
els are used to compare the results of different methodological
techniques in the study of mortality differences. Factors are
derived from the data to estimate the error which would result
from neglecting to consider the effects of smoking and residence
history.
There were greater differences in mortality rate within each
community due to smoking than between the two communities. The
higher mortality rates of smokers makes comparisons between popu-
lations with similar heavy smoking habits a more powerful test of
mortality differences due to air pollution than comparisons of
populations with mixed smoking habits. Increased length of resi-
dence in the highly polluted community was associated with in-
creased age-adjusted mortality. The reverse was true in the
control community, suggesting a process of self-selection in in-
migration.
The findings demonstrate that differences in the prevalence
of smoking or in length of exposure to air pollution could affect
the validity of studies not taking these factors into account.
The degree of the resulting error is estimated.
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1. Introduction
The Preamble to the Constitution of the World Health
Organization defines health as "...a state of complete physi-
cal, mental and social well-being and not merely the absence
of disease or infirmity." Although this definition is widely
accepted in principle, most assessments of health effects are
much more limited in scope due to the inability to develop
suitable measurements to determine the presence, Absence or
degree of "health." Mortality statistics are often consid-
ered too crude and non-specific for the analysis of health
effects, yet this non-specificity which may complicate ana-
lytical techniques supports their use as a measure of this
broad concept of health. As Taeuber suggests, mortality
rates reflect cumulative stresses, deprivations of place,
milieu, and life-style, along with insufficiencies of medical
care and health guidance[l],
2. Importance of Various Parameters ojf Mortality
Tabulations of deaths by cause often present difficul-
ties of interpretation due to unreliable and varying diagnosis
and artificialities introduced in the coding process. Infor-
mation on cause of death may help to estimate the impact in
terms of individual suffering and societal costs in the care
of terminal illness. It can also be useful in establishing
cause-effect relationships between environmental stress and
increased mortality. The fact that one population may have
more heart disease or more cancer mortality than another is
certainly useful in analyzing the impact of environmental
stress. But the major measurement of impact must bet does
one population suffer a higher expectancy of mortality within
the age groups that represent substancial losses to society.
As important as cause of death is in the epidemiology of dis-
ease, it is of only supplimentary value in the assessment of
health effects. Age at death is a much more important factor
in evaluating the impact on society than cause. The total
number of deaths within suitably narrow age groupings are also
available for most countries to a high degree of accuracy.
-------
679
3. Design of Mortality S
The design of epidemiological studies of the effects of
air pollution on mortality have been generally temporal or
inter-area comparisons. The availability of data from a
study of the effects of air pollution on respiratory function
done 13 years before by others [2] provided us with the oppor-
tunity to conduct retrospectively a long term follow-up of
mortality in two populations exposed to widely different air
pollution levels. Subgroups of the two populations were
established based on age, sex, smoking history, and residence
(exposure) history at the beginning of the observation period.
Unfortunately, data on occupation and socioeconomic status were not
available.
4, Analysis
Alternative approaches were taken to the analysis of
the data, The general approach was to compare the observed
number of deaths with the expected in each population sub-
group by means of the standard mortality ratio (SMR). The
expected deaths were determined by applying rates from the
1965 U.S. white population. Two methods were employed. The
first determined the expected deaths over the study period by
determining the effective population exposed to the risk of
death at each age level and multiplying by the standard popu-
lation mortality rate for that age. Mancuso and Coulter ex-
plain this method in detail [3], By reducing exposure to terms
of person-years, this method equates a small population ob-
served over several years to a larger population over one
year, allowing easy comparisons between populations with
different observation periods. It also allows easy inclusion
of information on individuals who enter or leave the popula-
tion at any time during the study. Since the mortality
pattern in the sample itself helps to shape the exposure, it
has some influence on the denominator as well as the numerator
of the SMR.
Table 1 presents the data developed for this analysis.
-------
680
SEWARD-NEW FLORENCE
Summary of Data by Smoking and Residence History
Length of Residence
SEWARD
Males
Never smoked
Intermediate
Heavy
Females
Never smoked
All smokers
NEW FLORENCE
Males
Never smoked
Intermediate
Heavy
Females
Never smoked
All smokers
Over
Person-
Years
375-5
669
605
1258
413-5
228.5
423-5
400
1098
429
20 Years
Obs
9
24
18
20
1
4
17
10
14
0
SMR
63
106
190
76
36
49
102
154
50
0
Less Than 20 Years
Person-
Years
122.5
395
512.5
771.5
431
227.5
286.5
375.5
656
182
Obs
0
9
4
3
4
2
5
7
8
0
SMR
0
99
65
47
215
62
101
186
60
0
Total person-years observedt 9860.
TABLE 1.
The second approach was to calculate the a_ priori
expection of mortality from the standard population life
table, based on age and sex at the beginning of the observa-
tion period. Mortality patterns during the study then are
independent of the expected number of deaths. This approach
also allows analysis of generational effects.
In addition to the use of SMRs, survival curves were
used as described by Cutler and Edera 4 . These curves show
the fraction of the population surviving over time. Numeri-
cal comparison of the slope of these curves (linearized on a
-------
681
semi-log plot) for different subgroups, of the population show
differences in the rate of mortality.
5. Findings and Discussion
The findings of the study indicate a significant 2-fold
difference in mortaliity rate between heavy cigarette smokers
and non-smokers in the adult male population! Differences
between smokers and non-smokers within each community were
greater than the differences between communities. An in-
creased mortality rate among long time residents of the
highly polluted area was suggested. Increased length of
residence in the high pollution community was associated with
increased age-adjusted mortality. The reverse was true in
the control community, perhaps suggesting a self-selection
process in in-migration.
The primary parameters influencing mortality rates are
age and sex. These must be taken into account when assessing
the differences in mortality due to other factors. Quanti-
tatively, the difference in mortality rate for long time
adult residents apparently due to air pollution between the
two communities was 3.8/1000, or 0.09/1000 per ug/iP total
suspended particulate air pollution difference. If it is as-
sumed that there is no effect in the more recently arrived
population (actually, the data indicate a. reversal of the ef-
fect in this group) than each unit increase in the percentage
of the population with more than 20 years residence leads to
an increase in the overall adult mortaliity rate of 0.04/1000
or 0.001/1000 for each ug/m^ difference in suspended particu-
lates. Under this hypothesis, 10 percentage points reduction
in the percentage of the population with over 20 years resi-
dence could reduce the mortality rate observed in the general
population by 20 percent.
Each unit increase in the percentage of heavy smokers
in a community resulted in a change in the general adult
mortality rate of 0.12/1000. Thus, a difference of 10 per-
centage units in the fraction of heavy smokers in the two
communities could result in a difference in mortality of
1.2/1000, or about one-third of the difference due to air
pollution.
-------
682
6. Conclusion
Mortality data provide a useful index of the total
health of a population. They are thus a useful tool in the
assessment of the health impacts of environmental stress on
a population. The fact that the stress may work primarily
through specific subgroups of the population and the action
of other concurrent forms of stress can confound the results.
Thus, differences in the relative size of the group primarily
affected by air pollution in the population can result in
spurious differences in the effects on the total population
mortality rate. Differences in the fraction of the popula-
tion that are heavy cigarette smokers, if not accounted for,
may mask or imitate air pollution effects.
By utilizing factors developed here, cohort studies
can be limited to groups for which high mortality rates are
expected. Significant differences due to air pollution,
where they exist, may then be obtained by the use of smaller,
but more carefully selected, samples. Of more general inter-
est, we believe that these factors can provide estimates of
the error inherent in studies which do not include data on
smoking or residence history.
7. Acknowle dgements
This paper is based on a doctoral dissertation sub-
mitted to the Graduate School of Public Health, University
of Pittsburgh. M.A. Shapiro was the major faculty advisor.
S. Morris was supported by a U.S. Public Health Service Trairx-
eeship during part of the research.
References
TAEUBER, I.E., "Mortality and Population", Am. J. of
Public Health 61, 2335 (1971).
-------
683
PRINDLE, R.A., je_t aJL, "Comparison of Pulmonary Function
and Other Parameters in Two Communities with Widely
Different Air Pollution Levels", Am. J. of Public
Health 53. 200 (1963).
MANCUSO, T.F. and E.J. COULTER, "Methods of Studying
the Relation of Employment and Long Term Illness:
Cohort Analysis", Anu _J. Pub]ic Health 49. 1525 (1959)
CUTLER, S.J, and P. EDERA, "Maximum Utilization of the
Life Table Method in Analyzing Survival", J. Chronic
Disease .8, 899 (1958).
DISCUSSION
SULAIMAN (Nigeria)
Most participants give details on the impact of smoking
and general air pollution. How can we remove the effect of
home environment factors from these atmospheric air pollution
MORRIS (U.S.A.)
This is a very important point and one on which considerablv
more study is needed. To take home exposures into account we
?«*™ ?? e Study P°Pul-ation into groups based on level of
indoor pollutants in the home. This could be done by questioning
S£i ?2 i^J Subjfcts as to the ^Pe of equipment and fuel 9
used for heating and cooking, presence of smokers in the home,
etc. Then a sampling of homes in each subgroup must be monitored
to determine variations in exposure. I was especially pleased
to see Professor Bouhuys present some data of this nature in
his paper.
WASSERMAN (Israel)
Some surveys are needed for (characterizing) identifying
particular sources of air contamination which may cause a high
incidence in respiratory illness. With reference to Solaiman's
remark, it should be observed that in some African houses, the
babies are carried on the vack of their mothers until the age of
two, while they are cooking in the home with green wood. These
children are inhaling high amounts of smoke, which - as observed
in cases of necropsy - impregnate their lungs. Thus, in a
survey, the rate of illness will be reported to cooking facilities
in the respective homes.
-------
685
L'INFLUENCE DES POLLUTIONS INDUSTRIELLES
DE L'AIR SUR L'ORGANISME WAIN
W, DOBRYSZYCKA, S, IWANKIEWICZ, K, JACYSZYN,
S, KOTLAREK-HAUS, E, OLEKSYK, K, STANKOWSKA
Institut Biopharmaceutique, Institut des Maladies Internes,
Clinique Otolaryngologique de l'Acad€mie Me'dicale de Wroclaw,
Pologne
RESUME
On a examine la population habitant d proximite dea fonde-
riea de cuivre et a titre de oomparaison - lea groupea de con-
trole de la population n'etant pas exposes aux pollutions indua-
triellea. Chez lea aujets exposes la concentration de plomb
dans le aang etait remarquablement elevee. On a oonetate une
diminution dea aotivitea de ALAD, LDH, Aid, t G-6-PD dans lee
grythrocytea ainai que le taux des haptoglobines eignifioative-
ment abaiese et celui d'alpha-1-antitripsyne - Sieve. Les exa-
mena hematologiquee ont demontre un taux abaiaee de Hb et le
nombre diminuS d'erythvooytea ainai que le pouvcentage plus
eleve dea reticulocytea dans lea groupea dea peraonnea expoaSea
au danger de pollution industrielle.
Aujourd'huit avea le brusque developpement de I'industriali-
sation, le probldme de la sante humaine menaoee par des facteure
physiques, chimiques et biologiquea devient de plus en plus actuel
et imminent. L'objectif de notre travail eat la determination
de I'influenae des oxydee de plombt des oxydes de ouivre et d'
autres faateura toxiquea de I'atmosphere eur I'Stat de la eante
et sur lea proceaaua m&taboliquea de la population habitant a
proximite dea fonderies de ouivre.
-------
686
ABSTRACT
Population groups living in the vicinity of copper foundries
were examined and compared with control groups composed of sub-
jects not exposed to industrial pollution. In the subjects
exposed to pollution the lead concentration in the blood was
remarkably high. We recorded a decrease in ALADj LDHt Aid.
and G-6-PD activity in the erythrocytes, a significant drop in
the number of haptoglobins and a high alpha-1-antitrypsin count.
The haematological tests revealed a decrease in Hb and erythro-
cytes and a higher percentage of reticulocytes in the groups of
people liable to exposure to industrial pollution.
In these days of rapid industrialization the problem posed
by the physical, chemical and biological threat to human health
is becoming more and more immediate. The aim of our work is
to determine the effect of lead oxides, copper oxides and other
toxic substances in the atmosphere on the health and metabolic
processes of population groups living in the vicinity of copper
foundries.
-------
687
MATERIEL ET METHODB3
Les populations suivantes constituent le materiel de nos recher-
che s:
- Groupe I t habitants aux environs de la fonderie de cuivre en mar-
che depuis 10 ans environ (122 sujets);
- Groupe II : habitants aux environs de la fonderie de cuivre en mar-
che depuis 2 ans environ {121 sujets);
- Groupe III : habitants d'une grande ville, dee homines, exposes profes-
sionellement aux gaz d'Schappement (92 sujets);
- Groupe IV : groupe de contr81e englobant les habitants de la campa-
gne, pratiquement non exposes aux pollutions induatriel-
les (134 personnes),
Au total, on a examinS 4^9 cas.
Les personnes en question ont passe des examens me'dicaiuc de
routine, en plus, examen radiologique du thorax, examen de 1'urine, ECO,
examen otolaryngologique. On a pr'oce'dS a une minutieuse enqufite concernant
les conditions de leur vie quotidienne, leurs indispositions subjeotivea,
leur travail professional, leur contact avec des produits chimiquea, leurs
maladies ant^rieures et defauts congenitaux, ainsi que leur charge familiale.
On a determinfi le taux d'hemoglobine (Hb), la valeur de I'hema-
tocrite, le nombre d'firythrocytes et de leucocytes, la formule sanguinef le
nombre de re'ticulocytes et le nombre de plaquettes sel. Rees-Eoker (1).
La concentration du plomb total du sang a 6t6 effeotufie d'apres Dutkiewioz
(2), celle du cuivre dans le s6rum sel.Chiesura (3)1 I'aotivit4 (1'aot.)
de cholinesterase (CHE) sel. la methods de Hestrin en modification de
Huerg-Yesinic-Popper (4), 1'act. de deliydratase de 1'acide delta-aminolfi-
vulinique (ALAD) sel. Bonsignore (4), 1'act, de gamma-glutamyl-transpep-
tidase (GGTP) sel, Orlowski (5), 1'act. des aminotransf^rases (ASPAT, ALAT)
dans le s^rum d'apres Reitman-Prank (6), 1'haptoglobine sel. Jayle (7)»
la c4ruloplasmine sel. Ravin (8), l'alpha-1-inhibiteur de tripsy syne sel.
Homolka (9). L'act. de la phosphatase alcaline (Ph.alc.) et acide (Ph.ao.)
a et4 dgterminee sel. Sigma Tech,. Bull. (10), 1'act. de 1'aldolase (Aid.)
dans les erythrocytes sel. Bruns (ll)f 1'aot. de la d^hydrogenase gluooso
-6-phosphate (0-6-PD) suivant Richterich (12), l*act. de la d^hydrog^naae
lactique (LDH) par la m^thode de Zimmermann et Weinstein (13), 1'electro-
-------
Tableau II.
Coraportement des indices biochimiques
/moyennes arythmetiques/.
Concentration de Pb o
dans le sang jUg/ 100 car
Concentration de Cu ^
dans le plasma, jug/100 cnr
Serum:
«.*>.» WW
Haptoglobines mg/100 cm^
Ceruloplasmine mg/100 cm-3
Pseudochol-est. I.U,
ASPAT " "
ALAT " "
Alk.phosph. " "
Acide phosph. " "
or-1 inhibiteur de tripsyne
4
grgthroogtes; /I.U.^g Hb^_
LDH
Aldolaae
G-6-P Deh.
Acethylcholest.
ALAD
ASPAT
,____«________«_________- __-.._--L
Groupe I
182
-——___— _
274
106,1
44,4
2,0
15,3
12,3
2,0
0,9
1344,0
,
23,6
0,99
4,0
6,0
3,3
0,83
i Groupe II
76
231
122,9
37,4
1,8
17,6
9,7
2,1
0,6
1559,1
20,0
0,75
2*9
6,6
8,0
0,93
Groupe III
______ _ _
114
116,2
40,7
1,2
41,5
34,0
0,91
1251,6
„_, _,_ „
~ — I
20,0
1,2
4,0
4,5
6,6
1,75
Groupe IV
42
___- ._— ______
138
„_____________
144,4
43,2
2,3
15,3
9,4
1,9
0,8
1302,4
— «,
27,3
1,14
4,2
7,2
16,6
0,75
00
CD
-------
689
phorese des iso-enzymes (LDH) sur gel polyacrilamidique sel, Davis (14)*
On a £valu£ les moyennes arithmetiques et les deviations-standard; les
differences entre lee moyennes ont ete determines suivant le test "t"
Student afin d'y trouver la difference valable.
LES RE3ULTAT3 DE RECHERCHE3 ET DISCUSSIONS
Dans les groupes I et II on notait souvent des plaintes centre
la fumSe, contra la mauvaise odeur dans 1'air, et dans les groupes de fern-
mes, contre les naua<5ea et les douleurs dans la region de 1'hypochondre*
Les groupes I et II pr^sentaient en general un etat de sant6 moins loon*
On a trouv^ dans ces groupes dee changements de la muqueuse de la cavite*
buccale et du nez (25 - 30 $) de type allergique aveo endonunagement de
l*odoratt ou bien des changements dans 1'avant-bouche et des genoives
ressemblant a la leucoplasie.
Les resultats de recherche hematologique ont 6t6 groupesdans le
tableau I.
Farmi les femmes du groupe I et du groupe II, le taux de Hb et
le nombre d'^rythrocytes etaient en effet plus bas que dans le groupe de
contrOle. Ce qui est frappant chez les homines du groupe I, o'est le taux
abaisse de Hb; le taux de Hb le plus elev£ et le plus grand nombre d'dry-
throcytes deoiontre le groupe urbain* Le nombre de plaquettes chez les fem-
mes etait le plus bas dans le groupe II ou le temps de menace dtait plus
court* Le pourcentage des retioulooytes dans les groupes I, II et III
etait significativement plus Sieve que dans le groupe de contrflle,
Dans le tableau II on a roontre le comportement de factcurs bio-
chimiques estimes par nous comme lea plus sensibles dans I1evaluation du
danger* La concentration de ploob dans le sang etait la plus dlevee dans
le groupe I et differait de maniere significative des valours moyennes
d'autres groupes* La concentration de cuivre dans le serum du groupe I et
du groupe II etait deux fois plus 5levee que dans les groupes de controle*
L'analyse des resultats de recherche sur les protelnes et sur les enzymes
demontre un abaissement du taux des haptoglobines statistiquement signi-
fioatif, ainsi que la diminution de 1'activite de CHE serique et 1'augmen-
tation du taux d'alpha-1-inhibiteur de tripsyne dans les groupes I et II*
-------
Tableau I. Resultats des examens hematologiques
/moyennes arithmetiques et deviations standard/
Groupe ,
d* examines sexe
/nombre de cas /
fern. /36/
masc./78/ '
fe'm./63/
masc,/54/
_ __ _ _
- —
gr.III masc./91/
fenu/41/
o-t* TV ___ — _____i
masc./9l/
Facteur examine I
^Taux
d hemoglobi
ne en g$
11,6
r — ^
12,8
.
F — - 1
11,2
r —~ i
14,5
L _ J
r — - -
15,6
12,2
I H
13,7
1,1
1,3
.
r - --i
1,1
r~ n
0,9
L _ _ _l
' ~
1,3
1,4
1,2
Hombre en mm-' / x 10^ /.
Erythro
%
3 952,0
i " 1
4 280,0
_ .
r — i
3 793,0
r~ ~"\
4 009,0
_ _ ,
4 703,0
4 181,0
4 532,0
Bytes
S
0,3
1
0,4
_j
- i
0,8
1,7
.
_
0,5
0,8
0,6
Leucc
X
6,3
7,4
—
6,9
6,0
_
-i
5,6
7,0
7,0
>cytes
5
2,3
2,2
j
1,5
1,6
L _ _l
1,3
2,7
2,3
Plaquettes
- 1 s
y j o
. **> _ ! _ _—
127,8 J33,8
i — i —
146,6 [40,6
L
- — r --i
105,4 j 52,6
130,1 i 43, 3
L J
i
150,1 | 50,1
i
127,6 | 40,1
123,6 { 46,0
i
i
r
Reticu
en i
X
10,5
i
10,5
_
_
11,4
9,6
—
8,0
6,4
6,3
locytes
^
S .
10,7
I r
6,3
L _ I
r —
5,4
| _____.f
5,3
. — j
•
4,3
3,5
3,3
1C
o
-------
691
On a constatS des changements nets dans lea enzymes erythrocytaires chez
les sujets exposes, a savoir: 1'act, ALAD e'tait remarquab lenient diminue'e,
a part cela, dans le groupe expos6 a 1'action des facteurs nocifs pendant
un court dSlai de temps 1'inhibition correspondait a 50 % et.dans le groupe
exposd au danger plus longteraps, elle correspondait 'a 75 %• La diminution
de 1'act. de ACHE e'tait la plus marquee dans le groupe urbain expose1 au
danger de vapeurs de plomb te'trae'thyle. Les activit4s de G-6-PD, d'Ald.,
et de LDH e'taient significativement. plus faiblea dans les groupes exposes,
L'Stude de 1'isozymie LDH d'e'rythrocytes a demontr6 des dfiplacements nets
orient6s vers LDH. et LDHC»
4 5
L1Evaluation de micro-intoxication des populations humaines est
un probleme complexe par rapport au nombre £lev£ des facteurs agissant
ainsi qu'au manque des manifestations cliniques nettes, N&mmoins, les
systemes specifiques d'enzymes sensibles au plomb et au cuivre, que nous
avons choisi pour nos recherches, semblent le mieux refle"ter le degr£ de
danger auquel est expose1 la population, d'autant plus qu'ils sont en cor-
relation avec les changements hematologiques* Les troubles enzymatiques
d'6rythrocytes peuvent Stre pr^curseurs ou blen accompagner les changements
morphologiques des Srythrocytes, elles peuvent e'galement temoigner des
troubles dans le metabolisms cellulaire d'autres tisaus de 1'organisme.
REFERENCES
1) KRAWCZYNSKI, J., OSINSKI T., "Laboratoryjne me tody diagnostyczne",
PZWL, Warszawa 196?
2) DUTKIEWICZ T., - Hed. Prncy 3, 183, (1951)
3) CHIESURA P., - La Medicina del Lavoro 61, ^37 (1970)
«t) BONSIGNORE D., CARTASEGNA C., VBRONAHO C., et al.- La Medicina del
Lavoro 59, *H9 (1968)
5) OHLOWSKI M., MEISTER A., - Bioch. Bioph. Acta 73» 679 (1963)
6) REITMAN S., FRANKEL S., - Am. J. Clin. Pathol. 28, % (1957)
7) JAYLE M.F. - Bull. Soc. Clin. Biol. 33, 876 (1951)
8) RAVIN H.A. - Lancet 1, 756 (1956)
9) HOMOLKA J. - Biochemia kliniczna, PZWL, III ed., 539 (1971)
10) Sigma Technical Bull. Nr 10^, March 1963
11) BRUNS F. - Biochem. Zeitschr. 156, 325 (195*O
12) RICUTBRICH R. - Chemia Kliniczna, PZWL, Warszawa 1971
13) ZIMMERMANN H., WEINSTEIN II., - J. Lab. Clin. Med. <46, 607 (1956)
DAVIS B. - Ann. N.Y. Acnd. Sci. 121,
-------
692
DISCUSSION
HERNBERG (Finland)
Les teneurs en plomb que vous avez trouve"es dans tous les
groupes sont si SlevSes qu'il ne peut s'agir que d'une tres
grave erreur me'thodologique. Des dizaines d'fitudes portant sur
les populations et utilisant des methodes controlees ont fait
ressortir des teneurs moyennes en PbB d1environ 15-30 ,mg/10Oml,
qui, meme au voisinage des sources de pollution, dfipassent rare-
ment 4Oying/lOOml. L1 erreur methodologique, qui doit etre, pour
le moins, d'un ordre de grandeur, ote, toute valeur a vos con-
clusions.
KOTLAREK-HAUS (Pologne)
La methode que nous utilisons est une methode de Dithizone
(diphenylthiocarbazone) effectuSe dans le sang total. Les
valeurs normales dans cette mSthode sont conutie celles pour notre
groupe de controle; elles sont un peu plus elevees en comparaison
a la me"thode AAS. Notre mSthode est neanmoins conforme aux
valeurs obtenues par d'autres autres utilisant cette methode,
dont les valeurs s'elSvent a 60-70 meg/100 ml du sang. (1) Crad-
wohls, Clinical Lab. Methods and Diagn. Ed. by Fraenkels.,
Reitman S., C^W. Mosby Comp. 197Oj (2) Kehoc R., Arch. Environ.
Health 1964, 8, 232* (3) Christian G.D.: Analyt. Chem. 1969, 41,
274.
Dans notre groupe de controle la teneur en plomb dans le
sdrum s'e'levait a 10 mcg/lOO ml.
ZIELHUIS (Pays-Bas)
Le tableau I fait ressortir des differences, par exemple
dans les niveaux d'hemoglobine et dans les nombre d'Srythrocytes.
La comparaison des groupes du point de vue des effets de leur
exposition a 1'environnement ne peut se faire que si les groupes
sont par ailleurs comparables en ce qui concerne les conditions
de nutrition, les conditions socio-Sconomiques, le milieu eth-
nique, les pressions baromfitriques, I1 age. Cette e"tude satis-
fait-elle a ces conditions?
KOTLAREK-HAUS (Pologne)
Les groupes examines Staient comparables par rapport aux
moyennes d'Sge, et a la distribution dans les classes particuli$-
res d'age; leurs conditions socio-ficonomiques et gfiographiques
n'etaient pas eVidemment les memes, mais pare!lies. Toutes ces
donn£es ont e"te evalufies par les statisticiens quand S. leur
validity.
-------
693
BERLIN (C.E.C.)
1. Dans votre communication il est indique que les taux de
plombSmie pour les personnes du groupe III (habitants des villes
professionnellement exposes) sont beaucoup plus Sieves que pour
ceux dv groupe II (habitants aux environs d'une fonderie de
cuivre en marche depuis 2 ans) . Que lie Stait la profession des
personnes du groupe III?
2. Vu ces taux de plombe'mie tres Sieve's quelles sont les
actions entreprises pour les require? A partir de quel niveau
de plomb6mie une action prophylatique est-elle mise en route?
KOTLAREK-HAUS (Pologne)
1. Les personnes du groupe III Staient des employes de stations
service et des membres de la police routiere travaillant dans
les endroits les plus pollue's de la ville.
2. Us ont 6t6 soumis a la surveillance speciale, m^dicale et
prophylactique .
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695
EPIDEMIOLOGY STUDIES OF DDi AND DIELDRIN RESIDUES
AND THEIR RELATIONSHIP TO HUM CARCINOGENESIS
J, E, DAVIES+, A, BARQUET+, C, MORGADE+
AND A, RAFFONELLl"1"1"
+ University of Miami, School of Medicine, Miami, Florida, USA
++ Florida State Division of Health, Miami, Florida, USA
ABSTRACT
In contrast to dieldrin residues serologic and adipose sur-
veys of the human DDT residue in the population of South Florida
exhibited significant demographic characteristics in both tissues,
Total DDT residues were higher in blacks than whites, were age
and social class dependent^ higher residues being found in the
less affluent in both races. Similarly, geographic distribution
differences have been identified. These demographic and geo-
graphic distribution frequencies emphasize the significant con-
tribution of non-dietary sources of DDT in incidental DDT pollu-
tion and the identification of clustering within homes and envi-
ronmental studies in a Bahamian island population implicated
household dust as a major contributant to this type of pollution
in tropical areas. When adipose surveys were stratified for
these differences no significant differences in Total DDT adipose
residue were observed in autopsy and biopsy comparisons from 122
cancer cases when compared with race, sex and social economically
matched controls. Similarly, in separate comparisons of these
residues in 24 lung cancers, 14 gastrointestinal cancers, 29
breast cancers, and 15 generalized metastatio carcinomas, no sig-
nificant differences were observed. The complexities of case-
control residue studies are described.
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696
1. Introduction
In contrast to the frequency distribution of human dieldrin residues
several studies of the frequency distribution of human DDT studies in
i»oth fat and blood indicated that this pesticide residue is not homo-
geneously distributed throughout the general population. Earlier reports
emphasize the homogeneity of this residue a frequency which reeraphasized
the concept that incidental human exposure was primarily dietary. Fre-
quency distribution studies of the UDT residue in the general population
cf South Florida, which enjoys a semi-tropical climate, exhibited signi-
ficant demographic differences in both adipose and serological surveys.
Total DDT residues were higher in blacks then whites and were strongly
age dependent in the first two decades of life but less so in subsequent
age groups, exhibited family clustering and was strongly correlated with
social class in the greater Miami area when this parameter was measured
by the Hollingshead Index, the census tract population density or the
census tract median income (Davies et al CO). These socio-economic
differences which were noted from a serological survey of 581 adults in
the general population suggested that higher DDT residues occurred in the
less affluent members of the general population in both races. Geograph-
ic frequency distribution of DDT but not dieldrin has also been recogniz-
ed in sizeable adipose DDT residues in the United States. In a preva-
lence survey of DDT and dieldrin adipose residues from 4,469 members of
the general population from 22 States in the United States in 1968, the
mean Total DDT residue was 4.85 ppm in whites residing in the cooler sta-
tes and 9.21 ppm from whites in the warmer states; in contrast the mean
dieldrin residue was 0.13 ppm in both areas of the continent. From adi-
pose reisudes of 804 blacks, the mean Total DDT was 7.68 ppm in the cool-
er states and 14,37 ppm in the warmer states whereas the mean dieldrin
level was 0.14 ppm and 0.13 ppm in the cooler and warmer states respec-
tively. These demographic and geographic distribution frequency differ-
ences for the human DDT residue emphasize the significant contribution of
non-dietary sources of DDT in incidental human DDT pollution, and the
socio-.economic differences, domestic clustering and environmental studies
in a Bahamian island which implicated the important role of household
dust, reinforce the importance of giving due consideration to the micro-
epidemiologic variables of the home environment in future epldemiologic
studies which may compare the organochlorine pesticide residues in health
and disease. Having these stratification requirements in man, adipose
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697
organochlorine levels in 122 cancer cases were compared with residues
from 122 controls obtained from persons coming to surgery or dying from
non-malignant cases, matched for age, race, sex and social class. This
paper reviews the results of this study and also describes the effect of
social economic class in this adipose organochlorine residue study.
2. Materials and Methods
Serological and biopsy fats were obtained from 122 cancer cases com-
ing to surgery or autopsy on account of the following malignancies: pri-
mary lung cancer, gastrointestinal cancer, breast cancer and generalized
metastatic carcinomatosis. Similar specimens were obtained from 122
matched controls coming to surgery or autopsy for non-malignant
conditions. Organochlorine levels were obtained by gas liquid chromato-
graphy studies on a wet weight basis using the modified Mills procedure
\2\. On the few occasions when the amount of fat was insufficient for
this method a micro-Mills analyses was performed and the results calcu-
lated on a lipid extractable basis. No statistical difference in resi-
dues by either method had been previously detected. In addition to
matching on the basis of race, sex and social class, and with few ex-
ceptions by age, within a 10 year period, information on significant
weight loss was categorized into to three categories. Category I -
moderate to severe (greater than 12 pounds or a pathologist*s report of
emaciation), Category II - less than 12 pounds or no weight loss, and
Category III - where weight loss was not recorded.
3. Results
In the comparison of adipose residues in 122 matched cancer patients
and controls the mean Total DDT was 7.8 ppm in the controls and 8.0 In
the cancer patients, a difference which was not significant at .05 level.
The mean dieldrin residue was 0.3 ppm in both cancer cases and controls
but as may be expected the degree of weight loss in the cancer cases was
significantly greater than that observed in the controls.
Table I compares the adipose residues in the four individual types
of cancers with matched controls. The data was further sub-divided as
to whether metastatic spread of cancer had occurred or not. Again,
there were no significant differences with regard to adipose concentra-
tions of Total DDT and dieldrin residues. Significant weight loss was
also observed in the individual cancer groups when compared to the con-
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698
TABLE I - Comparisons of adipose pesticide residues (ppm) in groups of match
cancer patients and controls. Dade County, Florida, 1972.
GROUP
PRIMARY LUNG CANCER
Controls
Cancer
"p" Value
PRIMARY LUNG CANCER
WITH METASTASIS
Controls
Cancer
"p" Value
PRIMARY GASTRO-
INTESTINAL CANCER
Controls
Cancer
"p" Value
PRIMARY GASTRO-
INTESTINAL CANCER
WITH METASTASIS
Controls
Cancer
"p" Value
PRIMARY BREAST
CANCER
Controls
Cancer
"p" Value
PRIMARY BREAST
CANCER WITH
METASTASIS
Controls
Cancer
"p" Value
METASTATIC CANCER
Controls
Cancer
"p" Value
n
24
24
22
22
46
46
4
4
29
29
10
10
4
4
X
Total DDT
8.9
8.5
NSD*
7.9
9.6
NSD
8.4
8.9
NSD
7.0
10.5
NSD
7.1
5.4
NSD
5.2
4.6
NSD
13.7
14.8
NSD
X
Dieldrin
0.3
0.2
NSD
0.2
0.4
NSD
0.3
0.3
NSD
0.3
0.2
NSD
0.2
0.2
NSD
0.1
0.2
NSD
0.6
0.4
NSD
Weight Loss
NSD
<.001
<.001
< .05
<01
NSD
NSD
*No significant differences at .05 level
-------
699
trols yet this weight loss differences had no effect on the average
pesticide residue concentrations.
The pesticide residue data in both cancer and controls was also
studied with regard to exploring socio-economic class differences
measured by the Holllngshead Index \_3_J. As had been previously noted in
our serological survey Total DDT residues were here again significantly
greater in the less affluent; dieldrin levels were greater in Social
Class 4 and 5 than in Social Class 1 and 2 in the controls (Table II).
4. Discussion
The fact that pesticide residue concentrations were not significant-
ly different in cancer cases when compared with matched controls can be
TABLE II - Total DDT and dieldrin residues (ppm) in adipose tissue by
social class groups. White cancer patients and controls,
Dade County, Florida, 1972.
SOCIAL CLASSES
Controls
I & II
IV & V
"p" Value
Patients
I & II
IV & V
"n" Value
n
35
72
43
81
TotaJLDDT (ppm)
Dieldrin (ppm)
5P
6.1
8.4
05>p>.01
S.D.
5.4
5.8
Range
0.5-25.0
0.7-29.0
X
0.2
0.3
<0.5
S.D.
0.2
0.4
Range
0-1.3
0-2.2
6.1 4.8
8.8 6.1
05>p>01
Tr.*-21.0
0.7-42.0
0.2
0.3
NSD**
0.2
0.3
0-0.9
0-2.6
*Trace Amounts
**No significant difference at .05 level
considered to be reassuring or to say the least, and in this small study
the data suggests that the incidental environmental exposure to DDT and
dieldrin was the same in these selected cancer cases as the matched con-
trols. Earlier reports of similar types of studies have reported con-
flicting results, with some studies finding significantly levels of
-------
700
Total DDT residues in cancer cases and others finding no significant
differences (Radomski, Deichman, Ray (4J, Dacre and Jennings Q>^,
Casarette, Fryer, Yauger and Klemmer GJ , Hoffman, Adler, Fishbein and
Bauer L7j . This study does not support a carcinogenic potential of
incidental exposure to DDT and dieldrin in this area in these cancers
and the data emphasizes that in future case control studies of persis-
f
tent environmental pollutants, due consideration will have to be given
to the more important geographic and demographic variables particularly
the socio-economic variable.
*This research was supported by the Division of Pesticides, Community
Studies, Office of Pesticides, Environmental Protection Agency, through
the Florida State Division of Health, under contract number 68-03-0088.
REFERENCES
1. DAVIES, J.E., EDMUNDSON, W.F., RAFFONELLI, A., CASSADY, J.C., MORGADE, C. "The
role of social class in human pesticide pollution" Amer. J. of Epid, 96, 335
(1972).
2. MILLS, P.A. Revised method. In Pesticide Analytical Manual, F.D.A., H.E W
(1968).
3. HOLLINGSHEAD, A.B. "Two-factor index of social position" A.B. Hollingshead nub*
New Haven, Conn. (1957). *
4. RADOMSKI, J.L., DEICHMANN, W.B., CLIZER, E.R. "Pesticide concentrations in the
liver, brain and adipose tissue of terminal hospital patients", Fd. Cosmet
Toxicol. 6, 209 (1968).
5. DACRE, J.C., JENNINGS, R.W. "Organochlorine insecticides in normal and carcino-
genic human lung tissue. Toxicol. Appl. Pharmacol. 17, 277 (1970).
6. CASARETT, L.J., FRYER, G.C., YAUGER, W.L., KLEMMER, H.W. "Organochlorine
residues in human tissue. Hawaii." Arch. Environ. Health 17, 306 (1968).
7. HOFFMAN, W.S., ADLER, H.t FISHBEIN, W.I., BAUER, F.C. "Relation of pesticide
centrations in fat to pathological changes in tissues. Arch. Environ. Health 15
58 (1967).
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701
EPSTEIN (U.S.A.)
The observations presented by the speaker, though of great
interest in themselves, do not appear to support his contention
that they are not consistent with the relationship of DDT and
dieldrin to human carcinogenesis. The studies reported were
designed to compare residues of these pesticides in relatively
small human populations with and without a variety of malignant
neoplasms. The studies clearly were not designed to investigate
the human carcinogenicity of dieldrin and DDT. To achieve the
latter objective, large populations with widely varying body
burdens of these pesticides would have to be followed for several
decades. Such studies, while feasible would be clearly compli-
cated by the ubiquity of environmental and human contamination
with both these persistant pesticides.
DAVIES (U.S.A.) (Editorial note: written answer submitted later)
I agree with the comments from Dr. S.S. Epstein. The ob-
servations though small at least might be considered reassuring
with regard to DDT and human carcinogenesis. All that can be
said is that in the sample size studied there was no increase
in residues of these pesticides in patients with these malignant
neoplasms.
As suggested by Dr. Epstein, prospective studies in high
and low exposure groups with sufficient years of exposure will
be necessary to settle the issue conclusively. With the better
understanding of the epidemiology of the DDT residue in the
United States and with special categorization of special factors
such as social class it is feasible to do retrospective studies
on populations of fairly large size. This would throw some
light on the issue but again one can foresee the interpretative
phenomena which would complicate such studies; these are: (1)
the concept of inducers and promoters in carcinogenesis and the
theory that two separate insults are required for human carcin-
ogenesis, and (2) is the issue that already, at least in Dade
County, insofar as DDT residues are concerned these are declin-
ing in the population and no DDT has been identified in air
samples taken in this area,for the last two years.
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702
DISCUSSION
(Editorial note: the paper was presented by J.A. Santoluaito (U.S,A
who answered the questions during the discussion).
WASSERMAN (Israel)
It seens valuable to compare levels of OCI in lipids ex-
tracted from the tumoral and presumably health tissues from the
same patients for an appropriate comparison of the storage pro-
cess.
SANTOLUCITO (U.S.A.)
Such comparisons would of course be interesting. However,
the levels of organo chlorine insecticides in adipose tissue
have been shown to reflect body burdens in relation to exposure
rate. Thus, while tumoral tissue concentrations of the insec-
ticides are not known, the observation still holds that body
burdens of DDT and dieldrin were not different between cancer
and matched non-cancer cases.
OLOFFS (Canada)
I think there is a misunderstanding between the speaker and
Dr. Wassermann. According to my interpretation of the paper by
Dr. Davies et al., his group did not look at adipose tissue
obtained from the cancerous areas, merely at adipose tissue from
another area of the body. Consequently, Dr. Wassermann's ques-
tion is irrelevant, although the problem he raised is of very
great interest and significance.
CHAMBERS (Ireland)
I notice that the results were presented for wet weight of
tissue. I believe that by now we should be attempting to pro-
duce our results as an expression of the dry weight of the tis-
sue as a matter of normal technique. This may be the case es-
pecially when tissues have been taken during surgery when there
can be great changes in wet weight during storage for example.
SANTOLUCITO (U.S.A.)
Your point is well taken provided, of course, that the
dehydration process did not result in losses of the material to
be measured. Implicit in the wet weight methodology is that care
must be taken to prevent or minimize water loss.
-------
TIERUNTERSUCHUNGEN
ANIMAL STUDIES
ETUDES SUR LES ANIMAUX
STUDI SUGLI ANIMALI
ONDERZOEKINGEN BIJ DIEREN
Vorsitzender - Chairman - President - Presidente - Voorzittev
L. COIN (France)
-------
705
ANIMAL MODELS FOR HUMAN DISEASE
D, E, GARDNER
Experimental Biology Laboratory, Environmental Protection Agency
Research Triangle Park, NC, USA
ABSTRACT
Epidemiology and animal toxicology together yield health
effects data which serve as the guidelines for formulation of
reasonable air quality standards. Epidemiological studies sug-
gest human health effects of pollutants in the ambient air, but
cannot provide direct "cause-and-effect" proof. Community
studies must cope with a host of complex oovariates, are restric-
ted to a limited range of exposures, and often provide information
too late for preventive measures. Toxicologiaal studies in
laboratory animals provide the opportunity to employ controlled
conditions, to utilize a wide range of pollutant exposures, and
to predict potential hazards. These two disciplines complement
one another in providing information suggesting the possibility
of a cause and effect relationship which can then be used to set
standards and do prospective studies. This paper will be dir-
ected toward the description, characterization, and evaluation
of currently available animal models which have been or could
be used as biological indicators of adverse human health effects
of environmental agents.
New technological innovations often produce a change in type
and quantity of pollutants. A variety of new substances could
be produced and distributed into the environment before their
effects on health are fully known. It is only through close
coordination between human and animal studies that environmental
-------
706
science can keep abreast of this vapidly changing challenge to
our environment.
Since it is not possible to present a detailed description
of all available animal models in the time allotted, this paper
will illustrate the contributions which some models have made
to our overall understanding of environmental toxicology and
indicate how these models can relate to epidemiological studies.
-------
707
Introduction
The synergistic interaction of epidemiologists, clinicians and animal
toxicologists has yielded an impressive body of scientific information
which points to the inescapable conclusion that contamination of
community air with environmental pollutants can produce profound health
consequences. While epidemiologic studies suggest human health effects of
pollutants in ambient air, such community studies usually cannot provide
"cause-and-effect" proof since they must cope with a host of complex
covariates. Furthermore, such studies are restricted to a limited range
of exposure, and often provide information too late for preventive
measures.
Toxicological studies using animal models provide the opportunity
1) to control some covariates, 2) to utilize a wide range of pollutant
exposures, 3) to evaluate a possible threshold for production of adverse
health effects, 4) to study effects of single agents as well as the inter-
action of several pollutants, and 5) to study the basic mechanism of inter-
action between pollutant and the living system. Most importantly, such
studies permit animals to be exposed to newly-introduced products and
serve as indicators of health effects before humans are subjected to
environmental exposure to new agents.
What is the best choice of animal models? The selection of the animal
model must reflect the need of the researcher. Some researchers prefer
normal random bred healthy animals while other studies are conducted with
modified, but healthy, animals, such as inbred, specific pathogen-free or
germ-free. In environmental health studies, specialized animals with
either induced or spontaneous specific diseases, such as emphysema,
genetic or infectious, may more sensitively reflect health hazards of
selected high risk individuals within the human population. Pregnant
animals offer the lexicologist the opportunity to study not only the
potential effects on the parents but extend the study to other generations.
The essential quality of a good animal model is its ability to reliably
and sensitively predict a disease process in man.
There are two general approaches that can be employed utilizing
animal models. First are those studies that have an immediate applied
goal centering around the detection of adverse health effects. The
parameters indicating injury may vary from mortality to very subtle
cellular and subcellular responses. This type of model seeks to identify
-------
708
the target organ or function which is the most sensitive to the assault.
The second approach has a different goal which is to study the
mechanism of the observed toxic effect. These studies could involve
alteration in structure or function of organs or individual cells or sub-
cellular functions. In this type of study it must be recognized that
while one seeks out a particular target one tends to lose identity with
normal physiologic influence of the whole animal.
With all animal studies there are certain biological factors which
must be considered before extrapolating the observation to human popula-
tion. Questions arise such as how to consider the life span of the
species and what segment of the animal's life to test. It can be mis-
leading to try to extrapolate years of animal exposure to years of human
exposure. Animals may not live long enough to develop diseases that
require a long latent period. What dose in the laboratory animal
corresponds to what dose in man? Does animal response always indicate
danger to man? Does the animal physically and metabolically handle
exogenous agents in a manner similar to man? The problem of small sample
size must also be considered. An adverse response with a relatively low
incidence (i.e. 1:1000) could be considered highly undesirable in the
community, although the usual toxicologic study which employs 100 or
fewer animals per treatment group would miss this effect. Table 1 lists
other more obvious problems that require consideration.
Table 1
Cautions When Using Animal Models for Predicting Effect in Man
1. Species differences.
a. metabolic
b. longevity
c. anatomic
d. physiologic
e. genetic
2. State of health.
3. Nutritional requirements.
4. Route and schedule of administration.
5. Small sample size.
6. Endogenous flora.
7. Micro-environment of test animals.
8. Spontaneous disease.
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709
Animal models have served to predict, substantiate or detect the
effects of environmental pollutants on alterations in lung function and
structure, resistance to disease, behavioral changes, and cancer.
Obviously this presentation cannot review all the animal models that have
contributed to our knowledge of environmental toxicology, but I would
like to discuss one in more detail as an example.
There is good agreement between epidemiologic and animal studies
concerning the effect of polluted air on the infectious disease processes,
and increasing emphasis is placed on the role played by pollutants in the
enhancement of morbidity from respiratory infections.
A model system which has been used in this type of study is to
expose animals to a pollutant either before or after a respiratory
challenge with an infectious microorganism. Table 2 depicts in outline
form the effect of the pollutant on increasing susceptibility which can
be measured by effect on (1) mortality rates, (2) survival time,
Table 2
Infectivity Model
Parameters of Response
Whole Animals
1. mortality rates
2. survival rates
3. pathology
Host Defenses
1. Pulmonary Defense Cells
Numbers
Viability
Phagocytic Capability
Bacteriocidal Activity
Hydrolytic Enzymes Activity
Clearance Kinetics
2. Immunological
Cell-mediated
Immunoglobulins
Antibody Levels
-------
710
Fiqure 1. A model for determining the effect of a test substance on
; defenses. Animals are exposed in separate chambers
to either the test substance or to clean air. They are then
combined and exposed to an aerosol of viable microorganisms.
The animals are then separated and various health indicators,
such as mortality, are studied.
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711
(3) phagocytic capability, (4) bacteriocidal activity, (5) hydrolytic
enzymes activity, (6) inflamatory response, and (7) alteration in immune
competence. Figure 1 is a schematic diagram of this model system.
Increased susceptibility to a number of microorganisms such as
Streptococci, Klebsiella, Diplococcus, and Influenza virus in mice,
guinea pigs, hamsters, and monkeys have been associated with exposure to
such environmental contaminants as 03, N02, S02, Mn, Ni, Pb, auto-exhaust,
and dust (Coffin [1], Ehrlich [2], Renters [3], Ardelean [4], Mai getter
[5], Hemphill [6], Port [7], Coffin [8]). Since this effect was first
noted the model system has been expanded to study the mechanism of the
observed adverse effects (Gardner [9], Coffin [10], Gardner [11],
Goldstein [12]).
At the present time other animal models are being developed which
may be applicable to community 'studies. Models which study the effect of
pollutants on humoral and cellular immunity; teratogenesis, mutagenesis
and post-natal development; acceleration of aging, shortening of life
span; and tolerance to the pollutant.
Within our society there exist certain high-risk individuals who are
predisposed to the adverse effects of pollutants. Research on the Inter-
action of environmental pollutants and established human disease 1s
currently inhibited because so little of the needed research can be done
on human subjects. But many diseases similar to those in humans occur
naturally in animals or may be deliberately induced on a predictable
basis. Some natural diseases of animals that mimic human diseases are
emphysema, autoimmunity, atherosclerosis and cataracts. A more complete
list of such models with references has been published (Jones [13]).
Obviously these animal models offer many advantages in the establishment
of controlled studies of the interaction of environmental factors and
pre-existing disease states.
In conclusion it would be appropriate to re-emphasize the Importance
of communication between the investigators using animals and those with
human subjects. By sharing ideas certain guidelines concerning
priorities, experimental design, and the practical application of the
data will be developed which will mutually aid each group in their
research. Such animal studies, coupled wi1:h human studies, provide a
noble example of a partnership between two disciplines seeking to
Improve human existence.
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712
REFERENCES
1 COFFIN, D, GARDNER, D., "Interaction of biological agents and chemical
air pollutants," Ann. Occup. Hyq.. 15, 219-234 (1972).
2 EHRLICH, R., "Effect of nitrogen dioxide on resistance to respiratory
infection," Bact. Rev., 30, 604-614 (1966).
3 FENTERS, Ju, FINDLAY, J., PORT, C., EHRLICH, R., COFFIN, D., "Chronic
exposure to nitrogen dioxide," Arch. Environ. Health, 27, 85-89 (1973).
4 ARDELEAN, L, "The action of atmospheric dust and gas pollutants on
infectious processes," Environmental Control Seminar Proceedings,
U. S. Dept. of Commerce, pp. 287-295 (1971).
5 MAIGETTER, R., FINDLAY, J,, FENTERS, Ju, EHRLICH, R., "Effect of
manganese dioxide on resistance to respiratory infection," Presented
at annual meeting Am. Soc, of Microbiology, Chicago, 111.,,
May 12-17, 1974.
6 HEMPHILL, R., KAEBERLE, M., BUCK, W., "Lead suppression of mouse
resistance to Salmonella Typhimurium." Science, 172, 1021-1032 (1971).
7 PORT, C., FENTERS, J,, EHRLICH, R., COFFIN, D«, GARDNER, £„, "Inter-
action of nickel oxide and influenza infection in the hamster,"
submitted to Environ. Research (1974)..
8 COFFIN, D.,, BLOMMER, Eu, "Acute toxicity of irradiated auto exhaust
indicated by enhancement of mortality from Streptococcal pneumonia,"
Arch. Environ. Health, 15, 36-38 (1967).
9 GARDNER, D., HOLZMAN, R., COFFIN, Du, "Effect of nitrogen dioxide on
pulmonary cell population," J. of Bacteriol,. 98, 1041-1043 (1969).
10 COFFIN, D., GARDNER, Do, HOLZMAN, R., WOLOCK, F., "Influence of ozone
on pulmonary cells," Arch. Environ. Health. 16, 633-636 (1968).
11 GARDNER, D., PFITZER, £„, CHRISTIAN, R., COFFIN, D., "Loss of protective
factor for alveolar mactophages when exposed to ozone," Arch. Intern.
Med.. 127, 1078-1084 (1971).
12 GOLDSTEIN, E., TYLER, W., HOEPRICH. P., EAGLE, C., "Ozone and the
antibacterial defense mechanisms of the murine luno," Arch. Intern.
Hed. Symposia, 9, 165-168 (1971).
13 JONES, T.,, "Mammalian and avian models of disease in man," Federation
Proceedings. 28, 162-169
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713
DISCUSSION
AUBERT (France)
I should like to ask the author which animal model he uses
or recommends in studying the phenomena of concentrations of
toxic chemicals, which increase the further one progresses along
the natural biological chains and which, beginning with infini-
tesimally small doses, may end in levels hazardous to the final
consumer - man.
Some years ago now we presented the findings of a study of
these phenomena in respect of the ocean, using an original
method whereby the experimental marine trophodynamic basins re-
created, in the laboratory, the main natural biocenoses: the
pelagic chain, the benthic chain and the molluscan or crustacean
neritic chains.
*
In fact we describe our method in the paper "Les chaines
trophodynamiques marines experimentales: methode d'fitude des
effets pathologigues consecutifs aux pollutions chuniques"
presented at this Symposium, to which reference may be made.
My question is thus as follows: did the author use this
method? Is he aware of similar findings by groups other than
our own?
GARDNER (U.S.A.)
Mo, I have not personally used such an elaborate system
as you and your colleague have described. Your model system
appears to systematically investigate the fate and potential
toxicity of chemicals as they pass through various biological
systems in hope to mimic what is occurring naturally. There
are, of course, many studies designed to follow the transferral
of pesticides via the food chain. These studies indicate that
many factors can directly effect the persistence, metabolism
and movement of such chemical agents through the ecosystem)
for example, the quality of water, abundance of plants and
animals, water temperature, species variability, etc. In order
to obtain the most information from your model systems, all of
these variables need close control. I will be looking forward
to seeing some of the toxicological data generated through the
use of your model system.
WASSERMAN (Israel)
Toxic effects in the food chain are studied with the aim
of detecting the most susceptible species, whose damage or
-------
714
distruction would disturb the ecosystem and in the last instance
the man.
The Ministry of Interior - Wildlife Research Group - is
developing such studies to organocholrine insecticides movement
in the ecosystem. Other aspects related towards this topic will
be reported in the Symposium on Biotoxicity which will be held
on July 1, 1974 at the Environmental Medicine and Biology Con-
gress in Paris.
BITTEL (Prance)
In my opinion, consideration should be given to the phen-
omena of concentration not on only the level of organisms and
tissues, but also on the sub-cellular level.
GARDNER (U.S.A.)
I think Dr. Bittel's point is a good one; that is, it is
desirable to use the most sensitive techniques we have available
for determining the concentration at the target site. In many
cases one may have to start at the tissue level but hopefully,
a more sensitive parameter would be developed to allow one to
study sub-cellular localization.
STARA (U.S.A.)
I believe that the previous questions have considered
another aspect of animal effects. There are two issues here.
The first is the use of animal models in public health appli-
cation i.e. toxicologic investigations to evaluate and predict
potential human health effects. The second is the studies
concerned with uptake of hazardous materials in various animal
species themselves resulting in a potential distruction of a
species and a disruption of an ecological chain. We must note
this difference. I believe that Dr. Gardner addressed himself
to the first aspect of toxicology.
GARDNER (U.S.A.)
I would like to add that although there are many different
applications for animal models in research, it was unrealistic
for me to try to review and discuss all those used in environ-
mental toxicology. With each research goal, the animal model
must reflect the need of the researcher. These needs vary gre-
atly. I devoted my time to those animal models that mimic
human diseases and can thus be used for predicting possible ad-
verse effects resulting from environmental pollution.
-------
715
DIE TUMORERZEUGENDE WIRKUNG FASERFORMIGER STAUBE
F, POTT, K, H, FRIEDRICHS, F, HUTH
Medizinisches Institut fur Lufthygiene und Silikoseforschung an
der Universitat Dusseldorf, BRD
KURZFASSUNG
Aus fruheren tierexperimentellen Vnterauohungen mit ver-
sohiedenen faaerfOrmigen und kOrnigen Stauben wurde geaohloaaen,
daae die kanzerogene Wirkun^g von Aabeat an die Faaerform gebunden
ist und nioht an seine ahemiaahe Zusammeneetzung. Weitere
Vereuohe mit UlCC-Chrysotil und aehr feinen Glaefaaern an Wiatar-
Ratten ergaben, daaa naah intraperitonealer Injekt-ion von nur
2 mg dieaer faaerfdrmigen St&ube Tumoren entatanden. Bei. dieaer
Doeia bildete aioh ke-ine deutliche Fibroae. Hiatolog-iaoh
handelte ea aioh bei den Tumoren meiat um aarkomcttdae Meaotheliome,
Die tierexperimentellen Befunde fuhren zu dem SohTuaa, daaa
die Doeia-Wirkungabeziehungen bei der Tumorinduktion duroh faser-
fOrmige Stdube von vier Faktoren abh&ngig aind: Faaersahl,
FaaerdurahmeaaeFf Faaerl&nge und Verueildauer am Ort der Virkung.
Be mu88 damit gereohnet uevden, daas extvem feine Glaafaaern
auoh beim Menaahen zu Tumoren ftthrent da die Kanzerogenitat von
Aabeat beim Meneohen ervieeen iatt und die Tumoren, die naah
intraperitonealer Injektion von Aabeat und von Glaafaaern bei
der Ratte diagnoatiziert uurden, aioh nioht voneinander unter-
Bohieden.
Der grdeate wirkaame Faaerdurohmeeeer uird auf 1 bie S ,VM
geeahdtzt. Glaafaaern mit einem Durohmeaaer von <5,um werden
in Deuteohland eret aeit 1961 hergestellt. Mit dem Auftreten
von Meaotheliomen beim Menaohen iat- frilheatena 20 Jahve naoh
-------
716
Beginn der Inhalation extrem feiner Fasern zu rechnen. Der
Beweis fiir die Ubertragbarkeit der tierexperimentellen Ergebnisse
auf die Humanpathologie wird daher kaum vor 1985 mVglioh sein.
ABSTRACT
Earlier animal experiments using various fibrous and granu-
lar dusts led to the conclusion that the carcinogenic effect of
asbestos is linked to its fibrous form and not to its chemical
composition. Further experiments on Wistar rats with UlCC-Chry-
sotil and very fine glass fibres revealed that tumours developed
after intraperitoneal injection of only 2mg of these fibrous
dusts. This dose did not cause any clearly-defined fibrosis.
Histologicallyf the tumours were in most oases sarcomatous
mesotheliomas.
The results of animal experiments lead to the conclusion
that, when tumours are induced by fibrous dustst the dose-effect
relationship depends on four factors: numbert diameter and
length of the fibres and length of contact with the affected
area. It must be expected that extremely fine glass fibres can
also cause tumours in humanst as the carcinogenic effect on hu-
mans of asbestos has been establishedj and the tumours diagnosed
in rats after the intraperitoneal injection of asbestos and glass
fibres did not differ from each other.
The largest effective fibre diameter is estimated at .7 to
3 .urn. Glass fibres with a diameter of <5 .urn have only been man-
ufactured in Germany since 1961. The appearance of meaotheliomas
in humans can be expected at the earliest 20 years after the
initial inhalation of extremely fine fibres. Thus until 1985
it will hardly be possible to prove whether the results of ani-
mal experiments can be applied to human pathology.
-------
717
Die tumorerzeugende Wirkung von Asbest 1st aeit etwa 40 Jahren
bekannt. In letzter Zeit wurden auoh andere faserige Staube im Tierexperi-
ment auf ihre Kanzerogenitat unterauoht. OTANTON und WRENCH (l) beriohte-
ten, dass naoh intrapleuraler Implantation von sehr feinen Glasfasern bei
Ratten Tumoren entetanden waren. Kornige Glaspartikeln fUhrten dagegen in
einer signifikant niedrigen Haufigkeit zu Tumoren. POTT und ERIEDRICHS (2)
kamen zu dem Ergebnis, dass naoh intraperitonealer Injektion von 100 mg
Glasfasern bei Ratten ebenfalls Tumoren entstanden. Auoh faserformiges
Magnesiumhydroxid mit der mineralogisohen Bezeiohnung Nemalith wirkte im
gleiohen Versuchsmodell kanzerogen. Demgegentiber induzierten kornige Mine-
rals taube, wie Biotit, Hamatit, Sanidin und Talk keine Oder nur verein-
zelte Tumoren.
Bei Weiterfiihrung unaerer Tierversuohe haben wir wiederum Qlasfasern
benutzt, da mit diesem Material ein Modellstaub gegeben war, an dem die
BeBtimmung der Langen- und Durohmesserverteilung dea Faserkollektivs mit
grosser Genauigkeit moglich ist. Ihr weiterer Vorteil gegenttber den Mine-
ralstauben beeteht darin, dass sie nioht wie z.B. Asbest duroh minerali-
sohe Begleitsubstanzen verunreinigt sind. Die Qrossenverteilung der be-
nutzt en Glasfasern ist in den Abbildungen 1 und 2 dargestellt. Hiernaoh
besteht die feinere Olaswolle MN 104 aus Flasern, die zu 50 % diinner als
0,2 Mikrotneter bzw. klirzer als 11 Hikrometer sind. Bel der groberen Qlas-
wolle MN 112 sind demgegeniiber iiber 50 % der Pasern diinner als 1 Mikro-
meter bzw, kiirzer als 28 Mikrometer. Die Staube wurden in 2 ml 0,9 Differ
NaCl-Loeung suspendiert und Wistar-Ratten intraperitoneal injiziert. Die
Versuohsergebnisse sind in der Tabelle 1 zueammengefasst. Naoh einem
Beobaohtungszeitraum von 24 Monaten zeigt sioh, dass bei dem (Jlasfasertyp
MN 104 eine Dosis-Wirkung-Beziehung besteht. Die angegebene Tumorhaufig-
keit je Tiergruppe ist jedooh nioht als endgtiltig anzusehen, da ein Tell
der Tiere nooh lebt. Ausserdem ist beim Vergleioh zwisphen der Glasfaser
MN 104 und dem Amphibot-Asbeet Krokydolith zu erkennen, dass die Turaorra-
ten bei gleioher Dosis ahnlioh sind. Die kanzerogene Wirkung der Olas-
wolle MN 112 aoheint demgegenttber der der MN 104-Faser niedriger zu sein.
Die hiatologisohe Diagnose der induzierten Tumoren ergab, dass es sioh in
fast 90 % der £&lle urn Mesotheliome handelt. Die tibrigen wurden ale
-------
718
99-i
90-
50-
10-
1
GLASFASER MN 104
O—O Durchmessar
X—X Ling*
01 02 05 1 2 5 10 20 50 100
Abbildung 1: Haufigkeitsverteilung von Lange und
Durchmesser der Glasfaser MN 104
-------
90-
50-
Ot
"3
719
GLASFASER MN H2
o
o
o
10-
o
J
O—O Durchmassir
X—X !.*•••
0,1 0,2 0,5 1 2 5 10 20 50 100
Abbildung 2: Haufigkeitsverteilung von Lange und
Durchnes&er der Glasfaser HN 112
-------
Tumors after intraperitoneal injection of fibrous glass, crocidolite and corundum in rats
(unfinished experiment about 2k months)
Dust
Fibre glass
MN 10k
ii
11
Fibre glass
MN 112
Crocidolite
DICC
Corundum
Dose
mg i.p.
in 2 ml
saline
2 .
10
2 x 25
20
2
?. x 25
Number
of rats
at the
start
80
80
80
ko
ko
ko
After
surviving
rats
38
21
-
9
17
25
2k months
% of rats
with tum.
10.0
33.8
68.8
27.5
lo.O
2.5
Histol. diagnosis
Mesothe-
lioma
7
23
k7
10
k
1
Spindle-
c. sarc.
1
3
6
1
-
-
.
o
• u
B tS
>> <0
i-4
/P '
fc O
-
2
-
-
-
•
o
h
Kt
O
1
-
-
-
-
•
O
• fc
o a
-H 10
4*
4> •
a o
1*
-
2*
-
2*
1*
Survival
time of
first rat
with tum.
days
576
210
19k
390
^52
5^5
-J
to
O
Spontaneous tumor, not valued
-------
721
Spindelzellsarkome oder polymorphzellige Sarkome eingestuft. Versuohe wit
Mausen ergaben zum Teil eine niedrigere Tumorrate naoh intraperitonealer
Injektion faserformiger Staube. Bei Goldhamstern und Meerschwoinohen wurde
bisher kein Tumor gefunden, weder naoh Injektion von Chrysotil noch von
Glasfasern.
Aufgrund unserer inzwisohen urafangreiohen Versuohsreihen ergibt sioh,
dass die tumorinduzierende Eigensohaft faserfSrmiger Staube massgeblioh
duroh die spezielle PartikeIform, nioht dagegen duroh die ohemisohe Zusara-
mensetzung bestimmt wird. Wir befinden Tins mit dieeer Sohlussfolgerung in
Uebereinstimmung sowohl mit ST ANTON und WRENCH (l) als auoh mit WAGNER,
B3RRY und TIMBRELL (3).
Die Dosis-Hi.rkung-Beziehung fiir die Tumorinduktion iat naoh dieser
Itypothese vorwiegend abhangig von der Paserzahl, der Paserlange, dem Paeer-
durohmesser und von der Verweildauer der Paser am Ort der Wirkungt
Eine Uebertragbarkeit der Brgebnisse von Tierversuohen auf den Men-
sohen 1st nioht ohne weiteres zulassig, derm alle hier benutzten Applika-
tionsverfahren, namlioh die intraperitoneale und intrapleurale Injektion
und auoh die intrapleurale Implantation tniieeen als unphysiologisoh ange-
fiproohen warden. Da jedooh die kanzerogene Wirkung von Asbest auf den Men-
sohen bekannt iet und da die im Tierversuoh mit Asbest und anderen faser-
fdrmigen Stauben induzierten Tumoren sioh nioht voneinander unteraoheiden,
ersoheint der Hinweis auf ein mdgliohes Gesundheitsrisiko ftlr den Mensohen
bereohtigt.
Die wirksame Dosis iet unter anderem abhangig von der Konzentration
atembarer J^sern. Die naohfolgend zitierten Konzentrationeangaben ttber Pa-
Bern in der Auasenluft Bind unter dem Vorbehalt zu wertenf dass der Be-
griff "Paser" hinsiohtlioh seiner KenngrSsse L/D international bislang
nioht einheitlioh definiert ist* So fand THAER (4) im Raum Prankfurt in
der Aussenluft 8000 Faaern/m3. SPURNY (5) ermittelte an versohiedenen Mees-
stellen zwisohen 1000 und 100.000 Fasern/iA Eigene Meesungen in Dtisseldorf
und Gelsenkirohen erbraohten Konzentrationen zwisohen 1000 und 8000 Fasera/
•» •
wr bei einem gewahlten L/D-Verhaltnia von 5 s 1. Bei den erwuhnten Hess-
programmen ist eine Untersoheidung hinsiohtlioh der stoffliohen Zusammen-
setzung bisher nioht erfolgt.
-------
722
Die Konzentration 1st demgcgeniiber an Arbeitsplatzen von Betrieben,
in denen Pasern hergestellt oder verarbeitet warden, vermutlich wesentlich
hoher. Am Beispiel der GlasfaserIndustrie muse ausserdem darauf hingewie-
sen werden, da.es seit oa. 15 Jahren zunehmend feinere Pasern hergestellt
werden (6). Per Anteil der atembaren Pasern steigt dementsprechend an.
Zusammenfassend 1st festzuhalten, daas aufgrund einer Vielzahl von
Versuohen mit Ratten die Kanzerogenitat faserfb'rmiger Staube naohgewiesen
wurde. Hieraus wird ein potentielles Gesundheitsrisiko fur den Menschen
abgeleitet, dessen Umfang erst duroh zukiinftige epidemiologische Untersu-
chungen ermittelt werden kann.
Literatur
1 3PAHTON, M.P. and C. WRENCH : Mechanisms of Mesothelioma Induction
with Asbestos and Fibrous Glass
J. Nat. Cancer Inst., 48 (1972) 797 - 821
2 POTT, P. und K.H. RR3EDRICHS : Tumoren der Ratte nach i.p. - Injektion
faserfSrmiger Staube
Naturwiss., 59 (1972) 318
3 WAGNER, J.C., ffiPfflY, G. and V. TIMBRELL : Mesotheliomata in rats after
inoculation with asbestos and other Materials
Brit. J. Cancer 28 (1973) 173-185
4 THAER, A.: pers. Mitt. 1973
5 SPURNY, K.R.: pers. Mitt. 1974
6 KEENAN» R*G. and J.R. LYNCH t Techniques for the Detection, Identifi-
cation and Analysis of Fibers
A.I.H.A. - J. 5 (1970) 587-597
-------
723
DISKUSSION
SCHLATTER (Schweiz)
Bestehen Anhaltspunkte dafur, dass faserformige Staube in
der Nahrung zu Tumoren fiihren konnen?
FRIEDRICHS (Bundesrepublik Deutschland)
Eine Tumorinduktion durch oral aufgenommene Fasern lasst
sich nicht ausschliessen, uns sind jedoch keine Befunde bekannt,
die eine solche Wirkung wahrscheinlich machen.
SCHLIPKOTER {Bundesrepublik Deutschland)
1. Das Auftreten von bosartigen Magentumoren ist bei peroraler
Applikation nicht bekannt, jedoch nach Inhalation sind Magentumore
beobachtet worden.
2. Die kanzerogene Wirkung der Fasern ist tierspezifisch. Da
jedoch Asbest bei Mensch und Ratte bzw. Mause kanzerogen wir-
ken und da das gleiche mit Glasfasern bei Ratten und Mausen
beobachtet wurde, muss in der Zukunft faserformigen Stauben
grbssere Aufmerksamkeit geschenkt werden.
3. Die von Pott und Mitarbeitern beobachteten Tumore waren
nicht nur Mesotheliome sondern es wurden auch Sarkome sowie
andere bosartige Tumore diagnostiziert.
OLOFFS (Canada)
Konnen Sie sich die negativen Resultate mit Goldhamstern
und Meerschweinchen erklaren?
FRIEDRICHS (Bundesrepublik Deutschland),
Die negativen Befunde bei Goldhamstern und Meerschweinchen
konnen wir vorlaufig nur mit dem allgemeinen Hinweis darauf
erklaren, dass die Empfindlichkeit gegeniiber Substanzen ln
vielen Fallen grosse artspezifische Unterschiede aufweist.
Moglicherweise sind die Unterschiede im Immunverhalten, die
insbesondere zwischen Meerschweinchen und Ratten bestehen, von
Bedeutung.
-------
724
REEVES (U.S.A.)
Ich glaube/ zwei verschiedene Prinzipien kbnnen bei der
Entstehung von Lungenkrebs und pleuralen Mesotheliomen nach
Asbesteinatmung eine Rolle spielen Das Lungenkarzinom scheint
eine relativ hohe Spezifitat als Agens zu haben; meines Wissens
hat bisher noch niemand die zuletztgenannte L'asion rait nicht-
asbesthaltigen faserfbrmigen Stauben hervorbringen kbnnen.
Paul Gross hat sogar nachgewiesen, dass Glasfasern nach Einatmung
in dieser Beziehung unwirksam waren. Andererseits hat das Meso-
theliom eine viel niedrigere Agens-Spezifitat und kann durchaus
von der Geometric der Staube abhangen, da sowohl Asbestfasern
als auch Glasfasern mit Erfolg von Stanton A Wrench verwendet
worden sind.
Weiter glaube ich, dass die offensichtliche Spezifitat bei
der karzinogenen Wirkung von Asbest mit der Bereitschaft der
Gasttiere verbunden ist, ferrogene Korper zu entwickeln. Die
Arten mit der ausgepr'agtesten Fahigkeit, ferrogene Korper zu
entwickeln (z.B. Meerschweinchen), sind anscheinend immun gegen
eine Asbest-karzinogenese, wahrend Arten mit einer entsprechend
geringen Fahigkeit hierzu (z.B. Ratten) am anfalligsten sind.
Die Bildung ferrogener Korper ist mbglicherweise eine Schutzmass-
nahme des Organismus.
FRIEDRICHS (Bundesrepublik Deutschland)
Sicherlich muss daran gedacht werden, dass die Entwicklung
des Asbest-induzierten Bronchialkarzinoms von anderen Eigen-
schaften des Asbests hervorgerufen wird als die Entwicklung des
AsbestAnduzierten Mesothelioms. Die Untersuchungen von Gross
erbringen jedoch unseres Erachtens nicht den Beweis dafur, dass
Glasfasern nicht zu einem Bronchialkarzinom fuhren kbnnen.
-------
725
TERATOGENIC, MUTAGENIC, AND CARCINOGENIC
EFFECTS OF INSECTICIDES
LAWRENCE FISHBEIN
National Center for Toxicological Research, Jefferson, Arkansas
USA
ABSTRACT
There is increasing concern over the possible toxicological
hazards posed by a spectrum'of environmental chemicals including
industrial pollutants, pesticides, food additives and drugs, ei-
ther considered singly as a class or specific agent or in combin-
ation. A number of insecticides, based on aspects of their
ubiquity, persistence, presence and/or concentration in the food
chain as well as their biological and toxicological properties,
constitute a major source of potential environmental hazard to
mammal species including man.
However, there exists considerable disagreement concerning
the scientific assessment of effects (notably teratogenio, muta-
genic and carcinogenic) of these agents, primarily because of the
variety of testing procedures employed within the three major
areas of toxioity, hence complicating comparisons within any one
area; complexity of unambiguous recognition and interpretation
of the toxic event and finally the extrapolation and relevance
of experimental laboratory findings to man.
To permit a more orderly assessment of the above effects
the possible interrelationships between teratogenioity, mutagen-
icity and oaroinogenioity will be cited as well as the most
salient features of the respective testing procedures employed
-------
726
with concomittant clarification of their principles, problems
and interpretations.
The teratogenic, mutagenic and carcinogenic insecticides
that will be discussed include the following:
chlorinated hydrocarbons (DDT and metabolites); cyclodienes
(dieldrin^ aldrin and endrin); miscellaneous chlorinated in-
secticides (benzene hexachloride and Mirex); organo-phosphorus
derivatives (dichlorvos (DDVP)S trichlorphonf malathiont para-
thion and methyl parathion) and aarbamates (aarbaryl).
-------
727
The major objectives of this presentation is to highlight the
principal factors in the assessment of the teratogenicity, muta-
geniclty, and carcinogenicity of a number of the commercially
important chlorinated insecticides, and secondarily, to consider
some of the consequences of their increasing replacement by a
number of organophosphorus and carbamate insecticides.
The complexity of unambiguous recognition of teratogenic,
mutagenic, and carcinogenic events is veil recognized. Table 1
illustrates a number of the principal factors Influencing carcino-
genicity testing. The cogent factors that must be vigorously
assessed in carcinogenesis investigations include: route of admin-
istration, dosage, frequency of exposure; species, strain and sex
of test animal; age of animal, at the start cf the test; basic diet
and possible dietary contaminants (e.g., pesticides, metals -
mercury, cadmium, lead, arsenic and selenium - mycotoxins, PCB's,
estrogens, synergists; levels which can change based on geographical
and seasonal considerations), immunologlcal status of the animal
and duration of the experiment; homogeneity of the test material,
possible Impurities in air, water, bedding (pentachlorophenol,
trace metals, etc.), housing conditions (crowding stress, inter-
current disease, and drug therapy). It should be noted that the
selection of doses, frequency of exposure, and duration of test is
still a matter of controversy.
No less controversial, and often conflicting, are factors
concerning the final assessment of carcinogen testing as exemplified
by Table 2. There is by no means common agreement as to assessing
either spontaneous or Induced tumor incidence in laboratory animals.
Diagnostic criteria applied to lesions and thoroughness of autopsy,
-------
728
TAME 1
PRINCIPAL FACTORS INFLUENCING CARCINOOENICITy TESTPIQ
1. Route of Administration, Dosage, and Frequenoing of Exposure.
2. Homogeneity of Test Compound (Purified Material; Commercial Prep;
Mixture of Isomers; Trace Contaminants).
5, Species, Strain, and Sex of Test Animals.
4. Age of Animal at Start of the Test.
5. Lmnunologlcal Status.
6. Diet, Nutrition, and Interactions.
7. Possible Diet Contaminants (Pesticides, Heavy Metals, Trace
Elements, Mycotoxlns, PCS'a, Estrogens, Enzyme Inducers).
8. Pollutants and Impurities in Air Water, Bedding (Pentachloro-
phenol, etc.).
9. Duration of the Experiments.
10. Housing Conditions (Crowding Stress; Intercurrent Disease;
Drug Therapy).
TABLE 2
FINAL ASSESSMENT OP CARCINOGEN TESTING
1. Analysis of Hlstological, Cytological and Electron Microscopic Aspects
of Pre-Malignant of Malignant Change.
2. Range of Precancerous and/or Hyperplastlc Lesions to Unequivocal
Malignant Change.
3. Confusion of Terminology ("Benign Tumor", "Hepatoma", "Nodular
Hyperplwia". "Benign Hepatoma").
4. Extrapolation from Experimental Results to Determination of Safety
Factor. (No Effect Level ?).
-------
729
with concomittant differentiation of benign and malignant tumors,
hyperplaeia, metaplasia, dyaplasia, and neoplasia being assuredly
fundamental in any valid assessment of carcinogenicity testing.
The difficulty in the assessment of carcinogenicity testing is
best exemplified in the case of DDT. Table 3 summarizes the results
of 18 studies with DDT involving a broad range of animal species,
strains, dosages, frequency of dosages, and duration of tests. Of
special note is the lack of uniformity of test material1 from one
study to another, which ranges from p,p-DDT of no stated purity, to
technical material which can contain a mixture of at least 16
components, ranging from the isomeric DDT'a and DDD's to varying
levels of synthetic precursors and degradation products as shown in
Table 4. Several multigenerational studies with DDT have also been
reported.
Studies in 5 generations from the inbred BALB/c mouse strain
were carried out by Kemeny and TarJan (1) with dietary DDT at a
level of 2.8 - 3*0 ppm. Chemical analysis of this basic diet
showed the presence of 0.2 to 0*4 ppn DDT as contaminant, possibly
accounting for the occurence of the few observed leukemia cases in
controls, as well as to the level of 12.496 of DDT fed animals with
leukemia and 21*.7% with tumors*
Tumor and leukemia incidences, generation by generation
calculated by Kay (2) and noted by Terracini (3) did not increase
with succeeding generations giving rise to the question as to
whether the age differences in the number of mice in generation
groups may have marked the outcome (Table 5).
Tomatis and co-workers (4) have been investigating the multi-
generational effect on technical DDT (70 - 7&% P,P - DDT, 2096 o,p -
-------
PAGE NOT
AVAILABLE
DIGITALLY
-------
TABLE 4
COMPOSITION CF TECHNICAL DDT
COMPOUND
l-Trichloro-2.2-bls (p-chlorophenyl )-ethane
(p,p'-DDT)a
l-Trlchloro-2-o-chlorophenyl-2-p-chloropheny-
lethane (o,p'-DDT)
1 , 1 -Dlchloro-2, 2-bis- (p-chlorophenyl )-ethane
(p,p'-DDD)
1 , l-Dichloro-2-o-chlorophenyl-2-p-chloropheny-
lethane (o,p'-DDD)
2-Trichloro-l-o-chlorophenylathyl p-chloroben-
zenesulfonate
2-Trlchloro-l-p-chlorophenylethanol
Bis- (p-chlorophenyl )-sulf one
tt-Chloro-i-p-ehlorophenylacetamide
dC-Chloro-4-o-chlorophenylacetamide
Chlorobenzene
p-Dichlorobenzene
1.1,1, 2-Tetrachloro-2-p-chlorophenylethane
Sodium p-chlorobenzene-sulfonate
Annonium p-chlorobenzene-sulfonate
Inorganic
Unidentified and losses
SAMPLE 1 SAMPLE 2
(SETTINQ POINT, (SETHNO POINT,
91.2 ), % 88.6 ), %
(a) 66.7, (b) 70.5,
(b) 72.9 (c) 63.5a,
(d) 64.5,
(e) 67.9
19.0 (c) 7.9,
(d) 15-3,
(«) 20.9
0.3 4.0
0.4 J^ 1.85
0.2
0.6 o.l
0.01
0.007
O.02
0.1 0.04S
6.5 5.1
SAMPLE 3
(SETTING POINT,
91.4 }, %
(a) 72.7,
(b) 76.7
11. 9b
O.IT"1
0.044
0.57
0.034
0.006
0.005
0.01fi
10.6
SAMPLE 4
(BY-PRODUCT
OIL), %
74. 8a
0.11
2.44
0.73
19.4
o,., "*«»tiv.ly studies. " Miscellaneous fraction
0.06* of p.p'-DDD isolated as such and 0.11* of the corresponding olefin e Isolated
r tt •"?7Zln8 f°r * ^^ °f W»CI*«- Ws -d^pr^e^1^ of
aJ'w ^ ^ T10' lead "^ IM«n"lun o«-bonstes were obtained. 8 insoluble In boiling 955^
Qualitative testa for ferric, ammonium, hallde and aulfate Ions were obtained.
-J
u>
10
-------
733
TABLE 5
CARCINOMA INCIDENCE AMONG BALB/c MICE FED 2.8 TO 3.0 PPM DDT OVER 5
GENERATIONS (CALCULATED FROM DATA OF TARJAN AND KEMENY ( 1)
.
Groups
Fl
F-
2
F
F
F
Total
Total
Age
mo
26
22
18
15
11
-
No.
of
mice
10
35
69
264
505
683
406
Tumors Incidence %
Male Female
10
26
9
11
10
11.5
0.49
20
23
22
13
20
17.5
•
2.71
Total
30
49
31
24
30
29
Controls
5.20
Leukemia incidence %
Male Female Total
10
5.7
1.5
2.7
3-3
3.1
total
0.74
30
0
14.5
10.6
7.5
9.4
1.72
40
5.7
16
13
11
12
2.46
Totaia
inci-
dence
70
55
47
37
41
41
5.66
Total of male and female, both types of carcinoma.
DDT, 0.5-1.556 P,P - DDD, and 0.5$ P,P - DDE) fed to CF-1 inbred
mice maintained aa specific pathogen-free at dose levels of 2, 10,
50, and £50 ppm. Liver tumors appeared earlier in the F- mice fed
50 to 250 ppm. Out of several hundred liver tumors in the DDT-fed
groups, only k gave metastases to the lung. Non-invasive, non-
metastasing liver tumors predominated. Difficulties of differentia-
ting benign liver tumors from hyperplastic nodules were reiterated.
There was little apparent evidence between P and F,, generations,
either for all tumors or only liver tumors which represented the
only tumor type (among 3 main types) to be dose related.
Confusion persists in the interpretation of the tumorigenicity
of aldrin, dleldrin, and endrin. Of four studies (Table 6) dealing
with the relationship between exposure to these chemicals and tumor
-------
TABIE 6
AEDRIN, DIEU3RIN. AND ENDRBI
AGENT
Aldrin, Dieldrln
Aldrin, Dieldrin
Dleldrln
Endrin
Aldrin, Dieldrin
SPECIES
Hat
(Osborne-Mendel )
Mouse
(CjHeB/fce)
Rat
(Carworth Farm
"E" Strain, SPP
Conditions )
Rat
(Osborne-Mendel)
Rat
(Osborne-Mendel )
DOSE
0.5, 2, 10, 50
100 or 150 ppm
(2 years)
10 ppm
(2 years)
0.1, 1.0, and
10.0 ppm
(2 years)
20, 50, 50 ppm
(51 months)
20, 50, 50 ppra
(21 months)
EFFECT
Pulmonary Itfmphosareoma
(Enhanced tumor incidence
in high survival-low
dosage group).
Hepatomas, Pulmonary
Adenoma, I^rmpoma, 17#,
1856 over-all tumor inci-
dence for Aldrin, Dieldrin
Non-Tumorigenl c
Inactive
Anti-Tumorigenic
REFERENCE
Fitzhugh, 0. 0., et al
Food. Cosmet. Toxicol.,
2 (1964) 551
Davis, K. J. and Fitzhugh,
Toxicol. Appl. Pharmacol.,
4 (1962) 187.
Walker, A. I. T. et al,
Toxicol. Appl. Pharmacol.,
15. (1969) ?45.
Deichmann, W. B., et al,
Ind. Med., 2i (1970) 426
Daichmann, W. B., et al
Ind. Med., 22 (1970) 426
•vl
U)
-------
735
Incidence, It was concluded in two studies by Fitzhugh and co-workers
that aldrin and dieldrln were tumorigenic in the rat and mouse.
(5, 6).
In a third study involving 2 year oral exposure of rats and
dogs, aldrin was found inactive by Walker and co-workers (7). In
a fourth study reported by Deichman and co-workers, aldrin and
dieldrin were judged to be antitumorigenic and endrin to be inactive
in the rar (8).
Walker et al (9, 10) reported neoplastic liver changes in CF-1
mice fed low doses of dieldrin, DDT, /J-BHC, f-BHC, and phenobarbitone
on a long-term basis.
Table 7 illustrates the spontaneous tumor experience with
Osborne-Mendel Rats of the FDA-strain and its University of Miami
substrain. The 1959 spontaneous tumor incidences with the FDA
substrain of Osborn-Mendel rats were 22#. Subsequently, the
incidence of this strain was much higher, for example reaching 30,44
and 48# in 1970. The highest incidence in aldrin treated rats in
the 1952 FDA experiment carried out by Fitzhugh (but not reported
until 1964) was 53# at the lowest level of administration, 0.5 ppm
aldrin (Table 8). This suggests according to Kay (2) that spontane-
ous variability might account for the apparent tumorigenlcity of
aldrin and dieldrin.
Table 9 illustrates the data of Deichman and co-workers (8)
and shows that the incidence of tumors in the aldrin and dieldrin-
fed rats was much lower at all levels of dosing than the incidrnce
of spontaneous tumors in controls, and this was the basis for the
authors evaluation of anti-tumorigenicity for these compounds.
However, this conclusion would be reversed and aldrin and dieldrin
considered tumorigenic if the control incidence had been as low in
-------
736
TABLE 7
SPONTANEOUS TUMOR EXPERIENCE WITH .JQSBCMJ&JJEMPELJWIS
OP THE PDA STRAIN AND
INVESTIGATORS AND
REFERENCE NOS.
Pitzhugh et al. (27)
Davis ft Pitzhugh (28)
Davis & Pitzhugh (29)
Davis et al. (50)
Fltzhugh et al. (3!)
Hansen et al. (32)
Hansen et al. (33)
Hansen et al. (33)
Hansen et al. (33)
Byron et al. (3*0
Byron et al. (3*0
DATE
POOD
1959
1962
1963
196*
1964b
1965
1966
1966
1966
1967
1967
ITS UNIVERSITY OF MIAMI SUBSTRAIN /FROM KAY (2)7
NO. OP
RATSb
AND DRIBS
224
50
50
24
17
50
50
50
50
50
50
665
University of
Radomskl et al. (35)
Radomskl et al. (35)
Delchroarm et al. (36)
Deichnann et al. (37)
1965
1965
1967
1970
60
100
60
163
SURVIVAL*
(2 YEARS)
ADMINISTRATION
55
42
26
54
50
38
no Info.
no Info.
no info.
42
36
Miami0
no info.
no info.
45
maximum
survival
27 months
NO. WITH
TUMORS
49
22
24
8
3
23
16
18
15
16
17
211
15
26
14
79
TUMOR INCIDENCE
%
22
44
48
33
18
46
32
36
30
32
3*
32
25
26
23
48
Starting age weanling or 3 weeks in all cases; diet Purina lab chow except
Refs. 27 and 31, ground commercial; Ref. 33 no Information. Duration of tests 24
months except Ref. 36,25 months; Ref, 37,2? months.
Half male, half female except Ref. 37.75 males, 88 females.
c Prom I960 and 1965 PDA stock.
-------
737
TABLE 8
SURVIVAL RATES AND TUMOR INCIDENCES FOR OSBORNE-MENDEL RATS FED ALDRIN
AND DIELDRIN-CALCULATED FROM
DATA OF FITZHUGH
ET AL. (5)
24 MONTH
NO. OF RATS
DIETARY LEVEL SURVIVAL RATE MICROSCOPICALLY
PPM %
Control
0 50
Aldrin
0.5 50
2 50
10 42
50 25
100 17*
150 4
Dieldrin
0.5 42
2 63
10 25
50 21b
100 13a
150 4a
SECTIONED
17
19
19
22
18
11
9
22
23
18
20
18
11
TUMOR INCIDENCE
%
17.6
53
37
36
28
45
36
35
22
20
17
0
aP ~ 0.01 for difference from controls.
bP = 0.05 for difference from controls.
-------
738
1970 as In 1967 (Table 7). Similarly, if the control incidence in
the Fitzhugh et al studies had been as high in 1952, as it proved
to be for the same colony in later years, then it could have been
concluded by these investigators that aldrin and dieldrin were antl-
tumorigenic rather than tumorigenic.
The salient factors suggested by Wilson in determining the
teratogenicity of drugs are outlined in Table 10 and include: the
chemical and pharmacological properties, the level - duration, and
material modulation of dosage, disposition within the conceptus and
susceptibility of species and individual. Table 11 is a summary
of teratogenesis its causes, suggested mechanisms, and manifesta-
tions.
Teratogenic effects of aldrin, dieldrin, and endrin in hamsters
and mice have been reported by Ottolenghi et al (11). Single oral
doses of aldrin, dieldrin, and endrin, (1/2 LDc0) in corn oil and
administratered to pregnant golden hamsters on day 7, 8, or 9 of
gestation caused a high incidence of fetal death, congenital
anomalies and growth retardation. (Most frequent defects were cleft
palate, open eye, and webbed foot.) Pregnant CD-I mice given equiy.
alent oral doses of each pesticide on day 9 of gestation, showed
similar anomalies without concurrent increase in fetal mortality or
growth impairment.
A crucial point concerning the evaluation of genetic risks by
chemicals is the choice of test systems on which to base practical
decisions and permit valid assessments. It has been further
suggested by Bridges (12) and others that the evaluation of the
potential hazard essentially Involves 5 stages of decision making.
These are: 1) Is the agent mutagenic ? 2) Is the agent likely to
be mutagenic to man ? 3) What dose of mutagen is being received or
-------
739
TABLE 9
TUMOR INCIDENCES IN OSBORNS-MENDEL RATS FED ALDRIN. DIELDRIN AND BNDRIN OVER
A MAXIMUM PERIOD OF 31 MO CALCULATED FROM DATA OF DEICHMANN ET AL.(8)
MALE
FEMALE
ALL
0 20 30 50 0 20 30 50 0 20 30 50
RATS ppm ppm ppm ppm ppm ppm ppm ppm ppra ppm ppm ppm
Aldrin
Histologically examined
% with benign tumors
% with malignant tumors
$ total tumours
75
7
19
26
45
5
7
11
46
2,
13
15
4i>
2 5
4
9
88
44
24
68
47
34
9
43
44
46
9
55
31
26
10
36
163
32
21
48
92
20
8
27
90
23
11
34
76
13
7
20
Dieldrin
Histologically examined
% with benign tumors
% with malignant tumors
% total tumors
75
7
19
26
48
2.1
6
8
38
5
13
19
44
2, 3
0
2.3
88
44
24
68
48
38
10
48
41
27
12
39
41
29
10
39
163
32
21
48
96
20
8
28
79
16
13
29
85
15
5
20
Endrin
0 2 6 12 0 2 6 12 0 2 ^ 12
ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm
Histologically examined 75 47 44 42 88 48 45 49
% with benign tumors 7 9 5 0 44 55 47 33
% with malignant tumors 19 15 9 24 24 21 11 22
% total tumors 26 24 14 24 68 76 58 55
163 95 69 91
32 33 26 18
21 18 10 23
43 50 36 41
TABLE 10
FACTORS DETERMINIKQ TBRATOQENICITY OF DRUQ3
1. Type of Drug (Chemical and Pharmacological Properties).
2. Level and Duration of Dosage.
3. Maternal Modulation of Dosage
4. Access to the Conceptus.
5. Developmental Stage at Time of Dosage.
6. Disposition Within the Conoeptus.
7. Susceptibility of Species and Individual.
-------
740
TABLE 11
SUMMARY OP TERATOGENESIS
CAUSES
MECHANISMS
Action of an agent
from the environment
on the embryo or the
germ cells,
e.g. Radiations
Chemicals
Dietary deficiency
Infection
Hypoxia, etc.
Temperature
Endocrine imbalance
Physical trauma
Placental failure '
Reaction within the
embryo or germ cells,
such as one or more
of following:
Mutation
Chromosomal
nondisJunction
Mitotlc interference
Altered nucleic acid
integrity or function
Lack of precursors,
substrates, etc.
Altered energy sources
Changed membrane
characteristics
Water-electrolyte
imbalance
Enzyme inhibition '
MANIFESTATIONS
Pathogenesis, Initiated
by one or more of followll|
Cell death
Mitotic rate change
Reduced biosynthesis
Altered differentiation
schedules
Impeded morphogenetic
movement
Etc.
- and leading to abnoiJ
tissue and organ develcf
ment which determine tb*
NATURE AND INCIDENCE
of FINAL DEFECT
-------
TABLE 12
MAMMALIAN TEST SYSTEMS
TEST
SYSTEM
ADVANTAGES
pi
;SADVAN£AGES
PRESENT
STATUS
FUTURE DEVELOPMENT
Host-mediat-
ed assay
Cytogenetic,
direct analy-
sis (mitotic)
Only feasible screening method
that detects point mutation, can
detect transient metabolities as
well as directly acting chemical.
Indirect detoxification as well
as potentiation by comparison with
direct test on indicators.
Simple, economical test.
Moderately skilled investigator
required.
Used to correlate carcinogenicity
with mutagenicity.
Applicable to a variety of hosts.
Direct observation of chromosomal
abnormalities.
Indirect indication
active metabolite
as to organ or tissue
may be difficult.
Practical
screening
procedure
over 100
compounds
Somatic and germ cells can be
analyzed comparatively low in cost
Moderately tine consuming.
Can be adapted to standard toxi-
cological protocol.
Needs highly trained
investigator.
Quantitative correl-
ation to point nutat-
ions not known.
Usually detects a
high percentage of
nonviable cells.
Chromatid aberrations
mainly detected.
Practical
screening
procedure
Indicators, such as mammalian
cells, that can divide in
host and mutate at same rate
as in vitro (i.e. mouse
system).
Localization of genetic
effect in various organs.
In bacteria, detection of
multiple genetic events
with a single indicator.
Anaphase determination.
Micro-nuclei determination.
Application of newer strain-
ing techniques.
Automation of chromosone
analysis.
-------
TABLE 12 (CONTINUED)
MAMMALIAN TEST SYSTEMS
TEST
SYSTEM
ADVANTAGES
DISADVANTAGES
PRESENT
STATUS
FUTURE DEVELOPMENT
Dcminant Genetic test conducted in variety
lethal of hosts.
Easy to carry out by trained
technician.
Fits into ongoing toxicity
protocol.
Moderate cost and time.
Determines stages of spermatogen-
esis affected.
Can be carried out on either
chronic, sub-acute, or acute
basis.
Test for lethality.
Indirect test for
cytogenetic abnormal-
ities.
Sensitivity presum-
ably less than viable
inherited abnormal-
ities.
Practical
screening
procedure.
Cytogenetic analysis of
live embryos.
Development of improved '
statistical procedures.
A higher precision in
timing of affected stages
of spermatogenesis.
-------
743
is likely to be received by the population or individual's at risk ?
4) What is the risk from being exposed to the agent and 5) What is
the acceptable risk ? Analogous considerations could also apply
for carcinogens and teratogens.
A number of procedures are presently available in mammals, the
majority of relatively recent origin, that can be used to determine
the mutagenic activity of chemicals. Table 12 lists what is
generally considered the three most useful test systems (e.g., host-
mediated assay, dominant lethal and in vivo cytogenetic) in terms of
their advantages, disadvantages, present status, and future develop-
ment. It is well recognized that other test systems are also
available or are being developed and refined, e.g., ranging from
bacterial, fungal, plant, insect, mammalian cell culture, and intact
mammal to reveal genetic damage, such as dominant lethality, trans-
locations, deletions and duplications and non-diejunction, chromosome
aberrations and forward and/or reverse, multiple specific locus, and
induced recombination gene mutations. Tier approaches to the
assessment of mutagenicity are also advocated (13) by investigators
such as Flamm, De Serres and Bridges.
Table 13 lists the cytogenetic andmutagenic effects of DDT that
have been reported, and once again illustrates the conflicting
results and the difficulty of assessment of the mutagenic status of
this most important insecticide.
With the apparent diminishing use of the chlorinated hydrocarbon
and cyclodiene insecticides (primarily DDT and dieldrin) in the U.S.
and many European countries, the organophosphorus and carbamate
Insecticides are assuming an increasingly important role. What are
the possible consequences of the enhanced use of these replacement
-------
744
TABLE 13
CYTOOQflC AMD MOTAGEMIC EFFECTS OP DDT
SPECIES OR SYSTEM
Mouse
Mouse
Rat
Rat Kangaroo Cell
Line in Culture
Chinese Hamster
Cell Line
Drosophila
Melanogaster
Mouse (Dominant-
Lethal Test)
Rat (Dominant-
Lethal Test)
Host-Mediated Assay
(S. Typhlnurium)
Wasp
(Bracon Hebe tor),
Brime Shrimp
EFFECT
Chromosomal Damage
Chromosomal Damage
Non-Cy togenet io
Chromosomal Damage
DDTi Non-Cytogenetlc ,
Non-Mutagenlo
DDE: Cytogenetic
Weakly Mutagenic
Mutagenlc
Marginally Mutagenlc
Weakly Mutagenic
Non-Mutagenio
REFERENCES
Johnson, 0. A., and Jalal, S. M. ,
J. Heredity, 63. (1973) 7
Markaryan, D. W., Qenetika, 2 (1966)
132
Legator, M. S,, Palmer, K. A., and
Adler, I. D. , Toxicol. Appl. phar-
macol., 24 (1973) 337
Palmer, K. A., Green, S., and Legator,
M. S., Toxicol. Appl. Pharmacol.,
22 (1972) 355
Kelly-Oarvert, P., and Legator, M. S.,
Mutag. Res., 1J (1973) 223
Vogel, E., Mutat. Res., 16 (1972) 157
Epstein, S. S., et al, Toxicol. Appl.
Pharmaool. 2J (1972) 288
Palmer, K. A., Green, S., and Legator,
M. S., Food Cosmet. Toxicol., 11
(1973) 53
Buselmaler, W., et al., Blol. Zentral.
Bl., 21 (1972) 311
Grosch, D, S., J. Econ. Entomol., 60
(1967) 1177. Qrosch, D. S., Science,
155 (1967) 592
-------
TABLE 14
ORGANOPHOSPHORUS INSECTICIDES
AQENT
SPECIES OR
REFERENCE
Malathlon
Chick Embryo
In Ovo
Wistar Rat
Human
Teratogenlc
Teratogenlc
Chromosome
Walker, N. E., Toxicol. Appl. Pharmacol., 19_ (1971) 590
Dobbins, P. K., J. Fla. Med. Assn., 54 (19&7) 452
Czelzel, A,, et al, European Env. Mutagen Soc. Meeting,
UPPSALA, Sweden, June 4-7 (1973)
Ul
Parathion
Chick Embryo
in Ovo
Rat
Teratogenic
Teratogenlc
Hunan
Rat,
Guinea Pig
Chromosome
Anomalies
Cytogenic
Changes
Khera, K. S., Toxicol. Appl. Pharmacol., 8 (1966) 545
Fish, S. A., Am. J. Obstet. Gynecol., 96 (1966) 1148
Klmbrough, R. D., and Gaines, T. B., Arch. Env. Hlth.,
16 (1968) 805
Czeizel, A., et al, European Env. Mutagen Soc. Meeting,
UPPSALA, Sweden, June 4-7 (1973)
Dikshith, T. S. S. and Datta, K. K., Bull. Env. Contain.
Toxicol., 9 (1973) 65; Dikshith, T. S. S., Env. Physiol,
Biochem., 2 (1973) l6l
-------
TABLE 15
OROANOPHOSPHORUS AND CARBAMAT5 INSECTICIDES
AGENT
SPECIES OR
SYSTEM
EFFECTS
REFERENCES
Methyl
Rkrathion
Diazinon
Carbaryl
(Sevin)
Rat, Mouse
Human
Chick Embryo
In Ovo
Rat
Human
Guinea Pig,
Dog
Hamster,
Rabbit, Mouse
Rat, Mouse
Rat
Rat
Guinea Pig
Drosophlla
Helanogaater
Embryotoxlc
Malformations
Teratogenic
Teratogenic
Chromosome
Anomalies
Teratogenic
Non-Teratogenic
Carcinogenic
Teratogenic
Non-Teratogenic.
Non-Mutagenic
Non-Teratogenic
Mutagenic
Tanlmura, T., et al., Arch. Env. Hlth., 1£ (196?) 6l?
Fish, S. A., Am. J. Obstet. Oynecol., 2§ (1966) 1148
Ogl, G., and Hamada, A., J. Jap. Obstet. Gynecol. Soc.,
17 (1965) 569
Marliac, J. P., Toxlcol. Appl. Pharmacol., 7 (1965) 490
Dobbins, P. K., J. Fla. Med. Assn., 5.4 (1967) 452
Czeizel, A., et al, European Env. Mutagen. Soc. Meeting,
UPPSALA, Sweden, June 4-7 (197?)
Robens, J. P., Toxicol. Appl. Pharmacol., 15 (1969) 152
Smalley, H. E., et al, Toxlcol. Appl. Pharmacol., 15
(1968) 592 —
Well, C. S., et al, Toxlcol. Appl. Pharmacol., 21
(1972)
Andrianova, M. M. and Alekseev, I. V., Vop. Pitan, 29
(1970) 71
Zabezhinsky, M. A., Vop. Onkol., 16 (1970) 106
Schtenberg, A. I. and Ozhovan, M. V., Vop. Pitan., 50 (1971 )
151
Weil, C. S., et al, Toxlcol. Appl. Pharmacol., 2_6 (1975) 621
Weil, C. S., et al, Toxicol. Appl. Pharmacol., 26 (1973) 621
Brzheskiy, V. V., Genetika, 8 (1972) 151
-------
747
Insecticides ? The organophosphoroue compounds such as malathion,
parathion, and methyl parathion currently in use, as well as the
carbamate insecticide Carbaryl (Sevin) are generally considered non-
persistent and have a margin of safety between their effective use
•
concentrations and their minimum toxic concentrations to mammals.
They are however not without their potential carcinogenic, mutagenic,
and teratogenic risk as shown in Tables 11* and 15.
In summary, some of the more salient characteristics of
teratogenicity, mutagenicity and carcinogenicity (particularly the
latter) have been examined and a number of conflicting, contradictory,
and ambiguous examples In regard to the above toxieities of several
organo chlorine, organo phosphorus, and carbamate insecticides have
been cited*
It is suggested that a major step could be taken to ameliorate
the possibility of ambiguity in toxicity testing if there were a
central repository of well characterized environmental, carcinogen,
mutagen, and teratogen chemical standards. Perhaps the WHO might
well serve as a focal point in this capacity.
-------
748
References
1. Kemeny, T. and Tarjan, R., "Investigation on the Effects
of Chronically Administered'Small Amounts of DDT in Mice",
Experientia, 22,748 (1966).
2. Kay, K., "Occupational Cancer Risks for Pesticide Workers",
Environ. Res., 7*,243 (1974).
3. Terracini, B., "Multigeneration Studies on DDT. Letters
to the Editor", Food Cosmet. Toxicol., 8,478 (1970).
4. Tomatis, L. , Turusov, V., Day, N., and Charles, R.T., "The
effect of Long Term Exposure of DDT on CF-1 Mice", Int. J.
Cancer, 10,489 (1972).
5. Fitzhugh, O.G., Nelson, A.A., and Quaife, M.C., "Chronic
Oral Toxicity of Aldrin and Dieldrin in Rats and Dogs",
Food Cosmet. Toxicol. 2,551 (1964).
6. Davis, K.J. and Fitzhugh, O.G., "Tumorigenic Potential of
Aldrin and Dieldrin for Mice", Toxicol. Appl. Pharmacol.,
4,187 (1962).
7. Walker, A.I.T., Stevenson, D.E., Robinson, J., Thorpe, E.
and Roberts, M., "The Toxicology and Pharmaco-Dynamics of
Dieldrin (HEOD): Two Year Exposures of Rats and Dogs",
Toxicol. Appl. Pharmacol., 15,343 (1969).
8. Deichman, W.B., MacDonald, W.E., Blum, E., Bevilacqua, M.,
Radomski, J., Keplinger, M., and Balkus, M., "Tumorigenicity
of Aldrin, Dieldrin, and .Endrin in the Albino Rat", Ind.
Med., 39,426 (1970).
9. Walker, A.I.T., Thorpe, E. and Stevenson D.E., "The
Toxicology of Dieldrin (HEOD), "Long-Term Oral Toxicity
Studies in Mice", Food Cosmet. Toxicol., 11,415 (1973).
1O. Thorpe, E. and Walker, A.I.T., "The Toxicology of Dieldrin
(HEOD). II. Comparative Long-Term Oral Toxicity Studies in
Mice with Dieldrin DDT, Phenobarbitone, /J-BHC and V-BHC",
Food Cosmet. Coxicol., 11,433 (1973).
11. Ottolenghi, A.D., Haseman, J.K., and Suggs, F., "Teratogenic
Effects of Aldring, Dieldrin, and Endrin in Hamsters and
Mice), "Teratology,.!, 11 (1974).
12. Bridges, B., "Environmental Genetic Hazards - The Impossible
Problem" EMS Newsletter, 5,13 (1973).
13. Flamm, W.G., "A Tier Approach to Mutagen Testing", First
International Conference on Environmental Mutagens",
Asilomar, Calif., Aug. 29 -Sept. 1. 1973.
-------
749
DISCUSSION
EPSTEIN (U.S.A.)
Suggested distinctions between tumorigens and carcinogens,
have been repeatedly examined bv a wide range of expert national
and international committees and have been shown to be totally
lacking in validity. Illustrativelv, it has been claimed that
DDT and Dieldrin should be recorded as tumorigenic because both
produce mouse hepatomas which are clained to be "benign tumors".
However, DDT induced hepatomas netastisizing to the lungs if
mice are not prematurely sacrificed, DDT also produces nalignant
lymphomas in mice. Dieldrin has been shown in a wide range of
studies including those recently published by SHELL Toxicologists
(Thorpe & Walker, 1972/1973) to produce hepatomas in mice which
metastasize to the lungs, and a wide range of extra-pulmonary
malignant tumors. Dieldrin also provides liver cancers and
malignants in other organs in rats (Thorpe & Walker, 1973). These
hepatic and non-hepatic neoscosms in mice and rats following
dieldrin treatment occurred in statistically significant inci-
dences. The uneauivocal carcinogenicity of aldrin and dieldrin
has been recently summarized in testimony, at recent (1974)
hearings in litigation between EPA and EOF vs SHELL, by Saffiotti,
U. , Farber, E., and Epstein; S.S.
FISHBEIN (U.S.A.)
The comments of Dr. Epstein on the carcinogenicity of DDT
and dieldrin are noted and appreciated. The "unequivocal car-
cinogenicity of aldrin and dieldrin" which was referred to by
Dr. Epstein was derived from calculations performed on the data
(on the incidence of liver-tumors of CF-1 mice) of Walker/
Thorpe, and Stevenson (Food Cosmet. Toxicol., 11, 415-432, 1972)
employing calculations as described by Mantel and Bryan (J. Nat.
Cancer Inst., 27, 455-470, 1969) using an assumed slope on one
probit per log dose, to calculate the dose corresponding to a
maximum risk of one per hundred million mice. The highest cal-
culated dose corresponding to a risk of 10~& was obtained with
the data at 1.25 parts per million of dieldrin in the diet fed
CF-1 mice for 128 weeks. The values for Pmax for the experimental
points and Pmin for the controls were based on two-tailed 95%
confidence limit binomial tables, and hence correspond to 97.5%
one-tailed confidence limits.
With regard to the comment of Dr. Epstein that dieldrin has
been shown to produce hepatomas in mice which metastasize to the
lungs, this was observed in the study of Walker et al (Food
Cosmet. Toxicol., 11, 415-432, 1972) in a male and a female mouse
(out of 60 animals) fed 2.5 ppm dieldrin for 128 weeks. The
lung lesions comprised massive secondary tumors and a small emboli
of tumor cells in pulmonary vessels.
-------
750
It is also important to note that a new criterion for
determining the carcinogenicity of a substance was used in the
Environmental Protection Agency (U.S.) decision of October 1,1974
suspend all major uses of aldrin and dieldrin pesticides. For
purposes of "carcinogenicity testing", tumorigenic substances
and carcinogenic substances are "synnymous" and a carcinogenic
substances is defined as "one that increases the incidence of
benign or malignant tumors in exposed animals, decreases the
latency period between exposure and onset of tumor, or results
in unusual tumors" (Chem. Eng. News 52, 13, 1974; Federal
Register, Oct. 18, 1974, p. 37246).
OLOFFS (Canada)
In view of the last comments, it should also be mentioned
that O^p' - DDD, administered orally to humans at doses as
high as 1O gr/day for several months, has been used in the past
for the treatment of adrenocortical cancer.
-------
751
TOXICOLOGY OF ATMOSPHERIC POLLUTANTS RESULTING
FROM FUEL ADDITIVES AND EMISSIONS ASSOCIATED
WITH THE USE OF AUTOMOBILE CATALYTIC CONVERTERS
J. F, STARA, W, MOORE/ A. W, BREIDENBACH
Environmental Protection Agency, National Environmental Research
Center, Environmental Toxicology Research Laboratory, Cincinnati
Ohio, USA
ABSTRACT
The purpose of this paper -La to present the definitive toxi-
cologic investigative approach employed in our laboratory for
testing automotive emissions, fuel additives, and emission com-
ponents related to the catalytic converter emission control
system.
A matrix for investigating the toxicology of individual
pollutants and emissions from mobile sources has been used to
determine the potential toxic effects. The presented data are
illustrations of the response of selected animal models to the
catalyst-treated exhaust and to individual components such as
platinum and palladium. The biological parameters studied were
markedly influenced by the use of the converter.
-------
752
Introduction
The Environmental Toxicology Research Laboratory, NERC-
Cincinnati is engaged in the testing, evaluating and defining
potential harmful effects of environmental pollutants from mobile
and stationary sources. The data obtained in experimental biologi-
cal models provide necessary input for the development of scientific
criteria, which in turn, serve in the process to establish realistic
environmental standards. During the past year the toxicological
investigations conducted in this laboratory dealt primarily with
studies of individual catalytic converter emission components, and
exposures of animals to whole emissions from engines equipped with
or without catalysts. Emphasis was placed in this area because
the United States automotive manufacturers have developed and will
install on many of the 1975 light-duty vehicles catalytic converters
to reduce concentrations of carbon monoxide and hydrocarbons in
exhaust emissions.
Since human exposure from this source is particularly related
to air pollution hazards, the route of animal exposure is primarily
by inhalation. In many instances, however, the pollutants e.g.
heavy metal compounds, are found in more than one environmental
media and to provide relevant information on their public health
impact, several routes of entry are investigated. In addition,
appropriate mammalian species of different ages (from embryonic to
aged models) are being used to optimize the possibility of reproduc-
ing the human response in the different segments of the population.
The experimental protocol for the definitive toxicologic investi-
gations is based on a matrix which is summarized in Table 1. It
describes three types of investigations conducted in this laboratory.
-------
753
Table 1
1
[ACUTI [xroiuii I
1 »D jo lciO lrc
? ftmiQLOGICAl
PUIMONAIV fUNCMON
NfUIOPHYilOlOGiC
tISPONSI IIC
3 PAlMClOC*
GIOU 1 IIC HI
4 ilNSlTIVIIY DUMAL
QCUlAi ETC
S IIOCHEMICAL UMIN
iU»ACUfl IXPOSUII
1 PHTSIOIOOT
PUIMONAIT
IfMAWIOtAl NIU
PH11IOLOGIC Al
•iPtODUcnvi
7 IIOCHIMIillY
INIYMII
MHAiOlllti
3 PAIMOIOGT
LIGHT AMD IM
4 MfIA»OL»M
KINHtCV tODV
•UKDIN IK
t
111! tIPOtTi
1OPIN LIIIIATUIf.
INTflNAL]
1
ICHIONIC IKPO&UIIJ
SflfCTIVI IIMV ItOM
WHICH IVlt Ol IHt
IO FOItOWING li DIIMI
won PHOPCIC nvi
1 *n if HIMU Al
1 PHrilOlOGICAl
3 PAIHOLOGICAl
4 KIMINIAl
ANMTSti
S OIHIt AS
APMOPItATI
/
/
'
.Fig. 1. A closed "nose only"
rodent inhalation chamber
-------
754
(1) The short-term acute exposure experiments in which high levels
of new compounds are tested to ascertain frank systemic effect;1 '
(2) Subacute exposure experiments of longer duration at lower
levels where specific bioeffect end-points are measured to provide
physiological, biochemical and pathological parameters of injury;
and (3) Chronic, long-term, exposure studies conducted at realistic
(2 ?)
environmental levels. >v The approach for any given pollutant
will vary somewhat from the general scheme depending on what are
the gaps in current knowledge concerning the compound under invest!-
gation.
A. Animal Exposure Systems
For the single pollutant inhalation exposure studies, several
aerosol and gas generating systems are presently in use. An
example of a closed, "nose only" inhalation animal chamber system
is shown in Fig. 1. The inhalation chamber permits exposure of
80 rodents simultaneously to high level aerosols or gases including
radioactive metal tracers. In addition to this system, the
Inhalation capability of the laboratory includes 2l\ large animal
chambers (approx. 3 • ea.) and 25 smaller portable chambers
(approx. 1/2 ir ea.), and among others, a single animal aerosol
generation system to measure pulmonary mechanics and cardiovascular
parameters in animals during exposure. Another development during
the past year includes the use of telemetry for measurements of
respiratory and cardiovascular parameters in large animals (dogs'
or primates) which are being chronically exposed to various
pollutants in the larger chambers. '
B. Automotive Engine and Exhaust Exposure Systems
The installation of two engines (Ford 1975 prototype engine,
-------
755
Table 2
IS MPH STEADY SPEED RUNS (TENTATIVE)
(Exhoust Emission** Normalized to 7.5/1 Dilution)
Indol.n. {0.05% S| Indol.i.. (0.105C S)
Gottoui: With Converter (C) With (C) Without (C)
CO, ppm
THC, ppm
Particular:
Total, mg/m
Sulfate, mg/m
Mitral*, mg/m
7
9
9,4
4.1
.01
1
9
3O.2
12.8
.01
522
99
3.2
0.5
.04
•Measured in N-l •xpoiur* chambers
Table 3
AVERAGE PERCENT CONCENTRATION
REDUCTIONS OF TAME J OVER I
(W/0 CATAIY5T)
CO 93X
THC -SOX
ALDEHYDES AND ACETYLENE 99+%
OLEFINS. 93%
AllPHATICS _ .59%
-------
Fig. 2.
756
G.M. engine with "pelletized"
catalytic converter
Fig. 3. 1975 Ford prototype engine
with monolith catalytic converter
-------
757
Fig. 4. Dilution chamber for studies
of exhaust emissions
Fig. 5. Irradiation chamber
-------
758
Fig. 6. Animal exposure chambers for
exhaust emission studies
Fig. 7. Schematic diagram for toxicologic
testing of automotive emissions
NOM-IIIADUTID
IIK>ilMI CHAMII*
INC INI
-------
759
Chevrolet 1973 production engine with control devices) equipped with
catalytic converters enabled the laboratory to conduct subacute and
chronic inhalation studies of the emissions with and without the
catalyst. Simultaneously, aerometry measurements were made of
the emission components as a part of any of the Toxicologic Assess-
ment of Mobile Emissions (TAME) experiments. Figures 2-6 provide
a pictorial view of the exposure system. They include the G.M.
and the Ford engines with the catalytic converters, a dilution pipe,
irradiation chambers for production of photochemical smog, and 28
animal exposure chambers connected to this system (approx. 1.25 nr
ea.). The schematic diagram of the system, as well as aerometry
sampling ports is presented in Figure 7.
C. Aerometry of the Catalyst Emissions
From the standpoint of potential health effects, the emission
components of greatest concern following the use of the catalyst
is the increased amount of particulates, particularly eulfates,
and the potential release of catalytic attrition products into the
tn\
atmosphere containing the noble metals platinum and palladium. '
In addition, a shift in the individual hydrocarbons is also of
potential interest. Table 2 summarizes the effects of the catalyst
control system in the 1973 G.M. engine on the exhaust emissions.
The data show that the oxidizing catalyst has successfully reduced
the levels of carbon monoxide and total hydrocarbons in the exhaust
stream; however, the amount of particulates was significantly
increased, especially the amount of 8ulfates. A striking increase
occurred in the acidity of the particulate. In one of our
experiments with the catalyst in place, the acidity of particulatee
were measured to be some 65 times higher than in the exhaust without
-------
760
Table 4
RELATIVE ACIDITY LEVELS OF
EXHAUST EMISSION PARTICIPATE AT 24,2 km/Hr. (IS MPH)
Opororing
Co ndilion
No Cololyti* Convener With Catalytic Canvor'or
High-S IndoUnt fo«l »«S Indolin. HlgK-S I
Rclativ* Atidily
ol Particulot*
Tolul Parliculol*
@ 7.S/1 Dilution,
mfl/m1
/.J
260
25 6
Fig. 8. Mort-Hlity rate in infant rats
TAMI
,
-c*
0 I
012)41*7
Dlfl ol Il
-------
761
Fig. 9. Body weight of lactating
female rats
ISO
300
MO
700
uo
TAMII (M fo*^»")
.CO
MO
0 1
TAMI J |
-------
762
catalyst; if additional sulfur were added to the gasoline, the
acidity increased 260-fold (Table 3).
D. Biological Effects of Whole Emissions
In this series of studies, TAME-I (without catalyst) and TAME-J
(with catalyst), the primary animal species used were rodents; rats
and hamsters. The animals were exposed to one of the following
atmospheres: 1) catalytic treated irradiated exhaust; 2) cataly-
tic treated non-irradiated exhaust; 3) irradiated exhaust;
-------
763
l;ig. 10. Hamster lung tissue exposed
to catalytic emissions
,-^v
V
l^v.
Fig. 11. Pulmonary changes in hamsters
exposed to irradiated exhaust
without catalytic converter
-------
764
exhaust exposed hamsters, the pulmonary changes were not as severe.
Pulmonary changes noted in the rats exposed to non-catalytic treated
exhaust paralleled those found in the hamsters. In both the
Irradiated and nonirradlated exhaust exposed hamsters (TAME I),
some vacuolar changes were found in hepatic parenchyma! cells after
5 days of exposure. Similar vacuolar changes in the renal tubular
cells were seen in the irradiated exhaust exposed hamsters. In the
catalyst-treated exhaust exposures, TAME J, no significant treatment-
related morphologic changes were noted in either the rat or hamster
tissues. In the clinical pathologic tests performed on animals
exposed to the catalytic-treated exhaust (TAME J), the only
statistically significant treatment effect was an increase in total
serum proteins. In the animals exposed to the noncatalytic treated
exhaust (TAME I), there were statistically significant treatment
effects in both nonirradiated and irradiated exhaust exposures on
total protein, platelet count, RBC and WBC counts, white cell
differential, alkaline phosphatase, HB, HCT, PTT, SCOT, and SQPT
levels. The irradiated exhaust also produced a treatment effect
on levels of BUN and fibrinogen. It should be noted that the
exhaust exposed animals in the noncatalytic-treated groups showed
a rather striking increase in hemolysia resistant RBC'a, which
necessitated manual determinations of WBC counts. In explaining
the effects, the RBC related changes and alkaline phosphatase
levels could relate to the high levels of CO; the WBC changes most
probably relate to the severe acute inflammatory response in the
lungs; the other changes could relate to hepatic and/or renal
dysfunction.
-------
765
Fig. 12. Retention of 103Pd and 103Pt
in rats
L-t.iti*« im
Fig. 13. Retention of 191PtCl4 and 191Pt(S04)2
in rats following inhalation
INHALATION
EXPOSURE
DAYS
-------
766
Table 6. Acute Lethal Toxicity of PdCl.
Species
Rat
Rat
Rat
Rat
Rabbit
Approx. LD50
5 mg/kg
70 mg/kg
200 mg/kg
6 mg/kg
5 mg/kg
Route
iv
ip
po
itr
iv
Table 7
OEOMAL lOXICItr OF PAUADIUM AND
HAIINUM COMPOUNDS IN •AHIIS
Of lOtt'/fO MAIM
iNICAtlvf CONIIOll
PAUAO'UM MONOKIM
IHOI
AMMONIUM
H)ii>cmoiorMi«o>il
HAIINUM OIOXIDI |PIO,I 0
HATINUM OKWOIIOI 01
IBCIjl
riATIhUMIITkACHlOIIDI III
1 HdHTICICIOHNIAWNtL 01
MANGANIII fftlCAMONVl
IMMII
04
Itlltl
01
GLACIAL ACEUC ACIO
-------
767
E. Biological Effects of Individual Components
Studies on individual components of catalytic converter emissions
are primarily directed to obtain needed toxicological data on the
sulfuric acid and sulfate particulate emissions, in addition to
/o\
data already available in the literature, ' However, the two
noble metals, platinum and palladium, which are used in the catalyti
material may be also emitted into the atmosphere particularly during
the period of catalyst degradation. There is an extreme paucity
of toxicologic data on these two metals. We have conducted a
series of acute and subacute experiments to obtain data on retention
of palladium and platinum after different routes of administration
(Fig. 12), and retention of various chemical compounds of platinum
after inhalation (Fig. 13). Additional experiments such as acute
toxicity of PdCl- (Table 6), dermal toxiclty of various platinum
and palladium compounds (Table 7), neurophysiological studies
utilizing the visual-evoked potential screen (Table 8), eye irrita-
tion screen, pulmonary, cardiovascular, biochemical and pathological
effects have been investigated. No significant amounts of Pt or
Pd were found in any of the tissues taken from animals exposed to
the whole catalytic exhaust for periods of one week to one month.
Conpendlum
The field of fuel additive toxicology including the exhaust
components of controlled systems such as catalytic converters is -
for a research toxicologlst - an extremely gratifying one. In
these investigations, the toxicology discipline should have as Its
primary goal the testing of substances that are considered for
commercial use and have not been as yet Introduced into the
environmental media. Toxicologic research on such materials
-------
768
Table 8
VISUAL-EVOKED POTENTIAL SCREEN
REPRODUCIBLE DOSE-EFFECT
METAL COMPOUNDS THRESHOLD (m,/lg|
COBALT 0.010
CADMIUM 0.10
CHROMIUM 0.40
PALLADIUM 0.40
BARIUM 2.0
MANGANESE 2.0
PLATINUM MINIMAL EFFECT
Table 9
Overview of Public Health Impact of Fuels
Fuel Additives
MQOMCtlON AMD CONIUMPTION IIAOM
IWl 01 lull AMITIVI
COKfOKINI
IJHMCH 1
AND U
IXVILOPMINll
Hoimmjiiox
«»D
VINOtNA
-JcantuupnoN
«HH»tio«
HUUIH MlluiatHCI MOUiltMt
IIHAICH *me«CHii TOIKOW*!
-------
769
provides the decision-making bodies with information on the
potential toxicity of new substances or compounds and In this way
fulfill a vital function. Table 9 summarizes the public health
overview and the role of toxicology in manufacturing, distribution,
and consumption of fuel additives. It lists the population sub-
groups which may be exposed to these compounds. It is in this
manner that toxlcologlsts can optimally coordinate their findings
with the epidemiologists and clinicians.
References
1. Annual Report-1972. Environmental Toxicology Research
Laboratory. National Environmental Research Center,
Cincinnati, Ohio. Environmental Health Effects Research
Series A-670/1-73-036, January, 1973.
2. Stephens, R.J., a. Freeman, J.F. Stara and D.L. Coffin.
Cytologic Changes in Dog Lungs Induced by Chronic Exposure
to Ozone. Am. J. Path. 73(3): 711-718, 1973.
3. Bloch, W.N., Jr., T.R. Lewis, K.A. Busch, J.O. Orthoefer,
and J.F. Stara. Cardiovascular Status of Female Beagles
Exposed to Air Pollutants. Arch. Environ. Health 21*: 31*2-351*,
1972.
if. Wieater, M.J., R. Iltis and C. Pfetzer. Respiration Measured
by Dae of an Implantable Strain Gage Sensor via Telemetry in
the Dog. (Abstract). Medical Instrumental 8(2): 183,1974.
5. Burkart, J.K., R.O. Hlnners, and M. Malancliuk. Catalytic
Converter Exhaust On leaions. Presented at the Air Pollution
Control Association 6?th Annual Meeting, Denver, Colorado,
June 9-13, 1974.
-------
770
6. Pierson, W.R., R.H. Hammerle, and J.T. Kummer. Sulfuric
Acid Aerosol Emissions from Catalyst-Equipped Engines. Presented
at the Automotive Engineering Congress, Detroit, Michigan,
February 25-March 1, 197
-------
771
STARA (U.S.A.)
Thank you for your compliment, Dr. Stupfel.
As to your first question: We have conducted an 8 year
study of air pollutant effects in beagle dogs - there the daily
exposure period was 16 hrs/day in order to compare at least
somewhat to usual heavier traffic condition in urban areas. In
this experiment the dogs were fed during the 8 hours rest period.
Presently we conduct exposure experiments in rodents, from one
week to two months duration on a 24 hours exposure basis. We
did have some problems with feed contamination and the animals
not eating it; we now exchange feed more often and the animals
are eating it as shown by weight gains similiarity between pos-
itive and negative (clean air) control groups.
I completely agree with your second comment dealing with
the metabolic rate of rodents during light and dark cycle and
the statement supporting 24 hours exposure of rodents. As a
matter of fact we have performed experiments of effects of the
light and dark cycle using mitotic rate index in the cornea of
the eye of rodents and found significant differences.
SCHLIPKOTER (Federal Republic of Germanv)
1. To what extent did you dilute vehicle exhaust emissions in
your excellent research inventigations?
2. How do you explain the high mortality rate in your acute
studies, if this was not caused by the CO concentration in the
exposure chambers?
STARA (U.S.A.)
1. For the present studies with the catalyst device emissions
we dilute the auto exhaust at a ratio of 1O : 1 because we
want to test the acute effects of auto emissions with and with-
out the catalytic converters. However,we have the capability
to lower the dilution down to realistic environmental levels,
e.g. 200 : 1.
2. The high mortality rate in the weanling rats was caused
by other emission components than the CO. The CO exposure
group was our positive control group and resulted in a negligible
mortality rate. The mortality was caused bv the combination of
total hydrocarbons, nitrogen oxides and perhaps CO together in
the raw exhaust (a total of 75% mortality rate), and additionalv
the oxidants in the irradiated exhaust (a total of 100% mortalitv
rate).
-------
772
CALANDRA (U.S.A.)
What is your assessment of the significance to the general
population of the safety of the catalyst and its attrition pro-
ducts as well as the increased emission of sulfates? This is a
health issue and not an economic one.
STARA (U.S.A.)
My answer must be based in this forum on scientific data.
Thus far, in our experiments of one week and one month contin-
uous exposure, we have not found overt deleterious effects in
rodents exposed to the exhaust from engines eauipped with the
noble metals oxidizing catalyst. That is not to sav that longer
chronic experiments may not show some positive results - perhaps.
But so far there is no technical evidence available to prove this
point either way.
Oh, vou wish to know my personal opinion? Well, I would be
happy to' discuss the matter further with you, but not from this
rostrum.
-------
773
QUANTITATIVE ESTIMATION OF THE EFFECT OF
VARIOUS PULMONARY IRRITANTS ON THE LUNG CELL
POPULATION
M, J, GRONSPAN
Medizinisches Institut fur Lufthygiene und Silikoseforschung an
der Universitat Dusseldorf, BRD
ABSTRACT
Polyvinylpyridine-N-oxide (PVNO) is a polymeric non-toxic
substance which is utilised in the treatment of experimental and
human ailiaoais.
After i-venoua injection in Rats, PVNO accumulated in the
macrophages of liver, spleen and bone marrow, but only poor in
the lungs.
This distribution was reversed by the intra-traoheal injec-
tion of Phosphatydilserine, Gama-Globulinet Freund Adjuvant and
the inhalation of N°n» an^ followed by a large accumulation of
PVNO in the lung tissue* We suppose that this increase is
caused by the migration to the lung of macrophages originating
from the HES of the body and which have previously pynocitosed
PVNO.
Counts of the total number of cells recovered from the lung
waehings from rate which were treated with Freund Adjuvant, showed
a good correlation between the progressive accumulation of
in lung tissue and the progressive increase of the number of
alveolar cells in the lung washings.
-------
774
As part of an organ which comes first in contact with atmo-
spheric pollutants and other pulmonary irritants, the alveo-
lar macrophages play an important role in lung defence,
which is accomplished by a certain number of alveolar macro-
phages. It is a still unresolved problem whether this defen-
ce is executed by an invariable amount of macrophages or if
various pulmonary irritants can increase or diminish the
number of alveolar macrophages in the lungs. The physiologi-
cal and pathological mechanisms which elicit the increase or
diminution of the alveolar macrophages are incompletelly in-
vestigated, tool. We suppose that because the lung macropha-
ges are part of the general RES, any augmentation of lung
macrophages suppose that supply is a secondary one inasmuch
as the cells are furnished to the lung from the RE organs as
a response to pulmonary injury.
The number of alveolar macrophages on the one hand probably
depends from the chemical nature of the pulmonary irritant,
its retention in the lung and the duration of the exposure,
on the other hand from the age of the exposed being; all the-
se relationships are in detail. In order to clarify this
problem we have developed 2 methods to test the influence of
various pulmonary irritants on the cell population of the
lung.
The first method
Young and old rats were exposed to gaseous noxious agents
that contaminate urban atmospheres, 24 h per day. At diffe-
rent times the animals were killed and the lungs were washed
with saline. The alveolar macrophages were obtained from 23
lung washings per animal, using 1 ml of saline for each
washing. The number of cells and the cell size was estimated
with a. Culter Counter, Model FN.
The second method
Rats were injected i-v which Polyvinylpyridine-N-oxide (PVNO)
a polymeric non toxic substance which accumulate in the RES.
By means of a quantitative method (GRt)NSPAN, 1, 2), we have
estimated the amount of PVNO stored in the liver, spleen,
-------
775
bone marrow, and lungs 1-2 months after the i.v, application
of PVNO, before and after the i-tracheal administrative of
various substances or the inhalation of NO^.
Following experimental groups are formed:
Group I - only PVNO i.v. (50 mg/kg body).
Group II - PVNO and 3 mg of Phosphatidylserin injected
i-tracheally.
Group III - PVNO and 3 rag of Gama-Globuline injected
i-tracheally.
Group IV - PVNO and 0,05 ml of Freund Adjuvant complet
injected i-tracheally.
Group V - PVNO and 0,005 ml of Freund Adjuvant complet
injected i-tracheally.followed by exposure to
N02 (20 ppm) for 48 hr.
Group VI - PVNO followed by exposure to NOo for 7 days.
Group VII - PVNO followed by i-tracheally injeccion of
0,5 ml NaCl 0,9$.
In the experiments with Freund Adjuvant we have made also a
lung lavage technique and the number and size of cells in the lavage
liquid was counted and correlated with the amount of PVNO found
in the lung.
RESULTS
1. Physiologic distribution of PVNO (50 mg/kg) in the normal
Rat:
When 50 mg PVNO/kg were injected i.v. to normal Rats 30
days after the injection," the liver contained 500 nig,
the spleen 180 /ug, the bone marrow 150 /ug and the lungs
only 50 yug of PVNO (fig. l).
2, The accumulation of PVNO in the lung following Phosphati-
dylserine is important, and was estimated at 140 /ug
{fig. 2).
3. The i-tracheal injection of Gama-Globuline caused an accu-
mulation of 100 /ug in the lung (fig* 3).
4. The greatest accumulation of PVNO in the lung occured 7
days after the i-tracheal injection of Freund Adjuvant
(200 ug) (fig. M.
-------
776
500
200
100
DISTRIBUTION OF POLY-VINYL-
PYRIDIN-N-OXID 21 DAYS
AFTER I.V INJECTION (SOmg/Kg)
(RAT 9 180-200g)
200
I
• iso
100.
so
LIVER SPLEEN LUNG BONE MARROW
Fig* 1. Physiologic disposition
of PVNO
ACCUMULATION OF PVNO IN THE
LUNG FOLLOWING PHOSPH. SERINE
INJECTED hTRACHEALLY
Ko
NaCI PhS
Fig. 2. Accumulation of PVNO in
the lungs following i-tracheal
injected Phosphatidylserine
200
'ISO
too
50.
ACCUMULATION OF PVNO IN THE LUNG 20°
FOLLOWING GAMA GLOBULN INJECTED §
t-TRACHEALlY 3
•ISO
Ko NaCI G.G.
Fig. 3. Accumulation of PVNO in
the lungs following i-tracheal
injected Gama-Globuline
100-
so.
ACCUMULATION OF PVNO IN THE LUNG
FOLLOWING FREUND ADJUVANT
INJECTED l-TRACHEALty
NaCI F.A.
Fig. *f. Accumulation of PVNO in
the lungs following i-tracheal
injected Freund Adjuvant
-------
777
200
|;50
100-
'
ACCUMULATION OF PVNO
IN THE LUNG FOLLOWING
I-TRACHEALLY INJECTED
FREUND ADJUVANT AND
48* EP05URE TO N02
(20ppm)
KO
';• Accumulation of PVNO in
the lungs after Freund Adjuvant
injected i-tracheally followed
by exposure to NO,, (?0 ppm) for
^8 hr. ^
CELLS
V6\
2,06
IJlfi
0,56
I
i
: •. ',
; M-•-....
CELLS IN LUNG
\ -k WASHING
QFiiS
IN
LUNGTISSUE
200
wo
so
S 8 10
30 DAYS
Fig. 6. Number of cells in lung
washings and PVNO in lung tissue
after i-tracheal injection of
Freund Adjuvant
KO == Controls
NaCl = NaCl 0,9#
GG := Gamma Globuline
P.S. Phosphatidylserine
F.A. Freund Adjuvant
i
The exposure to NO., determine an accumulation of PVNO
which is simular to those observed in the normal (fig.5).
In the Hats which have received i—tracheally Freund Adju-
vant, and consecutivelly NQ0 for 48 hr., we have observed
a large increase in the amount of PVNO in the lung tissues,
which was greater that the increase caused by Freund ad-
juvant alone (fig. 5).
Counts of the total number of cells recovered from the
lung washings from Rats injected i-tracheally with Freund
Adjuvants show a good correlation between the progressive
accumulation of PVNO in lung tissue and the progressive
-------
778
increase of the number of alveolar cells in the lung
washings (fig. 6).
The i—tracheally injection of NaCl not determine any accumu-
lation of PVNO in the lung tissue (fig. 2, 3, 4).
DISCUSSION
The results of this experiments demonstrated that it is pos-
sible to charge the RES of the Rat by means of Polyvinylpyri-
dine-N-oxide wich is pynocitosed in large amounts by the
macrophages from the liver, spleen, bone marrow and only poor
from the lung. By means of an i-tracheally injection of Phos-
phatidylserine, Gama-Globuline, or Freund Adjuvant alone or
followed by NOg, we can discharge the RES which mobilise the
macrophages loaded with PVNO to the lungs.
If the amount of PVNO found in the lungs of the normal Rat 30
days after the i.v.-injection is only 40-50 /ug, we assume
that this counted exprime a proportionally number of macro-
phages which have pynocitosed PVNO and which are present in
the lungs. We can accept that the amount of PVNO found in
the lungs is a mesure of the number of RE cells accumulated
in the lung as a direct consequence of an i-tracheally irri-
tation.
This irritation started from the lungs, reaches the fixed
cells of the RES which have previously pynocitosed the i.v.-
injected PVNO and determine this cells to migrate to the lung.
Our experiments demonstrate the existence of a mechanism
which suppose an intimate cooperation between lungs and the
RES of the body.
The fact that a component of the alveoles surfactant (Phos-
phatidylserine) and a substance involved in the host defense
of the lung (Gama-Globuline) are able to determine the migra—
tion of the fix cells of the RES is currently under investi-
gation.
Thus, the augmentation or diminution of the number of alveo-
lar macrophages following exposure to various noxious agents
can be quantified directly with the help of the Culter Coun-
ter or by measuring the PVNO content of Rat lung after label-
ling the cells with this nontoxic polymeric substance these
-------
779
estimations providing important insight into the defence of
the lung because the alveolar macrophage 1st involved both
into the phagocytosis of inhaled particles and into the pro-
duction of antibodies.
LITERATURE
JjlJ GRUNSPAN, M. und II. ANTWEILER: Biochemical and biophy-
sical reactions of rat lung tissue to quartz and corun-
dum with and without PVNO-oxide treatment.
In: Inhaled Particles III. Ed. by W.H. Walton, Vol. 1.-
Old Woking, Surrey: Unwin Brothers (1971), 373-378.
GRUNSPAN, M. et H. ANTWEILER: Methode zur quantltativen
Bestimmung von Poly-2-vinylpyridin-N-oxid in Blut und
Gewebe.
Int.Arch.Gewerbepath., 25 (1969), 338-346.
-------
781
LOCAL EFFECTS OF INHALED LEAD COMPOUNDS ON
THE LUNG
J, BRUCH, A, BROCKHAUS AND W, DEHNEN
Medizinisches Instltut fur Lufthygiene und Silikoseforschung an
der Universitat Dusseldorf, BRD
ABSTRACT
The effecta of inhaled lead particles on the lung parenchyma
are reported. The following findings were obtained;
1. The ultrastructure of the alveolar macrophages and the
pneumocytes type I indicate a cytotoxia action of the inhaled
lead aerosol.
2. The pneumocytes type I exhibit an increaoed H 3-thymidine
labelling index. This finding can be explained by elevated
turn-over of cells at the alveolar wall.
3. The determination of benzopyrene hydroxylase activity showed
a clear depression to 10% of the control values.
4. In a chronic 15 weeks experiment with alternate inhalation
of lead and titan dioxide aerosolst lung clearance was impaired
by 33 to 39%.
The lead concentrations used were in the range of 70 to
3
170 jug/m t i.e. close to the Vest German MAK-value.
On the basis of these investigations it must be assumed that
inhalation of lead compounds of the described or even lower con-
centrations produce local toxic effects in the lung.
-------
782
Introduction
Number and vitality of alveolar macrophages play an essen-
tial role in the defense mechanism of the lung against
live and inanimate noxious agents. After BECK and colla-
borators (1) could demonstrate that lead compounds exhi-
bit a considerable cytotoxicity in vitro comparative to
that of quartz, we investigated the local effects of in-
haled lead particles on the pulmonary alveolar macropha-
ges and the alveolar parenchyma.
For this purpose four different test methods were employ-
ed:
1) electron microscopy; 2) determination of the H-3-thy-
midine index; 3) studies of the benzopyrene hydroxylase
activity; 4) determination of the retention of an inert
dust following chronic lead exposure.
Lead exposure was carried out by the evaporation of lead
oxide at approximately 700° centigrade and subsequent
condensation of the fine lead particles in the cooling
air current. Lead concentrations were measured with the
aid of membrane filters and subsequent atom absorption
photometry.
Electron microscopic studies of_ the lung parenchyma
The electron microscopic investigations were carried out
on rats having inhaled lead oxide at an average concen-
tration of 150/ug/nr5 for four days. The lungs were fixed
with glutaraldehyde according to the instillation method
and embedded in araldite. The alveolar macrophages showed
partly discrete, partly pronounced alterations especially
of the endoplasmatic reticulum. The normally parallel
arrangement of the reticular membranes changes into a
vapid structure showing cisternae-like bulgings with con-
-------
783
comitant detachment of the ribosomes. With increasing
damage the membranes transform into myelin-like bands,
which may extend to the external nuclear membranes in a
striking manner (Fig 1). At the same time alterations of
the mitochondria can be observed which are characterized
by the condensation and clotting of internal structures.
The lysosomes, in contrast, exhibit no damage even though
in a number of organelles a myelin-like transformation
of the lysosomal matrix is noticeable (Fig 2).
The ultrastructure of the epithelial lining of the lead-
exposed lung cannot be definitely characterized by mor-
phological description. However, it can be said princi-
pally that numerous young cell forms are to be observed
besides damaged cells. As an instance, there are cells
whose epithelial extensions detach from the basal mem-
brane, projecting freely into the alveolar lumen. The
cytoplasm of these cells contains some clotted electron-
opaque mitochondria. On the other hand, cells are also
observed containing exceptionely numerous reticular mem-
branes (Fig 3). It is striking that many of the pneumo-
cytes type I super-impose their extensions over the api-
cal poles of type II cells. Under these extentions nume-
rous osmiophilic bodies collect which are regarded as
secretion products of type II cells (Fig 4). Another
striking feature are cells which are situated for the
greatest part underneath the pneumocytes type I and which
break through the epithelium with regular, coniform ex-
tensions. The cytoplasm of these cells is characterized
by abundant tonofibrils. However, mitochondria and other
cytoorganelles are sparse (Fig 5). According to locali-
zation and behaviour we are obviously dealing with young
cells which are migrating to the alveolar wall.
The ultrastructural findings permit the conclusion
-------
784
Exp. group
Control
Pb
No . labelled
type I-cells/
1000 cells
3,2
15,5
No. labelled 2
type I-cells/cm
inner lung surface
1,5 . 105
9,1 . 105
Tab 1
Treatment
Inhalation Ti02-conc,
time weeks mg/m3
Lead-cone.
Retention
TiO~ Increase
Tab 2
ge/control
I c
I Pb
II c
II Pb
15
15
15
15
18,8
18,8
27,7
27,7
0
82
0
168
0,40
0,53
0,69
0,96
0,10
0,10
0,18
0,29
33
39
-------
785
that the alveolar macrophages as well as the pneumocytes
type I in the alveolar wall are altered following inha-
lation of a aerosol as compared to normal animals.
Determination of the H-3-thymidine index of alveolar cells
In the second experimental series mice inhaled the
same lead concentration of 150/ug/nr for 14 days. 1 h be-
fore sacrifice the animals were injected 100/u Ci H-3-
thymidine/kg body weight. 1/u thick sections of the aral-
dite embedded lungs were coated with a L 4 emulsion, the
autoradiograms developed after 14 days, and the sections
stained with toluidineblue-azur II. The labelled cells
were counted and related to 1000 unlabelled cells or a
unit area of the inner lung surface. Tab. 1 shows the in-
crease of labelled epithelial type I cells in the lead
exposed animals. There was no change of the labelling in-
dex of the pneumocytes type II.
Determination of the benzopyrene hydroxylase in alveolar
macrophages
In a third test series guinea pigs inhaled lead oxide of
an average concentration of 150/ug/nr for 4 days. The al-
veolar macrophages of these animals were collected by
pulmonary lavage, and the, ability of the cells to meta-
bolize benzo(a)pyrene was determined by means of a method
by DIAMOND (4) modified by DEHNEN et al. (3). Immediately
after inhalation a drastic decrease of activity to 10#
of the original value is found. After an inhalation-free
interval of 3 days the activity returns to control values.
Influence of lead inhalation on lung clearance capacity
for inert dusts >' ' ,
In a fourth experimental series four groups of rats in-
-------
786
*
\
'
~>
'.7
*il
Fig. 1 - Alveolar macrophage of lead-exposed rat. Bulging of
endoplasmic reticulum with detachment of ribosomes (arrow)
Large phago lysosomes apparently containing lead.
1:25 000
Fig. 2 - Lead-exposed alveolar macrophage. Myelin-like transforma-
tion of endoplasmic reticulum (arrow). 1:20 000
-------
787
t ^
r.
'%>, r<
• - Mtf •
Fig. 3 - Lead-exposed pneumocyte type I with exceptionally well
developed endoplasmic reticulum. Some mitochondria
(arrow) exhibit condensad matrix and rarification of
cristae intermitochondrialee. 1:JO 000
Fig. 4 Extension of pneumocyte type I situated over the apical
pole of a pneumocyte type II (P2). 1:10 000
-------
788
haled titan dioxide dust in a concentration of 19 or 28
mg/m^ on 5 days of the week for 3 h. The dispersion of
the titan dioxide was performed with a Polly's generator.
Each day the fine dust concentration was determined gra-
vimetrically and, in addition, each hour by tyndalloscopy.
Two of the groups inhaled a lead aerosol of 82 or 168
/ug/nr for 5 h before the exposure to inert dust, while
the other two groups were not treated further and served
as controls. The inhalations were carried out for 15 weeks
in the described way, then the animals were killed, and
lungs and lymph nodes were examined for their content of
titan dioxide. The results of the analyses are presented
in Tab. 2 together with the inhalation data for all four
groups. In the lungs of the animals exposed to additio-
nal lead inhalation, significantly higher dust quantities
were found as compared with controls. The elimination was
impaired by 33 to 3996. With regard to the lymph nodes no
statistically significant differences were revealed.
Discussion
The effects on the* haemopoetic system and the central
nerve system have so far been in the foreground of the
discussion of toxic lead action. Our investigations de-
monstrate local effects in the pulmonary parenchyma fol-
lowing inhalation of lead aerosols. Both alveolar macro-
phages and epithelial cells are affected by the toxic
action, as demonstrated by a decrease in enzyme activity
in alveolar macrophages and an elevated turn-over of
type I cells. The biochemical and morphological findings
indicate that the primary point of attack of lead on the
cellular level is observed at the reticular membranes.
This ist in accordance with studies by SCOPPA and colla-
borators (5) who could demonstrate a reduction of the
activity of membrane-bound hydroxylases in liver cells
-------
789
Fig. 5 - Young cell form in the alveolar wall mostly covered by
theneighbouring cells. Coniform protrusions break
through the epithelial lining. 1:12 000
after oral lead poisoning. As result of the cellular da-
mage an impairment of lung clearance is found. Thus an
increased retention of exogenous substances must be ex-
pected following lead inhalation. Of further special in-
terest in the context of air pollution is the result that
carcinogenous hydrocarbons are metabolized to a lesser
degree, thus increasing considerably the period of time
these substances are present in the lung. The concentra-
tions chosen by us (70 to 170/ug/nr) are slightly lower
than the MAK values valid in the Federal Republik of Ger-
many but higher than the values normally measured in our
cities. In some cases, however, we measured 24 h averages
of 20 to 80 /ug/nr in the atmosphere of a West German city
in the neighbourhood of a lead plant (2). The pronounced
decrease of benzopyrene hydroxylase and the considerable
increase of labelled cells in the alveolar wall give rise
to;the supposition that even clearly lower concentrations
than 70yug/nr can lead to local injury in the lung. The
experiments are carried on in this direction.
-------
790
LITERATURE
1. BECK, E.G., N. MANOJLOVIC und A.B. FISCHER: Die Zyto-
toxizitat von Blei. "Die gesundheitlichen Aspekte der
Umweltverschmutzung durch Blei". Tagungsberichte In-
ternationales Symposium, Amsterdam (Nederland), Octo-
ber 2-6, 1972.- Luxembourg: Centre for Information and
Documentation - CID 1973, 451-461.
2. BROCKHAUS, A., H. WEISZ, K.-H. FRIEDRICHS und U. KRAMER-
Das Vorkommen von Benzo(a)pyren und partikularem Blei
bei unterschiedlichen Immissionssituationen.- Stutt-
gart: G. Fischer (im Druck),
= Schriftenreihe des Vereins fiir Wasser-, Boden- und
Lufthygiene, H. 43.
3. DEHNEN, W., R. TOMINGAS and J. RODS: A modified method
for the assay of benzo(a)pyrene hydroxylase.
Analyt.Biochem., 53 (1973), 373-383.
4. DIAMOND, L.: Metabolism of polycyclic hydrocarbons in
mammalian cell culture.
Int.J.Cancer, 8 (1971), 451-462.
5. SCOPPA, P., M. ROUMENGOUS and W. PENNING: Hepatic Drug
Metabolizing Activity in Lead-Poisoned Rats.
Experentia, 29 (1973), 970-972.
DISCUSSION
LYNAM (U.S.A.)
The speaker stated that air lead concentrations in cities
average 20 - 80/ug/m3. I know of no data to support such fig-
ures. In a study of air lead concentrations in seven major
US cities at 59 sampling sites, the annual average airborne lead
-------
791
concentrations measured for 24 hours a day for 1 ^ear ranged
from a low of 0.17/ug/m3 in a rural community in Los Alamos,
New Mexico to a high of 4.4/ug/m3 in Pasadena, California. In
a study reported by Prof. BoudSne, levels in Paris, France of
2 - 3/ug/m3 are reported. Data by Dr. Tsuchiya in Japan indi-
cate annual levels of lead in air in Tokyo of less than 2/ug/m3.
Data from the USEPA CAMP (Continuous Air Monitoring Program) in-
dicate lead levels in cities of a maximum of 1 - 4/ug/m3. Could
the speaker support his figure of 20 - 80/ug/m3 average in lead
concentrations in cities?
BRUCH (Federal Republic of Germany)
It is correct that the annual concentrations of lead obs-
erved in cities average 2 - 3/ug/m3. However, annual averages
of 8.3 - 11.9/ug/m3 were noted in areas of high traffic density
(1). The concentrations of 20 - 80/ug/m3 we quoted are for
24-hour levels measured by us on particular days in highly in-
dustrialized areas (2). Similar or higher levels were reported
from other areas having a lead industry (3) (Maximum level:
328/ug/m3j half-yearly average: 50/ug/m3), Since our find-
ings with respect to animals were obtained in some cases s -er
only a few days' exposure, the 24-hour levels seemed to us to
offer a more suitable basis for discussion than the yearly av-
erage.
References
1. COLUCCI, J.M. and Ch.R. BEGEMAN: Polynuclear aromatic
hydrocarbons and other pollutants in Los Angeles air.
In: Proceedings of the Second International Clean Air
Congress. Ed. by H.M. Englund and W.T. Beery - New York,
London: Academic Press 1971, 28-35.
2. BROCKHAUS, A., H. WEISZ, K.-H. FRIEDRICHS und U. KRAMT
Das Vorkommen von Benzo(a)pyren und partikularem Blei ~ji
unterschiedlichen Immissionssituationen - Stuttgart:
G. Fischer (im Druck).
= Schriftenreihe des Vereins ftir Wasser-, Boden- und Luft-
hygiene, H.43
3. FUGAS, M., B. WILDER, R. PAUKOVIC, J. HRSAK and D. STEINER-
SKREB: Concentration levels and particle size distribution
of lead in the air of an urban and an industrial area as a
basis for the calculation of population exposure. In:
Tagungsberichte Internationales Symposium "Die gesundheit-
lichen Aspekte der Umweltverschmutzung durch Blei", Amsterdam
Oct. 2-6, 1972 - Luxembourg: Centre for Information and
Documentation - CID 1973, 961-962.
-------
793
LOW LEVEL LEAD TOXICITY
ALAN M, GOLDBERG AND ELLEN K, SILBERGELD
Department of Environmental Medicine, School of Hygiene and
Public Health, Baltimore, Maryland, USA
ABSTRACT
Lead poi.Boni.ng in children is associated with several seri-
ous pathological and behavioral deficits. Studies in adult
animals have been unable to support the association between lead
intoxication and behavioral dysfunction. However, when suckling
mice are exposed to lead from birth they exhibit an increase in
spontaneous motor activity. In this study, mice were exposed
to lead acetate (2,5 or lOmg/ml) from birth indirectly through
their mothers' milk. At these doses, no classical signs asso-
dated with overt lead toxicity were observed. However, growth
of the offspring was decreased 10% and in measurements of spon-
taneous motor activity from 30-150 days of age, the lead intoxi-
cated mice were more than three times as active as ooetaneous
controls. To elucidate the basic mechanisms underlying this
lead-induced behavioral dysfunction, a combined, pharmacological
and neurochemical approach has been used. The results suggest
a defect in the interactions of central cholinergic and aminergio
pathways. This study emphasizes the necessity to examine effects
of potentially toxic compounds during critical stages of neuro-
logical development in order to assess fully the impact of these
compounds on humans.
This project was supported in part by the national Insti-
tutes of Environmental Health Sciences grants 00034 and 00454
and a Starter Grant to EKS by the Pharmaceutical Manufacturers
Association Foundation. EKS is a Joseph P. Kennedy, Jr. Fellow
in Neurosciences.
-------
794
Clinical studies have documented a relationship between
lead intoxication and behavioral disorders in children (Byers
and Lord [1]; Chisolm and Harrison, [2]; De la Burdee and
Choate [3]; David, et al, [4]). These disorders have been
described as irritability, restlessness and agressiveness.
Studies in adult animal models have not reproduced these
findings which supports clinical knowledge on the relationship
between age and susceptibility to lead. Recently, it has been
demonstrated that animals exposed to inorganic lead during
critical periods of neurological development (analogous to
early childhood exposure via pica) develop the behavior dis-
order of hyperactivity (Silbergeld and Goldberg, [5,6,7J;
Sauerhoff and Michaelson, [8]; Allen, et al, [9]). Hyperacti-
vity is seen in animals exposed to levels of lead below that
which produces severe growth retardation, kidney damage,
paraplegia, or convulsions.
THE ANIMAL MODEL - The intention of this animal model is to
provide an analogue to childhood lead poisoning in terms of
equivalent age, gastrointestinal route and chronic nature of
low level lead exposure. Inorganic lead (2,5, or 10 mg/ml
lead acetate) was administered to the mother in the drinking
water and through her to the nursing offspring which were the
subjects of study. These exposure levels of lead produced a
dose-related reduction of growth (about 10% at 5 mg/ml) and
a retardation of several developmental landmarks (Table 1).
The offspring have no signs of acute lead poisoning but in
analyses conducted in collaboration with Dr. J.J. Chisolm.
alterations in heme synthesis were found associated with a
blood lead level of 50-80 pg%.
-------
795
TABLE 1. DEVELOPMENTAL LANDMARKS IN
CONTROL AND LEAD-TREATED MICE3
Day of Occurrence
Landmark Controls Lead
Eye Openinj
Full Incidence
13
16
17
20
of Hair
Coordinated Walking
Weaning
18
22
25
30
Data from Silbergeld and Goldberg, [5].
Between 30 and 150 days of age, these animals were
individually tested for levels of spontaneous motor activity.
In all cases, the lead-treated animals were about 3 times as
active as coetaneous controls (Figure 1).
A number of points should be made about this animal model,
Increases in the level of lead exposure do not increase hyper-
activity but induce other sequelae of lead poisoning. Animals
whose lead exposure terminates at weaning also evince hyper-
activity as adults. However, animals exposed to lead only post-
weaning do not develop the behavioral dysfunction, suggesting
that the behavioral toxicity of lead is dependent upon the
accessability and immaturity of the developing nervous system.
-------
796
1750r
1500
125O
10OO
P 750
500
250
30 40 50 60 70
AGE IDAYS I
ISO
FIGURE 1.
Spontaneous motor activity (counts per hour) of lead-
treated and control mice from 30 to 150 days of age.
Activity was individually measured electronically;
results are shown for second hour of measurement. Open
circles and dotted lines indicate results of testing
lead treated animals; closed circles and solid lines
indicate controls. Points are means of 6-21 animals;
vertical lines are S.E.M. The activity levels of
treated mice were significantly higher at each day.
(p<0.001).
-------
797
Two major approaches were used to study lead-induced
hyperactivity. In the first, the behavioral effects of drugs
with known mechanisms of action were investigated. Alterations
in behavioral response to these drugs may indicate specific
changes in neurochemical pathways following lead treatment. In
the second approach, neurochemistry of lead-treated hyper-
active animals was studied by measuring transport and steady-
state levels of neuroactive compounds.
PHARMACOLOGY - Lead-treated hyperactive mice were found to
respond behaviorally with marked differences as compared to
controls to a number of pharmacological agents {Table 2). The
first observation to be made is that lead-induced hyperactivity
provides an animal model strikingly similar in its pharmacology
to Minimal Brain Dysfunction hyperactivity in children. In
addition, a general observation can be made that drugs which
act to increase CNS catecholamine-receptor stimulation exacer-
bate lead-induced hyperactivity {1-dopa, benztropine, and
apomorphine). Drugs which are thought to enhance central chol-
inergic function suppress lead-induced hyperactivity (physo-
stigmine, dimethylaminoethanol, and oxotremorine). Also, the
cholinergic antagonists, atropine and benztropine tend to exa-
cerbate the hyperactivity or reactivity of lead-treated animals.
However, several drugs which have been proposed as cate-
cholandnergic agonists do not exacerbate lead-induced hyper-
activity but rather suppress it. Amphetamine, methylphenidate
and fenfluramine are drugs which enhance catecholamine function
by either increasing transmitter release or blocking reuptake.
The apparently contradictory responses of lead-treated hyper-
active mice to these agents may be partly explained by their
action on non-aminergic sites. Amphetamine, in addition to
stimulating aminergic pathways can also enhance cholinergic
function.
-------
798
Table 2.
EFFECTS OF DRUQS ON LEAD-INDUCED HYPEHACTIVITY
a)
DRUG (Dose mg/kg)
EFFECT ON LEAD-
TREATED HYPER-
ACTIVE CONTROLS
COMMENTS
Or-nethylparatyrosine (50)
b)
d-amphetamine (10) —
dimethylaminoethanol (200)
fenfluramine (20)
methylphenidate (25, ^0)
oxotremorine (0«08) -
physostigmine (0.1-0.5)
apomorphine (10) 0
atropine (0.01) 0
benztropine (15) +
1-dopa (50) -M
phenobarbital (20) +4
4-
•H-
0
0
In treated, produces
"normal" response to
amphetamine} in controls,
abolishes amphetamine
response
In controls, some stereo-
typy
In treated, induces
stereotypic circling and
increases reactivity
In treated, increases
reactivity
In treated, increases
reactivity
In treated, increases
reactivity
a) Some of these data are reported in Silbergeld and Goldberg,
(6,7)* The rest is presented in material submitted for publi-
cation*
b) +«increase in activity; --decrease in activite; p«no change*
Number of symbols denotes qualitative amount of change; — in
treatedasuppression of activity to that of predrug baseline
of control animals*
-------
799
It is not presently known if the other aminergic
agonists methylphenidate and fenfluramine act similarly on the
cholinergic system.
NEUROCHEMISTRY - The transport of neurochemicals into neurons
has been proposed as an important step in the regulation and
turnover of suspected neurotransmitters and may be a valuable
method for studying CNS function (Kuhar, [10]). High affinity
transport systems are observed experimentally by the low con-
centrations against which neural tissue will accumulate the
compounds. Thus, changes in high affinity transport may reflect
alterations in neurotransmitter function. Many of the drugs
found to have differential effects on lead-induced hyperactiv-
ity are known to affect the high affinity transport of the
catecholamines and acetylcholine. In 40-60 day old Iead-.induca3
hyperactive mice the high affinity transport of dopamine,
choline and tyrosine were significantly affected (Table 3).
The transport of other suspected neurotransmitters and a non-
neuroactive compound, leucine, were not changed.
The steady state levels of acetylcholine, norepinephrine,
and dopamine were also measured in hyperactive animals.
Preliminary results indicate that only norepinephrine levels
are changed (25% increase). This is consistent with .the
increases in the transport of tyrosine, a precursor of
norepinephrine, and with the suppression of lead-induced
hyperactivity by a-methylparatyrosine, a compound which
inhibits the synthesis of norepinephrine (Table 2).
CONCLpSIONS - Low level lead exposure has been shown to inter-
fere with central nervous system function and to result in
retardation of development, in hyperactivity, and in altera-
tions in response to many pharmacological agents. The mechan-
ism of action is probably related to an inhibition of central
-------
TABLE 3.
HIGH AFFINITY TRANSPORT OF NEUROTRANSMITTERS AND OTHER COMPOUND BY BRAIN TISSUE*
COMPOUND
3
H -choline
H -dopamine
3
H -GAB A
14
C -glycine
3
H -5-hydroxy-
tryptamine
14
C -leucine
3
H -norepinephrine
14
C -tyrosine
b
FINAL CONCENTRATION N
IN ASSAY MEDIUM
4
1.
2.
7.
4.
4.
1.
3.
25
0
05
67
3
06
3
-7
x 10 M
x 10~6M
-7
x 10 M
-6
x 10 M
-ft
x 10 M
-6
x 10 M
-6
x 10 M
-fi
x 10 M
8
15
9
6
6
i
6
5
8
LEAD HYPERACTIVE
AS % OF CONTROLS
52%°
81%°
98%
95%
CO
105% °
88%
99%
f^
115%
Details of Methodology and Results are in Material Submitted for Publication.
N=number of animals assayed in each group.
'p<0.01, by Student's T test of the means for control and treated groups.
-------
801
cholinergic neurotransmission and concomitant alterations in
catecholamine pathways. Cholinergic inhibition by lead has
been demonstrated at the ganglion (Kostial and Vouk [11]) and
at the myoneural junction (Silbergeld, et al, [12,13]).
This animal model lends experimental evidence to support
recent findings that some cases of MBD hyperactivity are
associated with early exposure to lead (David, et al, [4]).
However, the question remains as to the significance of lead
exposure in the etiology of MBD hyperactivity.
The enhanced susceptibility of young children to the
effects of low level chronic lead exposure has been reproduced
in this animal model. This susceptibility may directly result
from the immaturity and vulnerability of the developing CNS.
In young rodents, the blood brain barrier is more readily per-
meable than in older animals. Additionally, neurotransmitter
pathways are rapidly changing in amounts of neurotransmitters
as well as in activities of important metabolic enzymes
(Benjamins and McKhann, [14]). Therefore, it is important to
carefully choose from populations at risk those groups likely
to possess increased vulnerability in order to assess fully
the potential hazard from environmental agents.
REFERENCES
1. Byers, R.K. and E.E. Lord. Late Effects of Lead Poisoning
on Mental Development. Amer. J. Pis. Child 66:471-494,
(1943).
2. Chisolm, J.j. and H.E. Harrison. The Exposure of Children
to Lead. Pediatrics 18;943-957. (1956).
3. De la Burdee, B. and M.S. Choate. Does Asymptomatic Lead
Exposure in Children Have Latent Sequelae? J. Pediat. 81:6,
1088-1091, (1972).
-------
802
4. David, 0., J. Clark and K. Voeller. Lead and Hyperactivity.
Lancet 11900-903, (1972).
5. Silbergeld, E.K. and A.M. Goldberg. A Lead Induced Behavioral
Disorder. Life Sci. 131:1275-1285, (1973) .
6. Silbergeld, E.K. and A.M. Goldberg. Lead Induced Behavioral
Dysfunction: An Animal Model of Hyperactivity. Exp. Neurol
£2:146-157, (1974).
7. Silbergeld, E.K. and A.M. Goldberg. Hyperactivity: A Lead
Induced Behavior Disorder. Env. Health Persp. ^7: , (1974) ,
8. Sauerhoff, M.W. and I.A. Michaelson. Hyperactivity and Brain
Catecholamine in Lead Exposed Developing Rats. Science 1^2;
1022-1024, (1973).
9. Allen, J.R., P.J. McWey and S.J. Suomi. Pathophysiological
and Behavioral Changes in Rhesus Monkeys Exposed to Lead.
Env. Health Persp. 7;, (1974).
10. Kuhar, M.J. Neurotransmitter Uptake: A Tool in Identifying
Neurotransmitter Specific Pathways. Life Sci. 13:1623-
1634, (1974).
11. Kostial, K. and V.B. Vouk. Lead Ions and Synaptic Transmission
in the Superior Cervical Ganglion of the Cat. Brit. J.
Pharmacol. 12:219-222, (1957).
12. Silbergeld, E.K., J.T. Fales and A.M. Goldberg. Evidence for
a Junctional Effect of Lead on Neuromuscular Function.
Nature 247:5435, 49-50, (1974).
13. Silbergeld, E.K., J.T. Fales and A.M. Goldberg. The Effects
of Inorganic Lead on the Neuromuscular Junction. Neuropharm.
In Press, (1974).
14. Benjamins, J. and G." McKhann, Neurochemistry of Development
In Basic Neurochemistry« Eds. Albers, et al. Little Brown,
Boston, pp. 269-298. (1972).
-------
803
DISCUSSION
KAMINSKI (U.S.A.)
1) What were the dose levels in the figures shown in your
presentation?
2) What was the lowest level at which you were able to observe
changes?
3) Since the highest level i.e. 10 mg/ml is eauivalent to a
lead intake in a 6O Ib child of 5Og per day do you consider this
to be low lead level?
4) Even the 5 mg/ml dose level of lead is 5OOO times the nor-
mal lead intake in our diet, this hardly is a low lead level.
GOLDBERG {U.S.A)
1) In the manuscript all doses are indicated. The data shown
for the developmental landmark- slide and the first drug slide
(amphetamines) relate to animals from the lOmg/ml dose. All
the other data were from groups exposed to 5mg/ml.
2) The lowest dose we have used is 2mg/ml. This dose produces
hyperactive animals.
3-4) One cannot directly compare exposure in animals and expo-
sure in man. Let me point out that these animals do not ex-
hibit the classical signs of lead toxicitv and have a blood lead
level of 50-80 ,ug %. At the present time I would have to con-
sider that these animals are at low-level exposure.
TER HAAR (U.S.A.)
If your idea of hyperactivity is correct, why are there
not evidences of increased hyperactivitv in the large group of
children in the United States who have blood leads in the
50-80 mg/lOOml range? While there is speculation in the liter-
ature that an increase from 25-30 mg lead/lOOml may cause
hyperactivity, there is no evidence of this fact in the large
group of children in the higher lead in blood level.
GOLDBERG (U.S.A.)
At this point in time I would not conclude that lead is
etiologic in hyperactivity but I would pose it as a question.
Obviously there are many other factors that one would have to
consider. Let me point out that the hyperactivity seen in
animals may be the first sign of lead poisoning and that other
sequelae develop at higher exposure.
-------
805
STUDIES OF LEAD ENCEPHALOPATHY IN THE DEVELOPING RAT
I, A, MICHAELSON, R, D, GREENLAND/ AND M, W, SAUERHOFF
Department of Environmental Health, University of Cincinnati,
College of Medicine, Cincinnati, Ohio, USA
ABSTRACT
Inorganic lead produces cerebral dysfunction and clinically
definable eneephalopathiee in man. The expected sequelae of
pediatria lead enoephalopathy consist of altered behavioral pat-
terns and learning disabilities. To date there have been feu
studies on the biochemical changes in brain following exposure
to inorganic lead. Studies correlating toxiaity to behavioral
and brain neuroohemical changes following lead exposure have been
hindered because adult laboratory animals are resistant to the
central nervous system (CHS) effects of lead poisoning. Such
studies have been impeded by lack of suitable experimental models
until Pentschew and Garro showed that brain lesions develop in
neonatal rats when a pregnant vat newly delivered of her litter
is placed on 4.0% lead carbonate containing diet. Lead passes
into the developing sucklings via maternal milk. Lead poisoned
newborns have pronounced retardation of growth and during the
fourth week of life develop paraplegia and severe signs of lead
enoephalopathy3 namely extensive histological lesions of the
cerebellum and brain edema.
We have employed this experimental model to study both sev-
ere and mild forms of lead encephalopathy in the developing rat.
In the former we have found a failure of cell multiplication in
the cerebellum. Howevert in the latter we have found that during
the fourth week of development young rate display hyper activityt
-------
806
tremor's and stereotype behavioral patterns. Pair-fed controls
ooetaneous to experimental groups do not display such activities.
Estimation of brain serotonin^ gamma-amino-butyric acidt nor-
epinephrine and dopamine suggests a disruption in catecholamine
metabolism during periods of altered behavior in lead exposed
developing rats. The data indicate that under conditions of
chronic ingestion of relatively low levels of lead from birth
to adulthood brain dopamine metabolism is either unchanged or
slightly slowed. On the other hand, norepinephrine metabolism
is increased by as much as 30% and leaves the brain (turnover
time) more rapidly than in control animals.
It is possible that our findings on increased motor activity
and changes in brain monoamine metabolism may correspond to early
responses to lead exposure before recognized overt signs of toxi-
oity.
-------
807
The brain is exceptionally sensitive to the effects of inorganic lead
poisoning anH it is primarily the young - from birth to about seven years
of age - who show the most serious damage following symptomatic lead poi-
soning. Brain manifestations of lead induced toxic episodes and cerebral
related sequelae of pediatric lead encephalopathy are well defined and
there is considerable knowledge about the histopathological conditions of
brain tissue of those lead poisoned individuals who do not survive. Our
laboratory raises three questions: (l) is there a neurochemical basis for
the cerebral and behavioral deficit as seen in some survivors of lead en-
cephalopathy and more important (2) whether a long standing minor degree of
elevation of blood lead concentration would be associated with future neuro-
logic malfunction and (3) is impaired brain function associated with lead
ingestion which is asymptomatic or produces symptoms less severe than en-
cephalitis?
Until recently experimental studies on lead encephalopathy has been
hampered by the inability to produce in laboratory animals unequivocal
neuropathological or behavioral changes characteristic of the human dis-
ease. Most adult experimental animals do not display manifestations of
brain dysfunction as usually seen in children with lead poisoning. This is
not the case when young animals are used. Experimental lead encephalopathy,
with histologic features characteristic of the human disease [1,2], can be
produced in suckling rats by feeding diet containing 4.556 lead acetate to
lactating mother rats after delivery of her young. The lead is transmitted
to the newborns through the mother's milk which contains lead. While the
mother rats tolerate the diet [l] the sucklings develop paraplegia and ex-
tensive vascular damage to the cerebellum, frequently leading to death.
We have used this model to study the biochemical interaction of lead
with brain to seek a chemical basis fir behavioral aspects of lead poisin-
ing as it relates to the CNS. We found that suckling rats ingesting lead
via the milk of mothers fed lead containing diet results in a pronounced
retardation in growth of the young and gross discolorization of the cere-
bellum as previously described [l,2]. There was an apparent reduction in
brain weights in the lead exposed animals and a diminution in DMA content
of the cerebellum suggesting failure in cell multiplication and/or matur-
ation in this organ. This appears to represent a working experimental
model of the more severe and fulminating forms of lead encephalopathy but is
much too destructive for the answer to the earlier question: Are there
brain effects associated with low levels of lead ingestion which are
-------
808
asymptomatic or which produce symptoms less severe than encephalitis or
usually looked for signs of lead poisoning?
Our initial finding suggested that low body weights of animals suck-
ling from mothers fed lead acetate and the cerebellar changes were similar
to those seen in undernutrition. Furthermore, it was observed that the ap-
pearance of paraplegia nad cerebellar pigmentation occurred shortly after
the time when the neonates were capable of gaining access to the mothers'
4-5$ lead acetate containing diet [3],
The question was raised whether access to the relatively greater
amounts of lead contained in the maternal solid food (24,OCX) pptn Pb) com-
pared to maternal milk could account for the histopathology observed in
reports using this regimen [l,2,3]. We devised a means of sampling the
maternal milk for estimation of its lead content. Figure 1 demonstrate?
I
1
0 , 58 13
DAYS POST-PARTUM
16
FIG. 1 Milk lead content (ppm) of lactating rats eating
4.5$ (2.4& 24,570 ppm) lead acetate containing
diet. Normal milk contains less than 0.02 ppm Pb.
-------
809
that a lactating rat eating 4.5$ lead acetate (2.4$ lead, 24,000 ppm Fb)
diet produces milk containing increasingly greater concentrations of lead
in relationship to length of exposure and increasing food consumption (nit
illustrated), 3y the sixteenth day they produce milk containing approxi-
mately 25 ppm lead, whereas milk from lactating rats eating normal diet
contains less than 0.02 ppm lead. During suckling the developing rat is
exposed to less than one thousandth (1/1000) the amount of lead their
mothers had been eating during the first 3 weeks of the nursing period.
Developing newborn rats suckling milk containing 25 ppm lead are cap-
able of climbing into mothers' food jars by 18 days of age and thereby
capable of consuming solid diet with a high lead content. In order to keep
the lead exposure of the developing rat constant, on the seventeenth day
the maternal diet was changed to one containing 25 ppm to 400 pmm lead,
comparable to, or sixteen times greater than the amount they had consumed
in the milk. There is a consequential acceleration of growth rate due to
change from a liquid maternal diet to solid food in both control and exper-
imental animals. There was no paraplegia and brains did noi^ contain^ pigmen-
tation as observed in earlier studies [1-3]. Neonatal rats exposed to
these relatively low levels of dietary lead for the first three weeks of
life exhibit hyperactivity, aggressiveness, and stereotype repetitive be-
havior. At 23, 2/» and 25 days of age, complete families of six siblings
from pair-fed controls or lead exposed groups were placed on a spontaneous
activity metering device for 24 hours. Appropriate pair-fed control and
experimental groups were tested on alternate days for a single 2A hour
period. Neonatal rats exposed to dietary lead for the first 3 weeks of
life exhibit a 40 to 93?& greater activity than coetaneous pair-fed
controls (Figure 2).
Exposure to lead via the regimen Described leads to a significant in-
crease in the amount of lead in brain within 5 days of the onset of ex-
posure (Figure 3). The brain lead content of exposed animals continues to
increase but the levels are merely one tenth that found when sucklings are
weaned to mothers' 4«5# lead acetate diet [3].
-------
I
g
•J
4.0T
3.5
3.0
2.5
2.0
1.5
1.0
0.5
.
10 15 20
AGE (DAYS)
25
30
FIG. 3 Brain lead content of neonatal rats suckling
mothers eating A.5$ lead acetate diets (0-10)
changed Of) to AO (0')> ^00 (:^)» and A,000
) ppm lead containing diet (17-58).
O" CD
O X
M "S
-
—
•
(E
3
sr
01
"8
B3 0!
r»- 3
w 01
o
-
-
RELATIVE PERCENTAGE
Q
:
-
3
no
o BJ
o
>^
C H- ^
3 <; CD
T ci m
° ^ no
M ^
~ ^
3 *< »
0 0 ±<
k<
i § CO
0? OP "^^ *
H
N CD
OJ 05
a a
U-
LJ
o o
I i
2 >
r-n
m
o
-------
811
We have now studied the metabolism of brain catecholamines using ani-
mals as described above except that sucklings were weaned to a diet con-
taining 400 ppm lead. Experimental and control rats were killed at dif-
ferent ages and their brains analyzed for norepinephrine and dopamine
content (Figure 4).
0.8
0.7
0.6
0.5
04
0.3
0.2
0.1
* ft'0.05
• NOREPINEPHRINE
• DOMMINE
CONTROL
LEAD
_L
_L
10 20 30
AGE DAYS
50
60
70
FIG. k Norepinephrine and dopamine in brain of leaded
and pair-fed control rats.
It is interesting to note that in this study there appears to be
slightly higher levels of norepinephrine and dopamine in lead treated rats
but the small number of animals employed make difficult a statistical
validation of this impression.
It is important to emphasize that this newer mode of developing lead
intoxicated rats results in subtle response (hyperactivity, aggressiveness,
stereotype repetitive behavior) and one might suspect similar subtle
changes in neurochemical components. Whereas steady state levels of tissue
concentration yield static datum; metabolic activity or turnover provides
mre meaningful data. We have studied norepinephrine and dopamine turnover
in brains of lead exposed developing rats. Since these chemicals in brains
are thought to be involved in motor activity and behavior, and because
earlier studies indicate increased activitv in the fourth and fifth week,
-------
812
we Pxamined the turnover of catecholamines in 32 day old rats receiving
MX) ppm lead.
We used the drug alpha—methyl tyrosine which is a specific inhibitor
of the enzyme tyrosine hydroxylase - the rate limiting step in catechol-
amlne biosynthesis. As the neuronal metabolic activity continues the non-
replenished stores of the neurotransmitters are depleted. The rate of
depletion is an indication of the neuronal activity.
The data indicates that under conditions of chronic ingestion of rel-
atively low levels of lead from birth to adulthood leading to increases in
brain lead content and altered states of behavior, that dopamine metabolism
is either unchanged or partially slowed; whereas norepinephrine turnover
is increased by as much as 30$, and that norepinephrine leaves the brain
more rapidly in leaded animals then in controls (Figure 5).
RATE CONSTANT AMINE LOSS Khr'1)
7
c
TURNOVER RATE n'v«n*
TURNOVER TIME (hr»)
C
031
0.168
«
Pt>
0.29
0.147
SS
pb/c
0.93
0.88
109
C
0.18
0.069
5.6
Pb
0.21
0090
4.8
Pb/C
LIT
190
0.86
1
5 ',
4 '
Pb
OOPAMINE (DM)
NOREPINEPHRINE (NE)
3 4 I
TIME (HOURS)
FIG. 5 Catecholamine metabolism in brain 32 day lead
exposed (....) and control ( ) rats re-
ceiving alpha-methyl tyrosine, 300 mg/kg.
-------
813
It is possible that our findings on increased motor activity and
changes in brain catecholamine metabolism may correspond to early responses
of lead exposure before the usually looked for overt signs of lead toxicity.
It may also be that the criterion of what is a toxic level of exposure may
have to be redefined.
REFERENCES
1. FENTSCHEH, A., GARRO, F. "Lead encephalo- myelopathy of the suckling
rat and its implications of the porphyrinopathic nervous diseases",
Acta Neuropathol. (Berl.) 6, 266 (1966).
2. THOMAS, J.A., DALLENBACH, F.D., THOMAS, M. "Considerations on the
development of experimental lead encephalopathy", Virchous Arch.
[Pathol. Anat.] 352, 6l (1971).
3. MICHAELSON, I.A., "Effects of inorganic lead on RNA, ENA and protein
content in the developing neonatal rat brain", Toxicol. Appl.
Pharmacol. 26, 539 (1973)-
DISCUSSION
STARA (U.S.A.)
I want to congratulate Dr. Michaelson and also the previous
speaker, Dr. Goldberg, for presenting data which are badly needed
to clarify an extremely important question of neurological eff-
ects of lead in growing organisms. I have two questions:
1. Why did you not expose the experimental animals through the
intra-uterine route - since the major portion of organogenesis
of CNS takes place during this period of development?
2. Studies by Kostial et al. in Zagreb, Yugoslavia, show that
up to 50% of ingested lead is absorbed during the lactation
period in the neonate, later, after weaning, the absorption
rate is rapidly reduced. Did you obtain data in these experi-
ments on the absorption rate of lead in time and on tissue bur-
den of Pb, particularly in the brain?
MICHAELSON (U.S.A.)
1. You may be correct about orgnogensis in a general sense
but I think you may be somewhat in error about the brain. Xt is
Sf Tj£St!,.0J l6£u °? the cent"l nervous system in which we
axe interested. The brain of the new born human is relat.1v*lv
-------
814
poorly developed and quite immature at birth. Relative to the
number of cells in the mature brain fifty percent of cortical
cells are present at birth and more significantly only 5 to 7%
of the cerebellar cells are present at birth. You therefore
have an organ system which is replicating very rapidly in the
human from birth to about 24 to 30 months of age. This corres-
ponds to the first three weeks in the newborn rat. This is
what makes this experimental model of lead encephalopathy, so
interesting and of potential value. Especially if you consider
that most human cases of pediatric lead encephalopathy coming
to hospitals at less than 7 years of age and indeed about 80%
of the cases suffering from bad encephalopathy are less than
4 years of age. You can therefore appreciate that if our con-
cern is the pediatric population then we should look at the
neonate and not at the embryo which is an entirely different
problem from that the which we address ourselves.
2. Within the time frame of our experiments there is a dose
related increase of lead in brain. At the higher exposures
where the animals are severly intoxicated and die the brain
contains about 1O to 12 ppm (wet weight) of lead. This is
within the range found in young humans who have died following
lead ingestion. In our relatively lower lead level exposure
studies in the absence of the overt signs of bad poisoning and
where the animals appear hyperactive there is about a five fold
increase in brain lead content over that of controls. Other
than the brain we have not studied tissues for their lead con-
centration other than blood. Both brain and blood levels remain
elevated for longer periods following removal of lead from the
diet.
SILBERGELD (U.S.A.)
We have begun work on effects of prenatal exposure to lead,
and the results suggest that the same decreases in growth and
alterations in behaviour as have been reported by us and by
Dr. Michaelson have also been observed on postnatal models.
EPSTEIN (U.S.A.)
Drs. Goldberg and Michaelson are to be warmly congratulated
on their presentation demonstrating behavioral changes and
learning deficits in young rodents exposed to relatively low
levels of lead. Sueh data are reflected in the recent and proper
decision of the EPA to phase out the use of lead as a gasoline
additive, a decision which is warmly endorsed bv the concerned
and knowledgeable scientific communities in the USA and else-
where. The authors have no reason to be diffident about the
low levels thev tested especially in view of alarming evidence
of very high lead levels in street dust and close-to-traffic
"corridors."
-------
815
Apart from the well recognized learning disabilities in
children recovering from acute lead poisoning, there have how-
ever been no adequate epidemiological studies on learning dis-
abilities in children with blood lead levels in the 6O-8O,ug/
lOOg range. Such studies are difficult, because children'at
risk often live in slums where poverty and other social factors
are complicating elements. Nevertheless, occupational studies
have recently demonstrated learning disabilities, altered
psychological performance and motor nerve conductivity distur-
bances in man exposed to lead with blood lead levels in only
the 60-8O .ug/10Og range.
-------
817
SUBCHRONISCHE ORALE TOXIZITAT VON CADMIUM BEI RATTE UND HUND
D, LORKE UND E, LOSER
Institut fur Toxikologie, Bayer AG, Wuppertal-Elberfeld, BRD
KURZFASSUNG
Ea wurde die aubohroniaahe orale Toxizitat (90-Tage-Teat)
von Cadmium bei Ratte und Hund unterauaht.
3-monatiger Putterungaver such an Ratten Je 20 mannliohe und 20
weibliohe Ratten evhielten Cadmium (ala CdCl ) in Dosierungen
£j
von 0., lt 3, 10 bzw. SOppm 3 Monate lang tdglioh mit dem Putter
verabreicht.
Bei den behandelten Ratten waren Aussehen, Verhalten, Futtev-
aufnahme, Waahetum und Sterblichkeit wUhvend der 3-monatigen
Veveuahszeit niaht beeinflusst. Die Verabreiohung von Cadmium
veruraachte keine Blut-t Leber- oder fUerenechadigungen.
Der eystolisahe Blutdruok way bei den behandelten Tieren
alley Vereuchsgruppen nicht erhOht. Sektionen und histo-patho-
logieche Untersuchungen ergaben keine Hinueiee fur Sohddigungen.
Cadmium uurde doeisabhangig in Nieren und Leber geapeiahert.
Z~monatiger Futterungevereuch an Hunden Je 2 mannliohe und 2
weibliahe Hunde erhielten Cadmium (ale CdCl-) in Konzentrationen
von 0, 1^ 3, 20 bzu. 30 ppm 3 Monate lang mit dem Putter verab-
reicht.
Bei den behandelten Hunden waren Aueeehen, Verhalten*
Futtevaufnahme, Waehetum und Sterbliohkeit in alien Veraucha-
-------
818
gruppen nicht verftndert. Blut-, Leber- oder Nierenschddigungen
wurden nach oralen Cadmiumgaben bis zu SO ppm im Putter nicht
verursacht.
Systolischer und diastolisaher Blutdruak war en bei alien
Versuchsgruppen bis 30 ppm normal. Sektionen und histo-patholo-
gische Untersuchungen ergaben keine Hinweise fur SchSdigungen.
Cadmium wurde vor allem in Nieren und Lebern dosisabha'ngig
gespeichert.
Doaen bis SO ppm Cadmium im Putter wurden von Ratten und
Hunden 3 Monate long schadigungsloe vertragen.
ABSTRACT
The eubahronio oral toxicity (90-day test) of cadmium in
rats and dogs was tested.
3-month feeding experiment on rats Cadmium (as CdCl9) uas ad-
J L * L _ __ •. »-j-. i — £
ministered in doses of Of 13 Zt 10 and SOppm daily to 20 male
and 20 female rats for 3 months with their food.
During the 3 months of the test, the appearance, behaviour,
food intaket growth and mortality rate of the treated rats re-
mained unchanged. The blood, liver and kidneys were unaffected
by the cadmium intake.
The systolic pressure did not increase in the treated ani-
mals in any of the test groups. Dissections and histopatholo-
gical teete yielde'd no evidence of damage. Cadmium accumulated
in the kidneys and liver in proportion to the size of dose.
-------
819
3-month feeding experiment ondoge Cadmium (as CdCl.) was ad-
ministered in concentrations of 0, lf 3, 10 and SOppm for S
months to 2 male and 2 female dogs with their food.
The appearance, behaviour, food intake^ growth and morta-
lity rate of the treated doge remained unchanged in all the
test groups.
The systolic and diastolic pressure in all the test groups
up to SO ppm was normal. Dissection and histopathologiaal
teats yielded no evidence of damage. Cadmium accumulated
chiefly in the kidneys and liver in proportion to the size of
dose.
Rats and doge withstood doses of up to SOppm of cadmium
with their food for 3 months without damage.
-------
820
1 . Einleitung
Schlieflt man die gewerbetoxikologischen Probleme beim Kon-
takt mit Cadmium aus der Betrachtung aus, so wird dieses
Metall praktisch nur oral vom Menschen aufgenommen. Es 1st
daher erforderlich, toxikologische Untersuchungen zur Ab-
schatzung der mb'glichen Gefahrdung durch den Umgang mit
cadmiumhaltigen Bedarfsgegenstanden nur bei oraler Verab-
reichung durchzufuhren.
Die in der Literatur beschriebenen Untersuchungen sind ent-
weder nicht mit oraler Applikation durchgefuhrt worden. und/
oder sie entsprechen nicht den Kriterien, die eine GefShr-
dungsbeurteilung mb'glich machen. Es erschien deshalb not-
wendig, weitere Versuche mit oraler Applikation durchzu-
f iihren.
2. Ergebnisse
Es wurde die subchronische orale Toxizitat (90-Tage-Test)
bei Ratte und Hund untersucht.
2.1. Ratten Je 20 mannliche und 20 weibliche Ratten er-
hielten Cadmium (als CdClp) in Dosierungen von 1, 3t 10
bzw. 30 ppm 3 Monate lang mit dem Standardfutter verab-
reicht. Als Kontrollen dienten 40 mannliche und 40 weib-
liche Ratten.
Bei den behandelten Ratten waren Aussehen, Verhalten und
Futteraufnahme sowie Sterblichkeit wMhrend der 3-monatigen
Versuchszeit nicht beeinfluflt. Die Gewichtskurven (Abb. 1)
der behandelten Tiere verlaufen etwa wie die der Kontrollen,
Die Ergebnisse der bei Versuchsende durchgefUhrten Blut-
untersuchungen lassen erkennen, daB durch die 3-monatige
Cadmiumapplikation bis zur Dosis von 30 ppm im Futter bei
keinem der Parameter ein scha'dlicher EinfluB auf-
trat. Auch im Differentialblutbild wurden keine pathologi-
schen Befunde erhoben.
Die Leberfunktionsprtifungen (Tab. 1) ergaben keinerlei Hin-
weise auf eine behandlungsbedingte Veranderung. Alle leber-
spezifischen Enzyme sowie Bilirubin und Serumproteingehalte
lagen im Bereich der Norm. In der elektrophoretischen Auf-
trennung der Serumproteine unterschieden sich die behan-
-------
821
330
300
250
200
150
100
50^
CADMIUM
Weeks —
0 :
Abbildung 1S'
Tiergewichtskurven von Ratten, die 3 Monate lang
Cadmium mit dem Futter aufnahmen
Liver-Function-Tests At 3 Month
i
Tabelle 1 :
Oosis
ppm
ALP
GOT
GPT
SDH
mU/hnl
Total
Bilirubine
Tfig/WOml
Total Serum-
Protein
g/lOOml
Male Rats
0
1
3
10
30
87,8
77,6
83,6
82,4
82,9
27,8
32,3
27,6
34,7
33p
11,5
14,1
13,0
15,6
18,6
2J
2,7
2,5
2,5
3,1
0,14
0,15
0,20
0,19
0,23
6,6
6,8
6,9
7J
7,4
Female Rats
0
1
3
10
30
71,3
83,7
73,8
66,9
86,0
27,3
32,5
29,4
29,1
33,8
13,6
13,4
12,0
U,5
16,8
1,7
2,4
1,8
1,8
2,5
0,15
0,20
0,18
0,22
0,19
6,7
6,4
6,9
6,9
7,4
Leberfunktionsteste bei Ratten, die
Cadmium mit dem Futter aufnahmen
Monate lang
Oppm
1 ppm
3 ppm
10 ppm
30 ppm
13
-------
822
delten Ratten nicht von der Kontrolle.
Die Nierenfunktion war bei behandelten Tieren nicht beein-
fluflt. Die Tab. 2 zeigt die wichtigsten Ergebnisse der
Nierenfunktionsteste. Auch hier hatten die behandelten Tiere
bis zur hochsten Dosisgruppe von 30 ppm stets physiolo-
gische Werte. Blutzucker- und Cholesteringehalte waren bei
alien Gruppen im Bereich der Norm. Die Messung des systo-
lischen Blutdrucks (Tab. 3) am Rattenschwanz mittels
Gartner-Manschette und Piezo-Element erbrachte sowohl nach
2- als auch nach 3-monatiger Versuchszeit bei den behan-
delten Tieren aller Versuchsgruppen keine hoheren Werte als
bei den Kontrollen. Diese bei 40 Tieren pro Versuchsgruppe
durchgefiihrten Messungen lieSen somit keinerlei durch
Cadmium bedingte erhohte Blutdruckwerte erkennen.
Die bei Versuchsende getb'teten Tiere wurden eingehend patho-
logisch-anatomisch untersucht, Hinweise auf Schadigungen
lietien sich nicht erkennen. Auch aus den Organgewichten
lassen sich keine Schadigungen ableiten. Bei Herz, Nieren,
Hypophyse, Gonaden, Harnblase, Uterus, Leber, Milz, Gehirn,
Schilddriisen, Nebennieren, Lungen, Thymus, Magen, Darme,
wurden aufgrund von histopathologischen Untersuchungen bis
zur hochsten Dosisgruppe von 30 ppm ira Putter keine Organ-
schadigungen festgestellt.
Cadmium wurde polarographisch in Leber, Nieren, Harn und
Faeces nach 1-, 2- und 3-monatiger Versuchszeit bestimmt.
Die Cadmiumgehalte nach 3 Versuchsmonaten (Tab. A) waren
in Leber und Nieren dosisabhangig erhoht. Diese Speicherung
1st, wie wir aus Analysen nach 1 und 2 Monaten wissen,
ebenfalls abhangig von der Dauer der Applikation. In den
Harnen waren dosisunabhangig nur Spuren von Cadmium nachzu-
weisen, wahrend die Hauptmenge des aufgenommenen Cadmiums
in den Faeces erschien.
2.2. Hunde Je 2 weibliche und 2 mannliche Hunde erhielten
Cadmium als Cadmiumchlorid in Konzentrationen ebenfalls von
0, 1, 3, 10 bzw. 30 ppm 3 Monate lang mit dem Putter verab-
reicht.
Bei alien behandelten Hunden waren Aussehen, Verhalten,
-------
823
Tabelle 2;
Kidney-Functkxi-Tests At 3 Month
Dose
ppm
Uric
acid
Urea
Crea-
tinine
mg/100ml
Serum
GOT
LAP
mUArt
Total
Protein
mg/UOml
Urine
Male Rats
0
1
3
10
30
1,3
1,5
1,8
1,8
2,0
28,4
32,4
31,6
31,6
33,3
1,02
1,03
1,05
1JD1
1,06
7,1
5,7
5,2
V
7,0
",7
12,5
13,0
15,2
13,3
24,6
29,7
32.7
30,7
33,2
Female Rats
0
1
3
10
30
1,2
I/
1,6
1,5
2,3
33,2
33,9
32,3
31,9
36,4
1,02
1,04
1,05
1,11
1,14
4,7
3,3
4,1
3,0
W
4,4
4,8
3,5
5,2
5,5
26,2
27,7
.28,1
28,8
30,7
Nierenfunktionsteste bei Ratten, die Cadmium
3 Monate lang mit dem Putter aufnahmen
Dose
ppm
0
1
3
10
30
Syst Blood Pressurefmm Hg), Rat Tdile Vein
At 2 Month
Male Rats
116,9
121,9
111,7
126,3
121,9
Female Rats
100,7
104,3
108,4
101,3
106,9
At 3 Month
Male Rats
121,4
123,9
121,4
125,8
121,1
Female Rats
106,2
103,8
103^1
101,1
104,2
Systolischer Blutdruck von Ratten, die Cadmium
J> Monate lang mit dem Putter aufnahmen
-------
824
Dose
ppm
Cadmium-Content At 3 Month
Liver
Kidneys
Urine (24")
Feces(24h)
ppm
Male Rats
0
1
3
10
30
0,05
0,17
0/1
3,18
8,77
0,13
0,49
1,06
4,98
12,06
0,04
0,04
0,05
0,07
0,07
1,03
0,71
1,90
11,63
46,96
Female Rats
0
1
3
10
30
0,05
0,59
0,39
2,96
8,91
0,13
1,10
1,17
5,07
12,54
0,03
0,04
0,06
0,05
0,08
0,69
0,87
2,55
22,87
54,13
Tabelle 4;
Cadmiumgehalte in Leber, Nieren, Urin und Faeces
bei Ratten, die 3 Monate lang Cadmium mit dem
Putter aufnahmen
-------
825
Futteraufnahme und Sterblichkeit durch die Zuraischung von
Cadmium zum Putter in alien Versuchsgruppen unverandert.
Auch das Wachstum war, wie die Gewichtskurven in Abb. 2
zeigen, unbeeinfluflt.
Die eingehenden Blutuntersuchungen bei alien Hunden (Tab. 5)
erbrachten keinerlei Hinweise auf behandlungsbedingte
Effekte. Auch im Differentialblutbild waren keinerlei Ver-
schiebungen im Anteil bei den einzelnen Leukozytenarten er-
kennbar.
Wie aus Tab. 6 hervorgeht, waren die Aktivitaten der leber-
spezifischen Enzyme in keiner Gruppe dosisabhangig erhoht.
Die Bromsulfophthaleinretention lag bei alien Versuchs-
gruppen im normalen Bereich. Auch Bilirubin- und Gesamt-
eiweifi-Konzentrationen waren nicht verandert. Bei der
Serumproteinelektrophorese ergaben sich keinerlei Verschie-
bungen im Anteil von Albumin und bei den einzelnen Globulin-
fraktionen.
Die Harnuntersuchungen sowie die klinisch-chemischen Be-
stimmungen (Tab. 7) zeigten keinerlei schMdigende Einflusse
durch die orale Cadmiumgabe. Auch aus Clearance-Totalunter-
suchungen von PAH und Inulin lassen sich keinerlei Nieren-
beeinflussungen erkennen.
Aus den bei nicht narkotisierten Hunden nach 3-monatiger
Versuchszeit durch Arterienpunktion gemessenen Blutdruck-
werten (Tab. 8) ergibt sich kein Anhaltspunkt dafUr, dafl
die mannlichen oder weibllchen Hunde nach der Cadmiumbe-
handlung erhohte Blutdruckwerte aufwiesen.
Aus anatomisch-pathologischen Untersuchungen und aus Organ-
gewichten lassen sich keinerlei Hinweise auf Organscha'di-
gungen ableiten. Die eingehenden histopathologischen Unter-
suchungen bei den in Tab. 9 aufgeftlhrten Organen erbrachten
lediglich normale Befunde.
Cadmiumanalysen in Leber, Nieren, Pancreas und Speichel-
driise zeigten dosisabhangig zunehmende Cadmiumgehalte in
diesen Organen. Die mittleren Werte finden sich in Tab. 10.
-------
: '
n.o
10.0
9,0
8,5; _
11,0
10.0
9,0]
8,5
CADMIUM
Weeks—
Oppm
1 ppm
3 ppm
10 ppm
30 ppm
0 1
Abbildung 2
II
13
Tiergewichtskurven von Huriden, die Cadmium 3 Monate lang
mit dem Futter aufnahmen
Blood Studies At 3 Month
Dose
ppm
Sed-rate
1h/2h
Hemogt.
g%
Hema-
tocr.
%
Ery.
K)6
Retics
%.
MCHC
T&
MCV
pm-*
Leucoc.
»3
Throm-
boc
103
Prothrom-
bintime
sec
Male Dogs
0
1
3
10
30
1/3
2/4
3/6
1/2
2/5
14,7
15,4
15,8
14,9
13,3
46
45
48
44
40
5,92
6,40
6,30
6,12
5,98
6
6
5
4
6
24
24
25
24
22
78
70
76
72
67
11
9
8
11
11
475
476
380
398
416
8
8
10
9
8
Female Dogs
0
1
3
10
30
2/4
1/2
1/3
4/9
1/3
16,8
15,8
15,8
15,6
16,4
48
43
46
44
45
7,23
6,27
6,14
6,29
6,72
9
6
5
6
7
23
25
25
25
24
66
69
75
70
67
8
8
7
7
8
532
463
426
476
591
10
10
10
10
10
Tabelle 5:
Blutuntersuchungen bei Hunden, die Cadmium
J> Monate lang mit dem Futter aufnahmen
-------
827
Liver-Function-Tests At 3 Month
Dose
ppm
ALP
GOT
GPT
SDH
LDH
GIDH
mU/ml
BSP
Total
Bilirubine
mg/IOOml
Total Serum-
Protein
g/IOOml
Male Dogs
0
1
3
10
30
63
50
59
54
56
12,4
9,2
9,2
12,3
11,8
16,8
7,3
8,4
8.9
7,0
1,9
1/
1,2
0,9
1,3
82
109
73
87
119
1,6
0,8
1,8
1,1
1,0
0,20
0,15
0,27
0,25
0,27
0,12
0,14
0,12
0,20
0,17
6,6
6,7
6,8
6,7
6,6
Female Dogs
0
1
3
10
30
64
61
62
68
48
15,7
10,1
13,7
16,2
14,7
6,0
8,1
9,9
10,2
10,6
0,7
1,6
0,9
1,0
I/
198
56
43
59
142
1,6
0,8
0,8
0,7
1,4
0,24
0,17
0,22
0,20
0,20
0,14
0,15
0,17
0,14
0,21
6,3
6,7
6,8
6,6
6,6
Tabelle 6;
Leberfunktionsteste bei Hunden, die 3 Monate lang
Cadmium mit dem Putter aufnahmen
Kidney-Function-Tests At 3 Month
Tabelle 7;
Dose
ppm
Urea
Uric
acid
Crea-
tinine
Total
Protein
mg/IOOmt
Serum
Urine
Male Dogs
0
1
3
10
30
0
1
3
10
30
18,1
19,2
16,7
16,2
15,2
2,0
1,7
2,0
1,9
2,0
1,04
0,89
0,92
0,96
1,10
Female Dogs
16,7
18,9
19,7
17,3
15,9
1,6
1,6
1,6
1,8
1,8
0,93
0,97
1,10
0,87
1,28
25,1
24,7
32,7
25,1
36,7
20,9
30,1
34,3
46,0
47,4
Nierenfunktionsteste bei Hunden, die 3 Monate lang
Cadmium mit dem Putter aufnahmen
-------
828
Dose
ppm
0
1
3
10
30
Syst./Diast. Blood Pressure (mm Hg)
At 3 Month
Male Dogs
185/123
203/ 125
178/118
208/135
195/135
Female Dogs
183/115
190/123
213/135
183/123
193/125
Tabelle 8;
Systolischer und diastolischer Blutdruck von nicht
narkotisierten Hunden, die Cadmium 3 Monate lang
mit dem Futter aufnahmen
Aorta arch
Lung
Heart
Thymus
Lymph nodes {cervical
and mesenteric)
Liver
Gall bladder
Spleen
Pancreas
Kidney
Urinary bladder
Prostate
Gonads
Uterus
Thyroids
Adrenals
Salivary gland
Oesophagus
Stomach (body and
antrum)
Duodenum
Jejunum
lleum
Upper colon
Lower colon
Skeletal muscle
Bone marrow (sternum)
Cerebral cortex "
Thatamic nuclet
Mid brain
Cerebellum
Medulla
Pituitary
Peripheral nerve
Eye
Optic nerve
Cervix
Tonsils
Central nervous
system
Tabelle 9:
Zusammenstellung der histopathologisch untersuchten Organe
-------
829
Dose
ppm
Cadmium-Content At 3 Month
Liver
Kidneys
Pancreas
Salivary
gland
ppm
Male Dogs
0
1
3
10
30
0,14
0,24
0,51
2,67
6,21
0,60
1,30
2,60
12/0
15,45
0,03
0,04
0,08
0,32
0,62
0,05
0,08
0,12
0,16
0,36
Female Dogs
0
1
3
10
30
0,08
0,25
0,56
2,21
6,37
0,38
1/5
3/0
7,19
17,15
0,02
0,03
0,10
0,24
0,58
0,06
0,10
0,12
0,25
0,39
Tabelle 10;
Cadmiumgehalte in Leber, Nieren, Pancreas und SpeicheldrUsen
bei Hunden, die Cadmium 3 Monate lang mit dem Putter
aufnahmen
3. Schlufibetrachtung .
Wahrend der Versuchszeit kam es bei den Ratten und Hunden
zu zunehmender Speicherung von Cadmium vor allem in Nieren
und Lebern. Trotz dieser Speicherung wurden weder die Lebern
noch die Nieren morphologisch oder funktionell geschSdigt.
Auch eine Veranderung des Blutes konnte nicht gefunden
werden. Selbst nach Dosen von 30 ppm Cadmium im Putter war
sowohl bei. der Ratte als auch beim Hund der Blutdruck nicht
erhb'ht. SchSdliche Einflilsse auf Reproduktionsorgane wurden
nicht beobachtet. Hiermit konnte gezeigt werden, dafl bei
oraler Gabe Cadmium selbst bis zu Dosierungen von 30 ppm
im Putter ohne SchSdigungen 3 Monate lang vertragen wurde.
Dieses Ergebnis veranlafite uns, weitere IMngerdauernde
toxikologische Untersuchungen zu starten.
-------
830
DISKUSSION
PISCATOR (Schweden)
1. Hat man Kadmium und Zink im Standardfutter bestimmt ?
2. Was 1st die Norm? Hat man nicht die 30ppm-Gruppen mit
Kontrollgruppen verglichen?
LORKE (Bundesrepublik Deutschland)
1. Cadmium wurde im Standardfutter bestimmt. Die Konzentra-
tionen lagen zwischen O,O1 und O,l ppm. Zink wurde im Putter
nicht bestimmt.
2. Der statistische Vergleich wurde mit der Individualkon-
trolle durchgefuflrt- In unserem Institut liegen jedoch auch
samtliche bei vieien anderen Kontrollgruppen bestimmten Werte
vor. Daher haben wir einen guten Ueberblick tiber die normale
Variation der untersuchten Parameter bei unsern Versuchstie-
ren. Alle im vorgetragenen Versuch gefundenen Werte lagen
innerhalb dieser Norm.
KJELLSTROEM (Schweden)
Die von Ihnen angegebenen Werte fflr die Cadmium-Konzen-
tration in der Niere waren selbst bei der Gruppe rnit der
hochsten Belastung urn das Zehnfache niedriger als die Pegel,
die nach allgemeiner Annahme zu Wirkungen fuhren. Auch nach
Berechnungen auf Grund der Dosis selbst entsprechen die Wer-
te fur die tagliche Aufnahme durch den Menschen 1OO-2OO ,ug
pro Tag, also Mengen, fur deren Wirksamkeit bisher noch kein
Beweis geliefert wurde. Ware es nicht ratsam, ein Belastungs-
kollektiv einzubeziehen, bei dem sich tatsachlich Auswirkun-
gen zeigten, so dass man weiss, dass Ihre Methoden richtig
sind? Warum fuhrten Sie diese Untersuchung durch, wo es dpch
so viele japanische und andere Untersuchungen gibt, die zei-
gen, dass man hohere Pegel braucht, um erste Wirkungen zu
erzielen?
LORKE (Bundesrepublik Deutschland)
Ihre Meinung, dass 30 ppm Cadmium in der Nahrung
schadigungslos vertragen werden, wird allgemein nicht ge-
teilt. In ersten Versuchen wollten wir no-effect-Dosen fur
Cadmium finden. Weitere Untersuchungen mit hoheren Dosen und
fiber langere Zeit sollen uns helfen, auch Ihre Frage klar
beantworten.
-------
831
LAUWERIJS (Belgien)
Sie ziehen die Schlussfolgerung, dass bei Tieren, die
in ihrer Nahrung 1 bis 3O ppm Cadmium erhielten, keinerlei
von Cadmium ausgehende Wirkung festzustellen war.
Mir scheint, aus den in Tabelle 7 dargestellten Ergeb-
nissen geht hervor, dass zumindest bei weiblichen Hunden
ein gutes Dosis-Wirkung-Verhaltnis zwischen Proteinurie und
Cadmium-Exposition besteht.
Konnten Sie diese Ergebnisse etwas erleUitern?
LORKE (Bundesrepublik Deutschland)
Eine durch Nierenschadigung bedingte Eiweissausschei-
dung beginnt oberhalb von 1OO mg/lOOml Urin, Alle bei unsern
Tieren gefundenen Eiweisswerte liegen erheblich niedriger
Mit den tiblichen Eiweissnachweismethoden (Essigsaure-Kochpro-
be, Sulfosalicylsaure-Probe) waren samtliche Urine negativ.
Es ist keine Seltenheit, bei unbehandelten nierengesunden
Hunden Eiweisskonzentrationen bis zu 10O rog/lOOml Urin zu
finden. Aus keinem der von uns gefundenen Werte lasst sich
eine Nierenschadigung ableiten, Auch die elektrophoretisch
durchqefuhrte Auftrennung ergab keinen Anstiecr der nieder-
molekularen Fraktionen.
-------
833
CRITERIA FROM ANIMALS EXPOSED TO KNOWN
CONCENTRATIONS OF NITROGEN DIOXIDE AND
OZONE WITH POTENTIAL USE IN EPIDEMIOLOGY
G, FREEMAN/ L, JUHOS, N, J, FURIOSI,
W, POWELL AND R, MUSSENDEN
Stanford Research Institute, Menlo Park, California, USA
ABSTRACT
A realistic concept of the pathogenesis of injury, induced
in animals by their residence in ambient concentrations of the
oxidizing gas, nitrogen dioxide (NO-)* hae been realised for the
rat. Also, observations have been made in the monkey, Maaaaus
speoiosa^. Physiologic and morphologic changes have been inves-
tigated starting immediately upon exposure of young animals and
then intermittently during an entire lifetime of exposure of
rats. Effects varied from an early significant change in res-
piratory frequency, during exposure to approximately 0.8ppm of
NO-, to the development of a fatal, chronic disease resembling
&
emphysema in man. Associated physiologic sequellae in terms of
respiratory function and polycythemia were studied. More re-
cently, similar observations have been made in rats exposed to
ambient concentrations of ozone (0-), a more injurious component
of smog, and to realistic mixtures of the interdependent oxidi-
zing gases, NO- and 0_. Clinical, pathologic, physiologic and
hematologic aspects have been studied in parallel.
Experimentally derived data will be discussed in relation
to detectable events that may logically be anticipated in man
exposed to reasonably equivalent circumstances* The problem of
selecting appropriate end points, or criteria, quantifiable by
-------
834
or -in man for epidemiologic purposes will be considered from the
viewpoint of responses elecited in animals through controlled
experimental studies. Included in the considerations will be
criteria for both cigarette-smoking and non-smoking populations
because smoking of tobacco is a fairly universal habit. The
intended evaluations will be in support of the development of
an acceptable spectrum of health-related effects in countries
with populations subject to exposure to atmospheres polluted
with photochemical oxidanta.
-------
835
Much has been learned about the responses of laboratory animals
residing in atmospheres polluted with either nitrogen dioxide
(Freeman et al. {L-^j ) or ozone (0,) (Stokinger et al. [5], Werthamer
et al. [6j , Freeman et al. [7], Schlipkbter et al . [19] ) » but applic-
ation to the epidemiology of disease in man is difficult. This is
due to the complexity of photochemical reactions themselves[8,9l , to
the variability of man's activities, and to the usual difficulties
in devising effective epidemiologic designs.
At least four special criteria are required in grouping
individuals for a meaningful epidemiologic study of the effects of
oxides of nitrogen (NO ) and oxidents. The first has to do with
residence in crowded, traffic-laden and industrialized areas and
alludes to the daily cycling of the toxic, interdependent, photo-
chemically induced atmospheric pollutants, NO^ and 0,, that occur
almost simultaneously during half of each day in varying ratios and
in fairly similar average concentrations [8,9]' Thus, epidemiologic
observations have necessarily been related to the impact of the mix-
tures. (Our experience with rats residing in atmospheres containing
either N02 or 0, reveals that, at least, in relatively short-term
experiments, 0, is about ten to twentyfold more injurious to the lungs
than N02 (Freeman et al. [lo]) ).
Whereas one may limit atmospheric components selectively to
NOp and 0, under laboratory conditions, peroxyacetyl nitrates also
exist as photochemical products [8], and independent pollutants,
such as sulfer dioxide (S0_) [ll] « appear frequently to complicate
interpretation.
The universal custom of cigarette-smoking is a second factor
in the epidemiology of NO -induced disease [l?]« Among the injurious
components of tobacco smoke, oxides of nitrogen are found in hundreds
of parts per million (Haagen-Smit et al. [l?])i of which an undeter-
mined portion is N0_. Of the large dose of NO carried into the lungs
with each bolus of inhaled smoke, almost all is retained (Freeman et
al. [l1*]). The close correlation between the prevalence of human
emphysema and chronic bronchitis with the amount of tobacco smoke
inhaled over a period of years [l2]is especially noteworthy and is
reminiscent of the disease readily induced in rats by exposing them
continuously to N02 in concentrations probably similar to
-------
836
those in the tobacco smoke that traverses the airways of the smoker [4],
As a corollary, nonsmokers are largely spared from chronic obstructive
pulmonary diseases regardless of their ambient environments.
A third, obvious factor is occupation. This concerns confined
concentrations of either NO2 or O3, or both, independent of concurrent
photochemical activity. This applies equally to NOX produced naturally
by bacterial action on crops stored in silos, causative of silo-filler's
disease in farmers, and to industrial processes such as welding and the
use of explosives in mines. High temperatures are reached in which the
oxidation of ubiquitous nitrogen is accelerated to form toxic oxides [9],
Similarly, certain occupational activities create high concentrations of
either N02 or 03 not achieved in photochemical reactions (Stokinger [15]).
The fourth factor is individual susceptibility, which includes the
age of initial exposure. As a rule, the very young are thought to be
poorly defended against exogenously induced disease compared with maturing
or mature individuals, but this may not be universal, as we shall see.
The elderly also are vulnerable due to the high probability of affliction
with cardiopulmonary diseases and possibly to aging itself. Individuals
prone to respiratory allergy are particularly responsive to atmospheric
irritants. With regard to chronologic age, the type of response to
exogenous insults may depend in part on whether an individual was born
into a polluted environment or was moved into it later in a virgin state,
relative to specific environmental components.
What is the evidence for this? Using a species sensitive to N02,
four-week-old rats increased their rates of erythropoiesis rapidly and
developed polycythemia during continuous exposure to the gas [3].
(Figure l) In contrast, animals exposed from birth generated red cells
at a rate less rapid than that expected of newborn rats when compared
with normal litters born in clean air. As a result, such exposed rats
were relatively anemic upon attaining seven weeks of age, and contrasted
sharply with the polycythemic rats of the same age that were exposed only
during the last two of the seven weeks [14],
Also, the mechanical compliance of excised lungs of rats exposed to
high subacute concentrations of NO2 <~15 ppm) from birth was not affected
by increasing the frequency of respiration. Lungs of animals of the same
age, exposed after they were a month old, developed frequency dependence
-------
837
O £ 40
h- "
i
;
I I I L_LJ J--L-1-1.
.
30 DAY OLD EXPOSED
CONTROL
NEWBORN EXPOSED
I i I I I I l_l I I 1 l_l 1 1 L
0 10 20 30 40 50 60 70
AGE ci.iv.
Fig. 1. Erythropoiesis in Immature Rats.
, ---
H Jli 48 60
Ml M'lllAIIONS I'lH MINIMI
JO.-'t'/l I mi •«•«-•-»""
Fig. 2. Frequency-Dependent Compliance (as percent of value
at lowest frequency).
u
Q
30 M) tO
AC,I
Fig. 3. Total Number of Alveolar and Ductal Air Spaces
-------
838
of compliance, presumably due to changes in the small airways 1 14 1 .
(Figure 2) Incidentally, growth of the lung—defined as the rate of forma-
tion of tiew alveoli in animals exposed continuously to N02 from birth —
was less than normal for the first ten weeks [14], (Figure 3) Thus,
the failure of animals exposed from birth to have developed small airway
disease, as judged by frequency dependence of compliance, may have been
determined by neonatal contact with NO2 . Doth the hematologic and respi-
ratory observations suggest altered responsiveness, reminiscent of the
* •*
immuno logic tolerance of animals exposed neonatal ly to antimetaboli tes
(Schwartz et al. [16]). Thus, the significance of very early exposure
relative to subsequent pathogenesis is interesting and deserves further
study.
Whereas smoking is clearly associated with chronic pulmonary disease
in man, ambient ozone has not yet been so identified epidemiologically fa].
What is its impact on the nonsmoking population? Its odor is known to be
detectable at a concentration of about 0.02 ppm; increased susceptibility
to infection in animals begins at about 0.08 ppm; and eye irritation to
peroxyacetyl nitrates is noticeable when the ozone concentration reaches
about 0.1 ppm [8], At that level, increased airway resistance also
becomes evident in some individuals, and it is readily apparent when
breathing a concentration of about 0.5 ppm in air for three hours on
consecutive weekdays. Exposure to an atmosphere of about 1.0 ppm rapidly
becomes intolerable to man
In contrast, there is little clinical evidence of effect on the
respiratory tract during experimental exposure to NOZ at levels below
1.0 ppm, although the odor is readily detectable at a concentration of
about 0.1 ppm [9]. To simulate maximum human exposure to the mixture of
NO2 and ozone on a cyclic photochemical basis, we exposed rats daily for
four hours and observed pathologic effects in the lungs that could relate
potentially to chronic pulmonary disease [10], Using a concentration of
about 0.9 ppm of each of the two gases, a lesion developed at the narrow
junction of the respiratory bronchiole and alveolar duct through which air
is conducted to and from the alveoli. (Figure 4 atb). Spithelial cells were
injured and replaced (Evans et al. flTj), often by hypertrophic cells.
The hypertrophic epithelium, supported by fibroblasts and chronic inflam-
matory cells, proliferated at the level of the smallest airways to form
denser tissue. Neither its effect on lung function nor its reversibility
-------
DEVELOPMENT OF LESIONS AT THE NARROW JUNCTION OF THE RESPIRATORY
BRONCHIOLE AND ALVEOLAR DUCT EXPOSED TO 0.9 ppm OF NO AND TO
0.9 ppm OF 0^
'• • • • • w
Fig. (4a. Control rat exposed to clean air,
$E~"*5*
Fig. '+b. Rat exposed to 0.9 ppm of NO and to 0.9 ppm of 0.
simultaneously for four hours daily.
-------
840
EMPHISHMATOUS LESION IN LUNGS OF RATS EXPOSED TO
0.9 ppra OF N02 AND 0.9 ppm of 0
Figure 5* « Normal rat with excised lunge at atmospheric pressure
Figure 5\> * R&"t after exposure continuously for three weeks to 0.9 of NO
and 0.9 ppm of 0
Figure 5° t EraphysematouB lesions in lung seen in Figure b
-------
841
RESPIRATORY BRONCHIOLE AND ALVEOLAR DUCTS IN RATS
Figure 6a I Normal terminal bronchiole and neighboring alveolar duct
,,
1
~r-'
/ ;
-------
842
has been established. As was the case with high subacute concentrations of
NO2 [l,4]t with continuous exposure to a mixture of ozone and NO2—each at a
concentration of about 0.9 ppm—rats developed an emphysema-like disease.
(.Figures 5a, b, c). Also, continuous exposure to a mixture of 2.5 ppm of NOg and
one-tenth that concentration (0.25 ppm) of ozone resulted in disease of the
alveolar ducts characteristic of the response to ozone rather than to NO2.
(Figures 6a, t>). Thus, it is clear that ozone is the primary cause of deep-
seated pulmonary disease in the experimental animal when mixtures of the
agents are presented in concentrations and on a schedule approximating man's
experience with smoggy atmospheres. Long-range studies are under way.
Shifting our attention to animal studies with higher subacute concen-
trations of N02, of the order of 14 to 17 ppm [3,4], the issue of smoking
and the epidemiology of chronic obstructive pulmonary diseases [12],
toward which NO2 may be contributory, come into focus. Extrapolation to
chronic human disease from the rat with a life span of about 4^ that of
man is hazardous. Nevertheless, the fortuitous similarity in the relative
time required for the development of emphysema in the rat, when compared
with its development in man, warrants attention [1,3,4], The clinical and
morphological features associated with habitual smoking of cigarettes are
consistent with the changes in the lungs of rats exposed to high, subacute
concentrations of NO2 for long periods [1,3,4]. It is noteworthy, also,
that ozone, which is absent from tobacco smoke, induces a similar disease
in the rat at approximately one-twentieth the concentration of N02 when
mixed with it in equal amounts [10].
In summary, it would not seem feasible, except in the case of high
occupational concentrations and in habitual smoking, to distinguish between
the specific effects'of N02 and O3, epidemiologically. As photochemically
interdependent gases, they must coexist and act almost simultaneously.
However, several recent studies suggest that biochemical changes in blood
of man (Buckley et al. [is]) may provide a handle by which to correlate
the atmospheric presence of these individual pollutants and, possibly,
nitric oxide (NO) with systemic effects [14].
This research was supported by Contract No. 68-02-1243 of the
Environmental Protection Agency (USA), Division of Health Effects Research,
Research Triangle Park, North Carolina, and in part by the National
Institute of Environmental Health Sciences, National Institute of
Health ( U.S.A.).
-------
843
References
1. FREEMAN, G., HAYDON, G.B., "Emphysema after low-level
exposure to N02/" Arch. Environ. Health, 8,125(1964).
2. FREEMAN, G., STEPHENS, R.J., CRANE, S.C., FURIOSI, J.H.,
"Lesion of the lung in rats continuously exposed to two
parts per million of nitrogen dioxide," Arch. Environ. Health,
17, 181 (1968).
3. FREEMAN, G., CRANE, S.C., STEPHENS, R.J., FURIOSI, N.J.,
"Pathogenesis of the nitrogen dioxide-induced lesion in the
rat lung: A review and presentation of new observations,"
Am. Rev. Respirat. Diseases, 98,429 (1968).
4. FREEMAN, G., CRANE, S.C., FURIOSI, N.J., STEPHENS, R.J.,
EVANS, M.J., MOORE, W.D., "Covert reduction of ventilatory
surface in rats during prolonged exposure to subacute
nitrogen dioxide," Am. Rev. Respirat. Diseases, 106, 563
(1972).
5. STOKINGER, H.E., WAGNER, W.D., DOBROGORSKI, O.J., "Ozone
toxicity studies: III. Chronic injury to lungs of animals
following exposure at low level," Arch. Industr. Health.
16,514 (1957).
6. WERTHAMER, S., et al., "Ozone-induced pulmonary lesions:
Severe epithelial changes following sublethal doses,"
Arch. Environ. Health, 20,16 (1970i
7. FREEMAN, G., STEPEHENS, R.J., COFFIN, D.L., STARA, J.F.,
"Changes in Dogs' lungs after long-term exposure to
ozone," Arch. Environ. Health, 26, 209 (1973).
8. U.S. Public Health Service, Air Quality Criteria for Photo-
chemical Oxidants, National Air Pollution Control Adminis-
tration Publication No. AP-63, Washington, D.C. (1970).
9. U.S. Environmental Protection Agency, Air Quality Criteria
for Nitrogen Oxides^ Air pollution Control Office Publication
No.AP-84,Washington, D.C. (1971).
10. FREEMAN, G., JUHOS, L.T., FURIOSI, N.J., MUSSENDEN, R.,
STEPHENS, R.J., EVANS,M .J., "Pulmonary pathology of
exposure to interdependent ambient gases (NO- and 0.,),"
in press, Arch. Environ. Health (1974)
11. U.S. Public Health Service, Air Quality Criteria^ for Sulfur
Oxides, National Air Pollution Control Administration
Publication No. AP-50, Washington, D.C. (1969).
12. U.S. Department of Health, Education and Welfare, The Health
Consequences of Smoking, Chapter 2, Public Health Service,
January 1973, pp.31-62.
13. HAAGEN-SMIT, A.J., BRUNELLE, M.F., KARA, J., "Nitrogen Oxide
content of smoke from different types of tobacco," Arch.
Industr. Health, 20, 399 (1959).
14. FREEMAN, G., et al. Unpublished data.
15. STOKINGER, H.E., "Ozone toxicology. A review of research
and industrial experience: 1954-1964," Arch. Environ. Health
10,719 (1965). ~~
-------
844
16. SCHWARTZ, R., DAMESHEK, W., "Drug induced immunologic
tolerance," Nature, 183, 1682 (1959).
17. -EVANS, M.J., STEPHENS, R.J., CABRAL, L.J., FREEMAN, G.,
"Cell renewal in the lungs of rats exposed to low levels '
of N02 ," Arch. Environ. Health, 24, 180 (1972).
18. BUCKLEY, R.D., HACKNEY, J.D., CLARK, K., POSIN, C., "Some
effects of ozone inhalation on human erythrocyte metabolism"
(Abstract), Fed. Proc. Abstracts, 33, 335 (1974).
19. SCHLIPKOTER, H.W., BRUCH, JOACHIM, "Funktionelle und
morphologische Veranderung bei Ozonexposition." Zbl.
Bakt. Hyg., I Abt. Orig. B 156, 486-499 (1973).
DISCUSSION
STUPFEL (France^
Do you relate the effect vou reported on ervthropoiesis
and the apparition of pulmonarv emphvsema?
FREEMAN (U.S.A.)
In growing and mature animals, we relate ervthropoiesis to
the hypoxemia of pulmonary disease (with or without the actual
development of emphysema). However, the reference to erythrop-
oiesis in the current presentation is to the difference in res-
ponse to exposure to NO., between neonatal (from birth) rats and
more mature neonatal rats. The former fail to achieve the level
of erythrogenesis of normal rats in the neonatal state, at least
to the age of 90 days, whereas the somewhat more mature neonatal
animals are already able to respond with exaggerated erythro-
genesis (polycythemia).
Although, it is presumed that the ervthrogenic response is
very sensitive and reflects some degree of oxygen-want, secon-
dary to the pulmonary effects of NO., room must be reserved also
for the real possibility that N02» or a reaction product, mav
penetrate the pulmonary-vascular barrier and effect an ervthro-
genic response on another basis also, for example, by affecting
red cells directly or the erythrogenic tissue. At present, there
is little or no evidence for this.
-------
STOFFWECHSEL
METABOLISM
METABOLISME
METABOLISMO
METABOLISME
Voraitzender - Chairman - President - Pveeid&nte - Voovzittev
D. BENINSON (Argentina)
-------
847
SOURCES AND METABOLIC PATHWAYS OF LEAD
IN NORMAL HUMANS
G, W, WETHERILL+, M, RABINOWITZ* AND J, D, KOPPLE"1"*"
+ Institute of Geophysics and Planetary Physics, University of
California, Los Angeles, Ca, USA
++ Veterans Administration Wadsworth Hospital Center, Department
of Medicine and School of Public Health, University of Cali-
fornia, Los Angeles, Ca, USA
ABSTRACT
Absorption, excretion and internal distribution of lead has
been studied in normal volunteer adult male subjects, maintained
under controlled diet and environment for periods of six months
•in a hospital metabolic unit. Stable isotopes of lead were
used to iaotopioally label lead from food. Thia permitted dis-
tinction of thia source from others (atmospheric and internal)
as body fluids and tissues exchanged "Lead internally and with
the external environment during the course of the experiments.
Lead concentration and isotopio composition in "blood, urine,
feces, hair, nails, sweat, bile, gastric and pancreatic secre-
tions and bone were determined by mass spectrometric stable iso-
tope dilution analysis.
It was found that the concentration of lead in the blood of •
these subjects can be simply related to measurable parameters
such as the fraction of food lead absorbed in the gut (8 to 14%),
the daily quantity of internal lead excreted in urine, hair and
secretions into the gut (38 to SO.ug), and the characteristic
(e-folding) residence time of lead in the metabolic pool of lead
which exchanges rapidly with the blood (cv35 days). Blood lead
concentrations could be predictably adjusted within the normal
-------
848
range of 0.14 to 0.27 .ug/g by controlling the dietary or atmos-
pheric input. Response of the blood lead concentration to
removal and introduction of atmospheric exposure to lead at
2
typical urban concentrations of 1 to 2 .ug/m showed that about
16 ,ug/day are added to the blood from this source, which there-
fore comprised about 1/3 of the daily intake from all sources.
These \same techniques were used to study the gastrointes-
tinal absorption of lead. While fasting^ absorption factors
as high as 50% were found. Differences between individuals
and chemical form were also observed.
-------
849
1. Introduction
The kinetics of the absorption, internal distribution, and excre-
tion of lead at normal concentration levels in normal humans is be-
ing studied with the use of stable isotope tracers. The use of
these nonradioactive tracers (20l*Pb, 206Pb, or Pb) permits label-
ling of dietary lead ingested over a particular period of time and
permits following the subsequent history of this lead as a consequen-
ce of physiological processes. This technique has the advantage of
allowing kinetic studies to be carried out under constant, steady-
state total lead concentrations, and also minimizes problems of lab-
oratory contamination in chemical analysis, as the sources of lead
contamination have isotopic compositions markedly different from
those of the isotopic tracers. Other important aspects of the meth-
odology are that all analyses are carried out using the accurate and
sensitive technique of mass spectrometric stable isotope dilution,
and that the human subjects are maintained on controlled diets and in
controlled environments in a hospital metabolic unit. Simultaneous
metabolic balance studies are carried out for other elements.
Results obtained on a first subject have been reported previous-
ly by Kabinowitz, Wetherlll, and Kopple [1] and preliminary results
have been given for a second subject [2]. In this work the subject's
normal diet was replaced with a low-lead diet. This diet was sup-
2QU.
plemented with Pb, so as to maintain the pre-study level of lead
ingestion. In both cases it was found (fig. 1 and 2) that ingestion
2QU
of a constant quantity of pb nitrate with meals resulted in a ris-
ing concentration of this isotope in the blood, which approached a
steady state after — 100 days. The e-folding time for the increase
2Q11
in Pb concentration is ~ 3S days. The increase of concentration
2QU
of Pb in the urine followed that of the blood closely, whereas
other fluids (bile, saliva, pancreatic and gastrointestinal secre-
tions) showed a delay in labelling. Hair, fingernails, and bone
were labelled more slowly.
These results can be expressed quantitatively by reference to a
3-compartment model (fig. 3) wherein compartment 1 represents the
blood and those tissues which exchange rapidly £$ 3 days) with the
blood. Introduction of compartment 2 permits representation of the
delay in labelling found for the bile and other secretions, and
-------
.30
CO
§ .20
g
fe .15
1
_j
« .10
I
1 .05
.01
WEKAffE
TOTAL LEW
LABELLED
WET/Ktlfffi
oo
Ul
O
0 10 20 30 40 50 60 70 80 90 100
: EATEN
110
X7
SPUG
120 130 140 150 160 170 180 190 200 210 220 230 240
-HO SPIKt •
•NORMAL DIIT-
Days of Diet
Figure 1. Blood lead concentration of Subject A, In the response to the diet containing
labelled lead, the blood lead showed the gradual appearance of labelled lead. However, not
all the blood lead will become labelled because of sources of_unlabelled lead, such as the
atmosphere and the skelton. When the dietary supplements of PB and 7PB are withdrawn,
the total lead concentrations in the blood decreases by a corresponding amount. This indicates
there is little tendency for the blood to maintain a constant lead concentration in this
concentration range.
-------
851
Blood Lead Concentration vs Time (Subject B)
•—Low Lead Diet + Norm ^Clean Air -*l
•I :
109 124 148 179
DURATION OP STUDY (days)
Figure 2. Blood lead concentration of Subject B, On day 109 a diet-
ary supplement of Pb was given in addition to the ' Pb, and at
the same time atmospheric lead was removed by filtering. The total
lead dropped slightly, reflecting the fact that the excess 207Pb did
not exactly balance the loss of atmospheric lead. On day 124, the
diut;iry supplements were discontinued. The only remaining lead
source was the low lead diet. On day 148 the atmospheric lead was
reintrodijci-ii, and the total lead started to rise. On day 179 the
iii.nn.il "high lead" diet was reintroduced. Detailed analysis of these
•in its calm!,itiun ui the atmospheric contribution given in
! .'Me 3.
-------
852
M
M2
20
Figure 3. 3-compartment model used to obtain the parameters of
table 1. M, is e short-lived c-ampartment consisting of the blooc'
and a similar mass of tissues and/or fluic's which exchange rapidly
with the blood. M- is a short-lived compartment, the labelling of
which is delayed and is the source of lead in gastrointestinal se-
cretions. M, is a long-lived compartment containing most of the
lead in the body, primarily in the skeleton.
compartment 3 may be identified with the long-lived lead pool of the
skeleton. Such a model is only a first approximation to the complex-
ity of human physiology. In fact, our data cannot be fitted exactly
to this model. Nevertheless this is a useful way of comparing the
principal similiarities and differences between different subjects.
If desired, most results of the work could be formulated in a less
model dependent way. The results of fitting the experimental data
for the two subjects are given in table 1. The parameters of the
model were obtained at a constant value of total blood lead. It is
found, however, that they are not strongly dependent on the value of
total blood lead, and have been successfully used to predict the re-
sponse of blood lead to decreases in lead input up to 7G% for per-
iods of times sufficiently long to cause the total blood lead con-
centration to decrease by 2Q96.
-------
853
Initial slope
Daily tracer
absorbed
Table 1
Model parameters
Subject A
.0024 + .0001
17.5 + .2
Subject B
. 00115 + 10 Mg/gr day
11.9 + .2
Compartment 1 mass
7.3 + .3
Lead in Compartment 1 1830 + 75
Lead in Compartment 2 260 + 100
\L
10
9.9 + .1 Kg
1825 + 50 us
900 + 100 pg
20
.035 + .002
.020 + .002
.010 + .003
.005 + .005
.07 + .02
.02 + .01
.05 + .02
.024 + .002 A/
.015 + .002
.006 + .002
.003 + .002
.03 + .01
.0002 + .0001
.03 + .01
di
n
tt
it
This report is not sufficiently long to allow adequate discus-
sion of all the data. However, some details will be given regarding
two aspects of the study, the contribution of atmospheric lead and
the gastrointestinal absorption of dietary lead.
2. Contribution of atmospheric lead
In these experiments it is possible to obtain a quantitative
value for the daily quantity of lead absorbed from a typical urban
atmosphere (2 jig Pb/m ) in several ways:
(1) The over-all lead balance during the course of the experi-
ment .
(2) The failure of the blood to become completely labelled.
The 206Pb/2(WPb ratio of the blood asympototically approaches a val-
ue distinctly lower than that of the food, indicating a non-dietary
-------
854
contribution of lead of normal isotopic composition to the blood.
(3) Response of the blood lead concentration to removal and re-
introduction of air lead by the use of atmospheric filters.
The first two methods are dependent on the assumption that
transfer of lead from the skeleton to the blood is equal to the
transfer of lead from the blood to the skeleton. Otherwise an ex-
cess transfer of unlabelled lead from the skeleton will mimic an at-
mospheric source of unlabelled lead. This assumption is avoided in
the third method, used for the second Subject (B). Results of the
three methods are shown in table 2. The good agreement found be-
tween the methods is evidence that the assumption of a steady state
for exchange with the skeleton is valid, an assumption supported by
other data. The value found, ~ 16 ;ig/day is somewhat less than
quantity absorbed da.ily from typical diets (2'f and 33 ;:g) . The re-
sult is in good agreement with estimates based on the measured con-
centration of aerosol arid vapor lead in the air breathed by the sub-
jects, estimates of daily respired volume and lung absorption data,
as measured by Booker et al L3] and Hursh and Mercer l>]
Table 2
Respired Lead Intake C'^day)
Subject
Balance Blood Labelling Response to Filtered Air
A 16 + 4 17 + 5
B 19+3 16+6 16+3
3. Absorption of dietary lead
During the two long-term (~ 6 month)studies, 204Pb was substi-
tuted for approximately £ the lead in the subject's pre-study diet.
The Pb was ingested as the nitrate in equal quantities with each
meal. Under the controlled conditions of constant diet, environment
and daily routine, the absorption of 2(Wpb by the gut, determined by
difference between dietary and fecal lead, was quite constant (8.5%
and 6.5% for Subjects A and B respectively). In contrast to the
case of dietary lead of normal isotopic concentration, absence of
-------
855
20'+
Pb in possible sources of contamination of the samples permitted
rather accurate measurement of the absorption factor. These factors
were different for the two subjects, but within the range reported
by Hursn and Suomela [5].
As discussed in the previous section, even in this urban envir-
onment, the most important single source of absorbed lead was diet-
ary. Furthermore the potentially available lead is much greater in
the diet, since 40-50% of the total atmospheric lead is already be-
ing absorbed, in comparison with less than 1096 of the dietary lead.
In assessing the significant sources of lead in humans it is there-
fore necessary to study the variability of the gastrointestinal ab-
sorption when the controls on this quantity are relaxed.
This was done near the end of the long term study with Subject
B. On two occasions the effect of fasting and chemical form were
studied. In the first experiment, the subject fasted for 8 hours
and then ate about 75 ;:g each of Pb nitrate, Pb cysteine, and
Pb sulfido and continued the fast for 6 more hours. This was
o nil o 07
repeated 35 days later with Pb sulfide and Pb nitrate. The
next day Pb cysteine was eaten with food. Absorption was meas-
ured in two ways: collecting and measuring unabsorbed lead in
fecus lor 10 days following ingestion and measuring whole blood iit 6
i
and 24 hours post -ingestion. Absorption could be determined from the
blood data because the response of the blood lead to absorption had
been well established during the long term study. The absorption
factors found are shown in table 3. The results by the two methods
may be considered to be in agreement considering they were based on
a single day's response. The striking thing was the high absorption
found while fasting. This greatly enhanced absorption (up to 50%,
i.e. 8-fold) was found in both experiments, and must be considered a
real effect.
In order to learn if this effect was peculiar to Subject B, a
similar experiment was performed on another Subject (C) . This sub-
ject had not participated in a long-term study and his blood re-
sponse was not previously calibrated. This was done in a single
measurement, using 206Pb nitrate, taken with breakfast. This was
compared with the fasting absorption of Pb nitrate and Pb sulfide
The absorption found for 2°'*Pb nitrate with food (14%), was higher
-------
856
Table 3
Effect of fasting and chemical form on lead absorption
Subject Mode Absorption Absorption
(diet-feces) (blood response)
% %
B fasting nitrate 40 35
B " " 28 25
B fasting sulfide 52 45
B " " 45 41
B fasting cysteine 30 22
B cystoine with food 6 6
B nitrate with food 6
C fasting nitrate 36 41
C fasting sulfide 14 14
C nitrate with food 14
A nitrate with food 8.5
than previously found for the other two subjects. While fasting,
much more lead nitrate was absorbed, whereas lead sulfide while
fasting was only 14% absorbed.
These experiments show that gastrointestinal lend absorption
varies between subjects, nnd is dependent upon chemical form. Most
significantly, there is a pronounced tendency toward greater absorp-
tion when lead is ingested without food. This effect should be con-
sidered in connection with evaluation of absorption following inges-
tion of lead from paint, putty, etc. by children. The variability
in absorption found in these experiments shows that the lead absorb-
ed by a given individual is more than simply a function of the total
lead ingested, and that much more information is required before the
relationship between dietary and absorbed lead is understood.
These results were obtained by Veterans Administration Project
No. 5016-02, and were supported by NSF KANN grant GI 38339.
-------
857
References
RABINOWITZ, M. , WETHERILL, G., KOPPLE, J. , "Lead metabolism
in the normal human: Stable isotope studies". Science 182,
725 (1973).
RABINOWITZ, M. , WETHERILL, G., KOPPLE, J. , "Studies of human
lead metabolism using stable isotope tracers", in press,
Environmental Health Perspectives (1974) .
BOOKER, D. V., CHAMBERLAIN, A. C. , NEWTON, D. , STOTT, A. N. B.,
"Uptake of radioactive lead following inhalation and injec-
tion", Br. J. Radio 42. 457 (1969).
212
HURSH, J. B., MERCER, T. T. , "Measurement of Pb loss rate
from human lungs", J. Appl. Phys. 28., 268 (1970).
HURSH, J. B., SUOMELA, J., "Absorption of pb from the
gastrointestinal tract of man", Acta Radio. £» 108 (1968).
DISCUSSION
MAGI (Italy)
I should like confirmation of the theory that 75% of the
blood lead is taken in with food whilst the remaining 25% is
absorbed from the atmosphere.
Was an accumulation of Pb found in the blood and at what
levels did this cease?
WETHERILL (U.S.A.)
Actually about 35% is absorbed from the atmosphere (when
the concentration is around 2yug Pb/m ) and about 65% from food.
Of course this will depend upon the lead content of the food
eaten, on the quantity of food and the lead concentration of
the atmosphere.
-------
858
As shown in out table 2, about 16 ,ug of Pb was absorbed per
day from the atmosphere. As explained in section 2 of the text,
this was measured in 3 ways. One of these was based on the fact
that the blood did not become entirely labelled (Fig. 1 and Fig.2).
204
The Pb accumulated in the blood during the first part of
the experiment and approached a steady state value of about
. 07,ug/g after about 10O days.
PFANNHAUSER (Austria)
In connection with the intake of lead from food, I should
like to ask if the relatively high level was also found with
aerosols, which are not metal lead but lead oxides?
WETHERILL (U.S.A.)
We do not have an accurate value for the absorption of Pb
from aersols for two reasons:
1. The atmospheric lead was not isotopically labelled.
2. The respired volume during the entire course of the experi-^
ment can only be estimated.
Reason (2) is the more serious.
As discussed in section 2 of the text, the daily quantity of
atmospheric lead absorbed (^16 ,ug/day) is in agreement with esti-
mates of a typical daily respired volume and the approximate
40% Pb absorption found by other v,*orkers.
COFFIELD (Belgium)
What was the length of time subject B was held in the no
lead atmosphere without changing any other experimental detail?
Did the subject come to equilibrium with regard to blood lead
level when there was no lead in air?
WETHERILL (U.S.A.)
The subject was maintained in the "clean air" environment
for a total of 39 days. During the first 15 days, a sufficient
quantity of Pb20^ was added to this diet to offset the anticipated
decrease in absorption of respired lead. This was very nearly
achieved, the total lead in the blood only dropping slightly.
Comparison of the increase in Pb in the blood with the decrease
in concentration of Pb of normal isotopic composition (not shown
in fig. 2) permits calculation of the quantity of respired lead
removed without waiting to achieve a steady state. During the
-------
859
2O4
remaining 24 days, all dietary isotopic supplements (Pb and
Pb207) Were removed, causing the total blood lead to decrease.
The decrease in lead of normal isotopic composition caused by
the removal of atmospheric lead continued as before, in agreement
with our previous finding that homeostatic mechanisms do not
operate to maintain a constant blood level. Upon reintroduction
of air containing Pb and return to diet of previous lead content
the blood lead concentrations increased as expected. The subject
did not come to equilibrium with regard to blood lead level during
this part of the experiment. However, this is not necessary.
The non-equilibrium response can be measured just as well, with-
out the considerable inconvenience to the subject which would
result from maintaining the atmospheric control for > 100 days,
as required to achieve a steady state. I would like to point out
that this experiment was designed to measure the contribution
of atmospheric lead at or near the subject's pre-study level of
blood lead concentration. This does not necessarily or logically
imply that the steady state blood lead concentration would be
proportionately lower, following the removal of atmospheric
lead. First of all, the contribution from deeper compartments,
primarily the skeleton, would continue. We estimate this to be
about 7yug/day, but the exact figure is uncertain.
Furthermore, it is possible that at sufficiently lowered
blood lead concentrations homeostatic mechanisms may operate
which tend to maintain a constant value of blood lead. We have
found no evidence for such mechanisms when the total blood con-
centration is lowered by up to 25% but it is conceivable that at
lower concentrations such an effect could occur.
BERLIN (C.E.C.)
In view of the attention presently given to atmospheric
lead and of the fact that your subjects were maintained indoors
in controlled atmospheres did you:
- sample regularly the indoor atmospheric lead concentration,
and if yes with what method?
- determine the granulometry of the lead aerosols present
in the ambiant indoor air?.
WETHERILL (U.S.A.)
Indoor atmospheric lead concentrations were continuously
measured by capturing particulate lead on a millipore membrane
filter (0.45,u HAWP), with a retention rate > 95% for particles
>_ .05 /u. Air was pulled through this filter at a rate of
approximately 1 m3/hr. for 5 day collection periods. The lead on
the filter was determined by stable isotope dilution. The sampler
hung above the subject's bed. During the "clean air" phase of
the study, the air lead levels in the subjects room were lowered
-------
860
to O.072/ug/meter (0.062 to O.087) from about 2 ,ug/m . No
detailed/examination of particle sizes was carried out. However,
an activated carbon scrubber supplied by Dr. Ter Haar of Ethyl
Corporation was placed behind the membrane filter to capture
"vapor" lead. This captured an additional 0.10 ,ug/m3 in outdoor
air and 0.05 ,ug/m3 in "clean air". We do not know the chemical
or physical form of this "vapor" lead except that it was able
to penetrate the membrane filter to be captured by the carbon.
It could perhaps be TEL vapor or small (<.05/u) particles of
lead salts. '
-------
861
SUL PASSAGGIO TRANSPLACENTARE DI INSETTICIDI
CLORURATI
C, GRASSO
Istituto di Igiene dell'Universita di Firenze, Italia
RIASSUNTO
Sono stati ricercati e titolati gli ineetticidi alorurati
presenti -in aampioni di sangue prelevati a puerpere e ai rispet-
tivi figli neonati, I prelievi nei figli sono stati eseguiti
prima ahe queeti aveesero assunto it primo pastof natufale o
artifioidle4 in manieva da titolave eiouramente soltanto gli
insettioidi clorurati pervenuti al figlio dalla madre, esaludendo
totalmente I'apporto di tali sostanse nel peviodo immediatamente
successive alia nascita.
Le titolazioni sono state eeeguite nel aangue in quanta e&eo
vappresenta la via di traaporto dagli ineettioidi ai vari teesuti.
I aampioni di aangue Bono stati pvelevati nella clinioa
Oetetrica dell'University di Firenze e nel reparto Pediatrico
dell'Ospedale di Arezzo, da donne di varia estrazione aociale,
lavoro, domiailiOj e dai riepettivi figli, alouni nati a tevmine
e sani (Firenze) e altri prematuri o affetti da qualahe forma
patologica (Arezzo), Compleaaivamente sono stati eeaminati 70
oampioni di aangue. Gli ineettioidi sono etati riceroati gae-
cromatografiaamentet con la seguente metodioa: - estrazione
degli ineettioidi dai aangue eon solvent-is - purifications
dell 'eluato in colonna di allumina; - analiei gascromatografica
con detector a cattura di elettroni (Stronzio 90). Tutti i
noetri oromatogrammi hanno preeentato piaahi di insettioidi olovu-
rati. In tutti i oampioni esaminati e presents il pp'DDT in
quantity one arrivano oltre le 0,6ppmt il DDD e il DDE. Gli
-------
862
altri insetticidi ricercati (lindano, eptacloro, eptacloro epos-
sido t aldrin, dieldrin) sono presenti nella maggior parte dei
campioni con eacezione dell ' eptacloro epossido e del dieldrin,
pr&senti rispettivamente solo in 4 e -in 9 campioni.
I cfomatogvammi degli insetticidi rilevati nel sangue d
madvi, e dei, rispettiwi, figli sono sinrili qualitativamente t ma non
quantitativamente , essendo sempve aupeyiori i tasai repeviti nel
sangue della madve riepetto a quelli dei figli.
Risulta dunque ahe almeno in parte gli insettioidi presenti
nel sangue della madt>e pasaano durante la gravidanza nel eangue
del figlio.
Pfesaooc'he per tutti gli ineetticidi, e tanto nel gvuppo
delle madri one in quello dei figli^ i taasi medi risaontrati
sono viaultati superiori nei soggetti di Arezzo rispetto a quelli
di Fivenze. Sono in oorso altre prove per valutare I ' eventuate
aorrelasione di queato reperto con lo stato di salute dei neonati,,
ABSTRACT
The chlorinated inaectioideo present in samples of blood
taken from puerperae and their children were investigated and
identified. The samples were taken before the infants had been
given their first breast or bottle feed, so as to be sure of
titrating only those chlorinated insecticides which had reached
the child from the mother, by totally excluding the introduction
of these substances in the period immediately following birth.
The titrations were carried out on the blood, since this -£8
the vehicle by which the insecticides reach the various tissues.
-------
863
The blood samples were taken at the Obstetrical Clinic of
the University of Florence and in the pediatrics department of
the Arezzo Hospital from women of various social categories,
working women and housewives, and from their offspring, some being
healthy children born at term, (Florence) and others born prema-
turely or with some pathological condition (Arezzo). 70 blood
samples were examined in all.
The insecticides were examined by gas chromatography, the
technique being as follows: - the insecticides were solvent-
extracted from the blood, the eluate was purified in an alumina
column, and a gas ohromatography analysis was made with an elec-
tronic capture detector (Strontium 90), All our chromatograms
showed peaks of chlorinated insecticides. In all the samples
pp'DDT was present in quantities exceeding 0.6 ppm and also DDD
and DDE. The other insecticides investigated (lindane, hepta-
ahlorine, heptachlorine epoxide, aldrin and dieldrin) were pre-
sent in most of the samples with the exception of heptachlorine
epoxide and dieldrin, which were found only in 4 and 9 samples
respectively.
The chromatograms of the insecticides taken from the blood
of the mothers and their children were similar in quality, but
not in quantity, the counts found in the mothers' blood being in
each case higher than those for the children.
This shows that to some extent at least the insecticides in
the mother's blood pass into the child's blood during pregnancy.
The average count of nearly all the ineeotioidee, in the
mothers as well as in the children, were higher for the subjects
from Arezzo than for those from Florence. Further tests are in
hand to see whether these results can be correlated with the
state of health of the newborn children.
-------
864
1. INTRODU2IONE
L'ormai accertata diffusissima presenza degli insetticidi cloru-
rati nell'ambiente (Grasso /1-2/)» e cons eguent entente anche in tutti gli
aliment! utilizzati dall'uomo (Grasso e coll., Mazzetti e coll. /3-4-5-6-£^,
ha portato a ricercare e titolare le quantita di tali insetticidi che 1'uo-
mo adulto accumula nel proprio organismo durante la propria vita (Grasso e
coll. /o/). Si dispone ormai di numerosi dati largamente sufficient! a di-
mostrare che queste sostanze si depositano in quantita apprezzabili nell'-
organiemo umano, soprattutto nel tessuto adiposo.
Si era eupposto da tempo che una certa quantita di insetticidi
clorurati potesse anche passare dalla madre al figlio durante il periodo
della gravidanza e fosse percio1 reperibile nei tessuti del neonato. Questa
ipotesi e stata controllata sia sugli animali da esperimento sia diretta-
mente sull'uomo, anche con lo scopo di indagare sugli eventual! effetti no-
civi che queste sostanze potrebbero produrre in tali circostanze.
II passaggio placentare degli insetticidi clorurati fu dapprima
dimostrato BU cani, eonigli, topi (Pinnegan e coll., Backstrom e coll.,
Mohn e coll. /9-10-11/). Successivamente furono eseguite ricerche anche
sull'uomo: furono analizzati tessuti di neonati di pochi mesi (Piserova
Bergerova e coll., Abbot e coll., Casarett e coll. /^2-13-1^7) ed in essi
furono reperiti insetticidi clorurati, a conferma di quanto osservato negli
animali.
In seguito furono condotte ricerche piu accurate su neonati mor—
ti subito dopo la nascita, confrontando i titoli di insetticidi clorurati
in essi rinvenuti con quelli dimostrati nella popolazione adulta vivente
nello stesso ambiente e, in alcuni casi, nelle rispettive madri. Zavon e
coll. ^15/1 Gravibesen e coll. /1^7, Charley e coll. /l^/ seguendo questo
indirizzo esaminarono i piu diversi organi e tessuti tra cui il sangue del
cordone ombelicale. Selby e coll. /IB/ ricercarono gli insetticidi cloru-
rati tanto nel sangue matemo quanto nel tessuto placentare delle stesse
donne.
I risultati di tutte le suddette ricerche concordano nell'indica-
re la possibility del passaggio placentare di tutti gli insetticidi cloru-
rati, dimostrando la presenza nei neonati di tali sostanze in quantiti
eguali o di poco inferiori a quelle riscontrate nella popolazione adulta
-------
865
della zona o addirittura nelle rispettive madri.
La nostra ricerca, traendo spunto dalle precedent!, si proponeva
di indagare nella maniera piu precisa e piu attendibile possibile il pas-
saggio degli insetticidi clorurati dalle madri ai rispettivi figli durarrte
la gravidanza. A questo scopo si e proceduto alia titolazione di tali sostan
ze sia nel sangue prelevato dalle puerpere ohe in quello prelevato dai
rispettivi figli, sani o portatori di una qualsiasi patologia, prima che
essi avessero assunto il primo pasto, natvirale o artificiale che fosse.
La metodica adottata, al contrario di quelle utilizzate nella
maggior parte delle ricerche precedent!, consente di titolare aicuramente
soltanto gli insetticidi clorurati pervenuti al figlio dalla madre, eeclu-
dendo totaibmente I1 influenza di qualunque apporto di tali sostanze nel pe-
riodo successivo alia nascita*
2. MATERIALS E METODO
Per eseguire le titolazioni programmate secondo i criteri sopra
esposti, e stato scelto come materiale da esaminare il sangue, sia in base
a considerazioni pratiche relative alia semplicita del prelievo, trattando-
si di bambini vivi, sia in base a oonsiderazioni relative al fatto che gli
insetticidi clorurati pervenuti nell'organismo, usufruiscono del sangue co-
me via di trasporto ai tessuti.
I campioni di sangue sono stati prelevati nelle provincie di
Arezzo e di Pirenze, da donne di varia estrazione sociale, domicilio, lavo—
ro, comunque tutte presumibilmente esposte allo stesso rischio di assunv
zione di insetticidi clorurati, e dai rispettivi figli, del quali alcuni
nati a termine ed altri prematuri. Tutti i neonati di Arezzo erano affetti
da qualche forma patologioa (ittero, broncopolmonite, emorragia intracra-
nica), mentre tutti quelli di Firenze erano sani al momento della nascita.
I campioni di sangue sono stati prelevati presso la Clinica Oste-
trica e Ginecologica dell'Uhiversita di Firenze e presso il Reparto di Pe-
diatria dell'Oepedale di Arezzo, ove i Primari e tutto il personale, medi-
co e non medico, ci hanno fornito una preziosa collaborazione.
La titolazione degli insetticidi clorurati e stata eaeguita per
mezzo di gas-cromatografia con detector a cattura di elettroni.
-------
866
Sono stati ricercati e titolati i eeguenti insetticidi: pp'DDT,
DDE, DDD, Lindano, Eptacloro, Eptacloro Epossido, Aldrin, Dieldrin.
II metodo utilizzato per 1'analisi gas-cromatografica e il se-
guente. I campion! di sangue sono stati trattati con EDTA per evitarne la
coagulazione; ml 1 del compione e stato messo in un irobuto separatore da
ml 10, ad esso sono stati aggiunti ml 3 di una miscela acetone-etere etili-
co 1:1, si 6 agitato il tutto per un minuto e ei sono lasciate separate le
due faai; si e poi ripresa la faee etere-acetone e tale operazione e stata
ripetuta tre volte. Gli estratti sono stati riuniti e mess! ad evaporare
fino a complete essiccamento; il residue e stato ripreso con ml 2 di ben-
zol o, passato in colonna di allumina ed eluito con ml 75 di benzolo. L'elua
to, evaporate fino a ml 1, 6 stato iniettato nel gas-cromatografo con si—
ringa Hamilton in quantita tali da poter evidenziare gli insetticidi pre-
sent!. II gas-cromatografo usato fe un RSCo con detector a cattura di elet-
troni (stronzio 9°)i c°n colonna di vetro (lunghezza m. 1,90| diametro in—
terno mm. 4t fase stazionaria QP 15$ su DC 200) e con registratore Honey-
well Electronik 0,1 mV. Le temperature di esercizio sono le seguenti:
iniettore 220°C, colonna 210°C, detector 22O"C. Gas di trasporto: azoto PP;
flusso 120 ml/min.
Solvent! usati: acetone, etere etilico, benzolo per spettrofoto—
metria ridistillati.
Tutti i nostri cromatogrammi hanno presentato picchi di ineetti—
cidi clorurati.
In precedenza avevamo fatte soluzioni standards dei diversi prin—
cipi attivi pur! al 99»9$ in beneolo puro per spettrografia, ridistillato.
f\
Dalla soluzione 1.10** g/ml, eseguita per pesata, si perveniva, per euocea-.
/J rj
sive diluizioni, alle conoentrazioni 1.10 e 1.10~' g/1. Di queste solu-
zioni e stata fatta una serie di iniezioni con siringa Hamilton da 1 a 10
ul e se ne e mieurata 1'altezza in mm. dei corrispondenti picchi.
In un grafico in cui in ascisse sono riportate le quantita iniet-
tate in /ul e in ordinate 1'altezza dei picchi dei cromatogrammi dei cam-
pion! di sangue con quell! delle soluzioni standards e applicata la formu-
la proposta da Hartman per la determinazione quantitativa:
V w h2
concentrazione in ppm
W v hi
-------
867
in cui V - volume estratto in ml
w » peso della soluzione standard in ng
v * volume dell'estratto iniettato in ml
H • peso del campione in g
M • altezza in mm del picco della soluzione standard
h2 • altezza in mm del picco del sangue
3. RI5ULTATI
I risultati delle titolazioni eseguite sono riportati in tab* 1.
I dati qui esaminati si riferiscono a campioni di sangue prelevati da 21
neonati fiorentini privi di manifestazioni patologiche e dalle rispettive
madri; e inoltre da 14 neonati aretini portatori di qualche manifestazione
patologica e dalle rispettive madri• Si tratta quindi, complessivamente,
di ?0 campioni di sangue.
L'osservazione dei dati riportati in tab. 1 consente le seguenti
oonsiderazioni principali:
1. In tutti i 70 campioni di sangue esaminati e presents il
pp'DDT in quantita relativamente elevate, fino ad oltre
0,6 ppm; anche il DDD e il DDE sono presenti in tutti i cam-
pioni, per quanto in ciascun caso in quantita notevolmente
inferior! a quelli del pp'DDT, ma senza consistent! diffe-
renze tra di loro.
2. Degli altri insetticidi clorurati ricercati, il Lindano,
1'Eptacloro e 1'Aldrin sono present! nella maggioranza dei
campioni di sangue, con frequenza e con concentrazioni decre
scenti nell'ordine sopra indicate; 1'Sptaoloro Epossido e il
Dieldrin, metabolit! rispettivamente dell1 Efctacloro e dell'-
Aldrin, sono al contrario present! solo in un numero estrema-
mente esiguo di campioni (rispettivamente 4 e 9)•
3* I risultati appaiono sostanzialmente simili nel gruppo delle
madri e in quello dei neonati* Per quanto nella maggioranza
de! casi tanto i valor! singoli che quell! medi riscontrati
nelle madri (tab. 2) siano di poco superior! a quell! dei fi-
gli, tali difference dimostrano infatti ben di rado una si-
gnificativita statistica (tab. 3): soltanto in un caso
-------
TABELLA 1.
Taasi di inaetticidi clomrati riscontrati su campion! di sangue di madri e di rispettivi
neonati delle provincie di Arezzo e Firenze,
Prove-
nienza
x
J
4
5
FI
X
X
X
X
X
X
X
X
X
X
AR
-
Categoria
Ma-
dri
X
X
X
X
X
Pigli
non
Pat.
X
X
X
X
X
Pat.
Tassi ematici ppm 10
pp'DDT
3.470
3.810
10.000
3.300
6.110
6.650
3.290
7.410
4.420
9.610
DDE
611
515
109
513
115
763
398
773
954
619
DDD
343
750
850
312
375
550
449
899
500
608
Lindano
121
113
246
-
779
974
168
196
137
784
Epta-
cloro
1.230
1.160
597
119
652
583
714
574
510
838
Eptacloro
Ep.
-
-
-
835
_
-
-
-
-
-
Aldrin
tracce
tracce
tracce
tracce
tracce
tracce
tracce
tracce
tracce
Dieldrin
-
-
-
-
-
-
-
-
-
-
CO
a*
CO
-------
TABELLA 1. (Seguito)
6
7
e
o
10
11
Prove-
nienza
FI
X
X
X
X
X
X
X
X
X
X
X
X
AR
Gategoria
Ka-
dri
X
X
X
X
X
X
Pigli
non
Pat.
X
X
X
X
X
X
Pat.
Tassi ematici ppm 10 •*
pp*DDT
5.000
6.300
6.530
6.300
1.500
4.400
4.800
4.400
1.600
1.600
2.330
4.000
DIE
716
482
750
438
839
312
575
475
409
287
359
196
DUD
388
515
611
343
662
272
391
501
200
280
266
280
Lindano
1.220
459
186
156
1.750
2.300
6.920
480
1.410
162
528
337
Epta-
cloro
1.080
691
652
521
1.500
892
550
450
-
-
804
-
Eptacloro
Ep.
-
-
-
-
-
942
-
-
-
-
-
-
Aldrin
tracce
tracce
tracce
tracce
119
85
71
450
28
26
73
42
Dieldrin
-
-
-
-
-
-
-
-
-
-
-
-
oo
-------
TABELLA 1. (Seguito)
Prove-
nionza
19
13
14
15
16
17
PI
X
X
X
X
X
X
X
X
X
X
X
X
AR
Categoria
Xa-
dri
X
X
X
X
X
X
Pigli
non
Pat.
X
X
. X
X
X
X
Pat.
Tassi ematici ppm 10 "^
pp'DDT
2.500
1.830
2.000
1.830
847
847
1.700
1.000
2.450
2.350
2.000
7.500
DUE
441
233
315
378
227
227
400
183
406
362
306
125
DDD
366
233
433
400
266
266
235
205
324
388
272
181
Lindano
4.660
112
450
574
1.080
1.820
297
106
894
142
257
138
Epta-
cloro
687
-
-
-
1.310
847
709
554
1.560
550
-
-
Eptacloro
Ep.
-
-
-
-
-
-
-
_
-
-
-
-
Aldrin
773
-
71
140
99
132
425
300
206
47
-
-
Dieldrin
-
-
-
-
-
-
-
-
-
-
-
-
GO
vj
o
-------
1. (Seguito)
Prove-
nienza
1ft
';'
9O
9\
O
PI
X
X
X
X
X
X
X
X
AR
X
X
X
1
Cat ego ri a
Ka-
dri
X
X
X
X
X
X
Pigli
non
Fat.
X
X
X
X
Pat.
X
X
Tassi eoati-ci ppm 10 "^
pp'DDT
1.000
2.330
1.660
2.500
2.900
3.540
2.940
13.900
44.300
16.400
30.100
61.900
DDE
312
375
566
383
375
312
541
879
4.840
3.860
3.230
4.760
HUD
227
357
250
285
400
300
231
833
1.510
333
616
2.840
Lindano
185
608
425
398
659
411
381
165
720
1.000
930
312
Epta-
oloro
787
206
1.510
1.450
1.310
800
854
449
1.650
630
4.520
870
Eptacloro
Ep.
-
-
-
-
-
-
-
-
-
-
-
-
lldrin
418
134
115
862
67
48
595
-
120
210
240
87
Dieldrin
-
-
-
-
-
-
657
-
-
-
_
-
-------
TABELLA 1. (Seguito)
Prove-
nienza
f\M
24
25
26
27
28
29
PI
AH
X
X
X
X
X
X
X
X
X
X
X
X
Categoria
Ka-
dri
X
X
X
X
X
X
Pigli
non
Pat.
Pat.
X
X
X
X
X
X
Tassi ematici ppm 10
pp'DDT
11.100
7.500
7.140
4*400
3.170
9.440
3.330
8.330
2.820
7.770
3.900
9.760
DIE
1.300
1.080
866
1.540
392
730
459
3.490
439
3.120
609
2.880
DDD
857
840
571
400
347
400
282
923
391
300
400
789
Linda.no
8.580
650
4.700
780
418
675
156
160
4.600
5.260
3.710
375
Epta-
cloro
1.940
1.000
6.220
1.100
600
893
372
3.870
647
175
941
675
Eptacloro
Ep.
-
-
-
-
-
-
-
-
-
-
-
-
Aldrin
122
140
122
100
-
tracce
50
370
tracce
tracce
71
362
Dieldrin
348
208
370
375
-
-
-
-
-
-
294
-
00
^J
to
-------
TABKT.T.A 1. (Seguito)
30
31
32
33
34 .
35
Prove-
nienza
PI
AR
X
X
X
X
X
X
X
X
X
X
X
X
Categoria
Ka-
dri
X
X
X
X
X
X
Piffli
non
Pat.
Pat*
X
X
X
X
X
X
Tassi emati-ci ppm 10 •*
pp'DDT
36*600
34.700
30.300
7.070
46.000
31.000
9.130
11.300
12.600
7.540
5.710
20.750 ,
HIE
4.500
5.660
4.070
5.160
4.600
4.170
714
656
1.710
2.660
452
1.490
DDD
760
6.250
2.400
125
5.010
710
500
»
454
1.770
280
400
710
Lindano
2.280
2.590
7.690
2.080
2.030
1.160
592
141
1.270
2.320
192
96
Epta-
cloro
3.650
3.110
3.510
2.830
2.960
1.500
800
200
4.090
371
404
120
Eptacloro
Ep.
-
—
-
-
-
1.500
—
-
1.010
-
-
-
Aldrin
—
203
tracce
176
tracce
2.300
tracce
tracce
530
223
—
tracce
Dieldrin
-
-
—
250
-
280
-
-
2.150
-
-
-
CO
-J
u>
-------
874
TABELLA 2
Valori medi relativi ai tassi del singoli ineetticidi clorurati riscon-
trati in campion! di sangue di madri e dei rispettivi neonati delle
provincie di Arezzo e Pirenze
i
Insetticida
l pp'DDT
DDE
DDD
LIND.UTO
EPT..CLORO
EPTACLORO EP.
ALDRI1I
DIELDHIN
Valori medi su ppra 10
Campioni di Firenze(n.2l)^
Uadri
3.287^.210
463+ 222
382* 163
1. 083-d. 665
810+ 376
-
145* 219
31 + 143
Neonati
4.543+3.S60
420+ 204
417+ 206
496+ 581
508* 409
84+ 267
107* 207
-
Campioni di Arezzo (n.14)
Uadri
17.514+16.^20
2.012+ 1.800
1.129+ 1.281
2.704+ 2.778
2.307+ 1.7P2
72+ 270
89+ 146
225+ 571
Neonati
17.030+15.900
2.946+ 1.660
1.006+ 1.622
1.252+ 1.388
1.239+ 1.185
79+ 390
262+ 589
79+ 137
TABELLA 3
Significativita statiatica delle difference dei valori medi di inaettioidi
cloruxuti riscontrati su campioni di sangue di madri e dei rispettivi neo-
nati delle provincie di Arezzo e Pirenze
Insetticida
Pirenze
Tladri
Arezzo
Madri
Neonati
ArezzoJ Firenze
Lladri
pp'DDT
0,027-t>0,01
0,02>t>0,01
DDE
0,02>t>0,01
0,001>t
0,02 > t>0,01
DDD
0,10>t>0,05
0,10> t>0,05
LUTDuL'O
0,20>t>0,10
0,20>t>0,lO
0,20> t>0,10
EPTACLORO
0,001>t
0,02>t>0,01
o,05> t > 0,025
EPT^CLORO EP.
0,30> t>0,25
ALD2IN
0, 60. -'t?- 0,50
0,001>t
0,503*
DIELDRIir
0,60 *
0,30>t>0,20
0,20 ^ t>-0,10
-------
875
(coppie di Firenze, Eptacloro) la significative,ta statistica
supera il valore di t - 0,001; in quattro casi poi (coppie di
Pirenze: pp'DDT; coppie di Arezzo: Lindano, Eptacloro e DDE)
il valore di 7 e compreso fra 0,02 e 0,01.
4. Pressoche per tutti gli insetticidi, e tanto nel gruppo delle
madri che in quello dei figli, i tassi medi riscontrati sono
risultati superiori nei soggetti di Arezzo che in quelli di
Firenze (tab. 2); tuttavia una concreta significativita sta-
tistica della differenza dei valori medi (t < 0.001) ha potu-
to essere evidenziata, per quanto riguarda i neonati, solo
per il DDE e 1'Aldrin; per quanto, invece, riguarda le madri
il valore di t e il risultato inferiore a 0.001 soltanto per
il pp'DDT (tab. 3).
5. I neonati aretini presentano i quadri patologioi piu vari e
non 6 pertanto possibile, dato il basso numero di osservazio-
ni, tentare di correlare i tassi ematici degli insetticidi
clorurati con le manifestazioni morbose dei neionati. Anche
la ricerca di un eventuale rapporto tra taeso ematico di in-
setticidi clorurati e peso alia nascita {ipotesi giustificata
da talune osservazioni sperimentali relative all'azione di
tali sostanze sul netabolismo del Ca e quindi presumibilmente
nel processo di ossificazione) non conduce ad alcun risultato
concreto per analoghi motivi.
In conclusione, le rioerche in questa sede riferite dimostrano
con sicurezza la costante presenza di inaetticidi clorurati nel sangue di
neonati prima dell'assunzione del prime pasto e sembrano pertanto convali-
dare eenza riserve, data la sicura esclusione di un'origine alimentare
post-natale dei suddetti insetticidi, la possibility del pasuaggio per via
placentare di tali sostanze, che in effetti si trovano costantemente anche
nel sangue materno.
II reperimento di tasai ematici piu elevati di insetticidi cloru-
rati in neonati portatori di qualche manifestazione patologica e generati
da madri ease pure presentanti tassi di tali sostanze relativaraente maggio-
ri lascia aperta I1ipotesi di una eventuale correlazione fra tali eventi
(elevato livello ematico materno—> elevato livello ematico neonatale —>
presenza di patologia neonatale).
-------
876
Tuttavia il fatto che cio' si sia verificato esclusivamente nei
campion! di sangue provenienti da una sola delle due circoscrizioni terri-
torial! prese in esame lascia adito a talune perplessita. Lreventuale
esistenza di una tale correlazione richiede quindi un piu approfondito con—
trollo, sulla base dell'analisi di un maggior numero di campion!, di un piu
chiaro riferimento a talune situazioni patologiche neonatali e d! una piu
estesa e piu precisa distrlbuzione topografica dei campion! prelevati.
BIBLIOGRAFIA
1) GRASSO C., BERNARDI G.t MARIOTTINI E.: Ann. San. Pubbl., 2gf 453, 1968.
2) GRASSO C., BERNARDI 0., MARIOTTINI E.: Ann. San. Pubbl., £g, 1029, 1968.
3) GRASSO C.: Atti del Convegno sugli insettioidi. Firenze 24-25 novembre
1962.
4) MAZZETTI G., GIACHETTI A., GRASSO C.: Agricoltura n. 12, 1968.
5) GRASSO C.: Atti III Convegno sulla Qualita. Perugia 25-27 maggio 1964.
6) GRASSO C.( LANCIOTTI E., BERNARDI G.s n. Ann. Ig. Microb., 22, 434, 1971.
7) GRASSO CM BERNARDI G.t BARONTI L.: N. Ann. Ig. Microb., 23_, 29, 1972.
8) GRASSO C., BERNARDI G., MARIOTTINI E.: Ann. San. Pubbl., 23, 1593, 1968.
9) PINNEGAN J.K., HAAO H.B., LARSSON P.S.I Proc. Soc. Exp. Biol. Med.f
I£, 357, 1949.
1C) BACKSTROM J., HANSSON E., UlLBERG S.: Toxic, Appl. Pharmacol.,
1, 90, 1965-
11) MOHN M.H., WILSON R.A., ISE G.H.: Pish. Wildlife Giro. (167), 47, 1963.
12) PISEROVA BERGEROVA V., RADDMSKI J.L., DAVIES J.E., DAVIS J.H.,:
J. Ihdustr. Med. Surg., 36, 65, 1967.
13) ABBOT D.C., GOULDING R., TATTON J.O.: Brit. Med. J.f 3_ (5611), 146, 1968.
14) CASARETT L.J., PYER O.C., YANGER W.L.Jr., KLAMMER H.W.: Arch. Environ.
Health, Jj[f 306, 1968.
15) ZAVON M.R., TYE R., LATORRE R.: Ann. New York Acad. Science, 160. 196, 196q
16) GRAVIBESEN C.( CHESEL I., UNTERMANN H.W.: Obetet. Gynecol., 19, 161, 1971
1971. .
17) CURLEY A., COPELAND M.FM KIMBROUGH R.D.: Arch. Environ Health,
J2, 628, 1969.
18) SELBY L.A., NEWELL K.M., HAWSER G.A., JUNKER G.: Environ Res.,
2, 247, 1969.
-------
877
DISCUSSIONE
ROSIVAL (Cecoslovacchia)
Dai risultati delle nostre ricerche (Rosival, SzAkolay,
Uhn6k) emerge una prevalenza del DDT totale sul BHC totals a
livello dell'utero nel secondo e terzo mese.di gravidanza, ed
inoltre una concentrazione di DDT totale e dl isomeri $ e y di
BHC piu elevata inregioni pianeggianti che in region! di media
montagna. II tasso del DDT 6 pixl elevato di quello del DDE
(il livello medio del DDT in regioni pianeggianti 6 di 0/128
ppm; in regioni di media-montagna 6 di 0,028 ppm> il livello
raedio di DDE in regioni pianeggianti 6 di 0,046 pr™, in regioni
di media-montagna di 0,OO6 ppm).
Cio1 denota una diversity nella capacity di penetrazione
dei residui nel caso del latte materno in rapporto alia costanza
della correlazione fra DDT e DDE nel grasso umano, ed alia pos-
sibilita che la placenta svolga una funzione d'organo metabolico
attivo.
La prevalenza del DDT sul DDE,indica sia breve durata all1
esposizione che material! a basso tenore in lipidi.
WASSERMAN (Israele)
I composti OCI e PCB attraversano la placenta e sul piano
qualitative li troviamo nei tessuti del neonato come pure nelle
varie fasi della vita intrauterina. La piu elevata esposizione
all'OCI e PCB si verifica quando il neonato viene alimentato
unicamente con latte materno, poiche" quest'ultimo contiene ali-
quote piu elevate di composti OCC che non 11 latte vaccino.
I dati forniti dalla D.ssa Grasso meritanto attenzione vista
iHmportanza di considerare le donne incinta come indice dell'
esposizione tossica ambientale.
DANIEL (Gran Bretagna)
Ha la D.ssa Grasso costruito altri parametri per spiegare
le osservazioni relative ai neonati di Arezzo?
Erano le donne dei due gruppi della medesima condizione
sociale? Avevano esse il medesirao tenore alimentare? Nei gruppi
considerati vi erano delle fumatrici?
E1 forse premature attribuire gli effetti unicamente ai
livelli di insetticidi clorurati present! nel sangue materno.
-------
878
GRASSO (Italia)
Le condizioni delle donne del due gruppi erano le piu'
simili possibili.
Non ho attribuito gli effetti agli insetticidi clorurati
reperiti: ho detto che gli studi proseguono per approfondire
1'osservazione fatta.
-------
879
MODIFICATION OF THE HOMOLOG AND ISOMER COMPOSITION
OF A POLYCHLORINATED BIPHENYL MIXTURE DURING
PASSAGE THROUGH TWO BIOLOGICAL SYSTEMS
B, BUSH/ F, D, BAKER/ C, E, TUMASONIS/ FA-CHUN LO
AND C, L. HOUCK
Division of Laboratories and Research, New York State Department
of Health, New Scotland Avenue, Albany, NY, USA
ABSTRACT
The polychlorinated biphenyl (PCB) mixture Aroclor 1254 was
administered to breeding populations of hene and vats at 6 mg/kg
body weight for 6 Desks, The populations Here observed then and
during the subsequent 20-week clearance period. Egg yolks and
tissues of adults, embryos and fetuses, chicks and pups were ana-
lyzed; and the fate of 16 individual homologs and iaomers of
Aroclor 1254 was observed during the build-up and decline periods.
The toxicity of the PCB mixture in the hen can be attributed ei-
ther to a metabolite of 3,4,2*,3',6'-pentaahlorobiphenyl or to
the intrinsic toxicity of the more persistent 3,4,2 ',4 ',5 '-penta-
chlorobiphenyl or 2f3f4,2',4'i5t-hexachlorobiphenyl. In the rat*
however, both of these pentachlorobiphenyla were eliminated rela-
tively vapidly. Hence 4,4'- substitution is move important in
determining persistence in the chicken than in the rat.
Analyses of rat fetuses showed that there was little placen-
tal transfer of PCB. However, PCB was passed from mothers to
pups as soon as suckling began. By contrast, the chick embryo
was exposed to PCB present in egg yolk from its earliest develop-
ment.
-------
880
Analyses of geese and duck collected in New York State con-
firmed results of laboratory studies with regard to PCB persis-
tence. The laboratory results indicate that the concentrations
of PCB in eggs of some wild birds, which as reported by other
workers are frequently greater than 10 ,ug/gt may quite possibly
be a factor in the reduced reproductive success of these species,
-------
881
1. Introduction
The toxicity of commercially produced polychlorinated biphenyl (PCB)
mixtures has been fairly well demonstrated and reviewed in recent years
(e.g., Fishbein [17, Hammond et al. [2."] ). The toxicity of the mixtures
themselves and of several components of typical mixtures has been
determined in a wide variety of living organisms. In long-term low-level
experiments, the concentrations of PCB in various tissues of populations of
animals have been determined. In short-term experiments, metabolic products
of several individual PCBs have been isolated and characterized. In some
studies with commercial mixtures, metabolic changes in composition of the
ingested mixture have been noted) both changes with time and differences
between organs in the same animal have been observed. A more thorough
investigation of such changes was one of the primary aims of the present
work.
The toxicity of PCB mixtures first drew wide attention in the poultry
industry because newly hatched chicks are highly susceptible to the
compounds* Since this rediscovery (the toxicity had been clearly
demonstrated in 1937 by the U.S. Surgeon General CSee 2j), residues of PCB
found in wildlife have been blamed for reduction in the reproductive
success of various species of birds and of some carnivorous mammals, e.g.,
otters and mink. Another aim of the present work, therefore, was to
investigate the effect of a low-level intake of a common PCB mixture
(Aroclor 1254) on the young of an avian and a mammalian species.
2. Experimental
Aroclor 1254, kindly donated by Monsanto Chemicals, St. Louis, Mo.,
was dissolved in the detergent Tween 80 (Sigma Chemical Co., St. Louis,
Mo.) and then emulsified with water. The concentration was adjusted to
give the animals a daily intake of 6 mg/kg body weight.
Twelve white Leghorn hens and two roosters were used for the first
experiment. Details of the experiment and some conclusions have been
reported by Tumasonis et al. [31 and Bush et al. [4] . Wistar rats (125
male, 125 female) were used in the second experiment.
Extraction of PCB from tissue samples and from eggs was carried out
by the usual methods (e.g., Bush and Lo [5] ). Possible residues of DDT-
related insecticides derived from animal feed were eliminated by
oxidative clean-up with chromic acid in acetic acid.
Analysis was carried out with 7600A Chromatographic System (Hewlett-
Packard, Avondale, Pa.) using electron capture detection* The stationary
phase was Apiezon L (2# on Gaschrom Q, 80*100 mesh), and the areas of the
eighteen peaks, as identified by Sissons and Welti f6,7Jfwere integrated.
For quantisation, the area of each peak was divided by the area of the
corresponding peak in the chromatogram produced by a known mass of Aroclor
1254. This ratio permitted a rough quantitatlon of the peak in pg/g* We.
have called each such value the notional concentration of the PCB compo- .
nent in the tissue extract. The mean of these notional concentrations
gives an estimate of the total mass of PCS present in the tissue* The
notional concentrations and their means were displayed as logarithmic bar
charts using the Wang Programmable Calculator and Data Plotter (Wang Inc.,
Tewksbury, Mass.). An unmodified residue would give a set of 19 equal
-------
882
bars (18 peaks plus the mean). The value of each mean is printed above it.
3. Results and Discussion
3.1 The hen experiment
Embryonic mortality reached 100% in eggs laid by hens which had been
exposed to the PCB intake for 3 weeks. Exposure was terminated 6 weeks
after the commencement of the experiment. Chicks started to hatch again
from eggs laid in the 15th week of the experiment, i.e., 9 weeks after
termination of intake.
100.0
i IOO.OJ- CHICK, 5 DAYS
-------
883
an exposure terminated not more than 4 or 5 months earlier (liver
composition and egg composition are highly correlated, Bush et al. C-O ).
Moreover, such a residue would be more toxic to embryos than an older
residue containing relatively more of the hsxa- and heptachlorobiphenyls.
IO
<\J
X «
o
I
4 • 12
WEEK
Figure 2. Change in the ratio of the notional concentration of peaks
12:9 in egg yolks with time.
Extrapolation to avian wildlife in general to indicate the absolute
potency of a particular residue burden would not be valid. The 12t9 ratio
might, however, provide a valuable indication of the age of a residue as
one parameter when interpreting the data from surveys of wildlife
populations.
3.2 Wildlife survey
In a limited survey of avian wildlife in New York State, two species
of duck have been found to be most contaminated by PCBi the greater
scaup ( Avthya marila nearctica) and the white-winged scoter (Melanitta
ij. T
deqlandij. The 17 samples of scaup had a mean level of 5 ± 7
(mean ± SD) in the liver, with values ranging from zero to 32 (ig/g.
Eight samples of scoter had a mean breast-muscle level of 7 ± 67
with values ranging from 1.5 to 50
Figure 1 shows the residue patterns of three samples of duck liver.
All show contamination by PCB mixtures other than Aroclor 1254, as
evidenced by the high relative concentration of some of the later peaks
and, in the second scoter sample, of several early peaks also. However,
there is a distinct resemblance between these patterns and those of hens
contaminated with Aroclor 1254. The ratios of peaks 12»9 in the duck
samples are 6, 2 and 10 respectively, indicating that the first two
probably result from relatively fresh exposure. Thus the mean notional
concentration of 14 ug/g in the first scoter sample may be more embryo-
toxic than the mean of 50 ng/g in the second sample.
3.3 The rat experiment
The reproductive success of the rats was not affected by the level of
PCB intake in this experiment. Intake was terminated after 9 weeks, and
the breeding population was observed for a further 16 weeks. Analysis of
the tissues of adults, fetuses and pups (both sucklings and weanlings)
showed a marked difference in elimination efficiency when compared to the
hen. (A detailed report on this study is in preparation.)
-------
884
Figure 3 shows typical PCB patterns after 3 weeks of exposure and 4
weeks after termination of exposure, i.e., in week 13. Peak 12 does not
persist, and only material eluting after that peak could be implicated in
any biological effect on the rat. Two liver microsomal enzymes, aniline
hydroxylase and cytochrome P450, were significantly activated by the
exposure.
WEEK 9
IOO.O- RAT, ADULT FEMALE : LIVER
WEEK 13
IOO.O - RAT, AWIT FEMALE : LIVE!
_ FETUS,FEMALE: CARCASS (COMPLETE)
i LL .ii.il Li..
12 19 18
MEAN
Figure 3.
MEAN
PEAK
PCB residue patterns in rats after 3 weeks of intake of Aroclor
1254 and 4 weeks after termination of intake.
The major components of Aroclor 1254 are the components of peaks 9,
12, 13 and 14 (Bush et al. [4] ) and of these, only peaks 13 and 14 remain
in the rat in the 13th week. These represent 3,4,2*,3',4'-pentachloro-
biphenyl plus 2,4,5,2',4',5'-hexachlorobiphenyl (peak 13) and 2,3,4,2',4',
5'-hexachlorobiphenyl (Sissons and Welti [6,7J ). It is probable that the
first constituent of peak 13 is eliminated because it contains the same
substitution pattern on one ring as peak 12; hence the two main components
of the residue would have the substitution pattern 2,4,5, and both have
4,4'-substitution. The minimal effect of the PCB intake on the rat and
its young may be due to the rat's ability to eliminate all but these two
compounds, which may themselves be relatively innocuous.
Analysis of the fetus, however, showed that the lack of effect on
fetal viability is probably best explained by protection of the developing
fetus from PCB contamination by the placenta. The mean PCB concentration
in fetuses was 2 ng/g, in sucklings 50 p,g/g and in weanlings 20 ng/g
during the experiment. Comparison of these levels with the concentrations
in the mothers at the same time (e.g. Figure 4) indicates that little PCB
reached the fetus. Only the sucklings became contaminated, and by that
stage any possible phase of development which was sensitive to attack by
PCB must have passed.
4. Conclusions
The work reported here indicates that there are subtle differences in
metabolic efficiency for pentachlorobiphenyls with differing substitution
patterns between.a mammalian and an avian system. It suggests also the
importance of the placenta in protecting the fetus from lipophilic agents
such as PCBs. It offers the possibility of evaluating the importance of
PCB residues of unknown origin found in wildlife specimens by comparison
with model laboratory systems.
The levels reported by other workers in a variety of avian species,
if they reflect mixture patterns similar to those reported here, may pose
a threat to the early stages of development of the species and hence to
their reproductive success.
-------
885
REFERENCES.
FISHBEIN, L., "Chromatographic and biological aspects of polychlorinated
biphenyls", J. Chromatogr. 68, 345 (1972).
HAMMOND, P. B., NISBET I. C. T., SAROFIM, A. F., DRURY, W. H», NELSON, W.
RALL, D. P., "Polychlorinated biphenyls - Environmental Impact",
Environ. Res., 5, 249 (1972),
TUMA90NIS, C. F., BUSH, B., BAKER, F. D., "PCS levels in egg yolks
associated with embryonic mortality and deformity of hatched chicks",
Arch. Environ. Contain. Toxicol., 1, 312 (1973).
BUSH, B., TUMASONIS, C. F., BAKER, D. F., "Toxicity and persistence of
PCB homokgues and isomers in the avian system", Arch. Environ. Contain.
Toxicol.. (in press).
BUSH, B., LO, FA-CHUN, "Thin layer chromatography for quantitative
polychlorinated biphenyl analysis", J. Chromatogr.. 77, 377 (1973).
SIS90NS, D., WELTI, D., "Structural identification of polychlorinated
biphenyls in commercial mixtures by gas chromatography", J, Chromatoqr.,
60, 15 (1971).
WELTI, D., SISSONS, D., "The proton chemical shifts of polychlorinated
biphenyls", Org. Mag. Resonance, 4, 309 (1972).
DISCUSSION
DANIELSON (Sweden)
1. How precisely do you draw conclusions about relative toxicity
of the various chlorinated biphenyls from your experiments?
2. Does not the technical PUB used in the experiments contain
contaminants?
BUSH (U.S.A.)
1. Embryo-toxicity of the residual PCBs mixture deposited in
the eggs by the hens 9 weeks after the termination of exposure
was more potent than the residue deposited during the build-up
phase of the experiment. Thus 50% embryonic mortality was
caused by approximately SO.mg/g PCB at the beginning and by only
10/mg/g during the clearance period. Toxicity in the hen at the
latter period must be attributed to the components of peaks 12,
13 and 14, these being the only PCB present at this time. The
rat eliminated peak 12, and hence any toxic effects observed
must be attributed to peak 13 and 14 material.
2. The technical PCB used in these studies was free of mat-
erials such as the dioxins to the best of the author's know-
ledge.
-------
887
INFLUENCE DES PESTICIDES ORGANOPHOSPHORES SUR LE
METABOLISME DES GRAISSES NEUTRES CHEZ LE RAT
J, P, BUCHET, R, LAUWERYS ET H, ROELS
Unitfi de Toxicologie Industrielle et MSdicale, UniversitS Catho-
lique de Louvain, Bruxelles, Belgique
RESUME
Le probleme toxicologique pose par I'utilisation sans cesse
ar>ois8ante dee pesticides organophoaphor&s eat I'effet a long
terme d'une exposition continue a de faiblee concentrations, pay
exemple sous la forme de residua alimentaires. Outre I'inhi-
bition den cholineateraaeat I 'inactivation de nombreuses hydro-
loses peut §tre imputes aux eaters organophoaphorea; c'est
notamment le aas d'enzymes importantea impliquees dans le meta-
bolisme des lipides.
Des Etudes effectueee in vitro ont permia la caracterieation,
au moyen d'eatera organophoaphorea, de diveraee activites gly-
c&ridaaiquea dana troia tiaaua du rat (coeurt intestin, et tiaeu
adipeux). Dans le but de verifier si I'inhibition de oes ao-
tivite's enzymatiquea obaerve'e in vitro ae reproduiaait in vivo
et} en mSme temps, d'en examiner lea oonsSquenaea eventuelles,
dee animaux ont ete aoumia (lea unat 3 moia; d'autres, un an)
d un eater organophoaphor£ melange a I'alimentation (normale ou
enrichie de graisae). AprSs sacrifice, diveraes activitea en-
zumatiques (Upases et cholinesteraaea) ont ete determineea;
divera dosagea de eonatituanta des graiaaes neutrea (acidea graa,
glyc£rol, cholesterol) ont en outre ete effeotues.
Cea travaux prSliminairee permettent de mettre clairement
en evidence I'interference d'un eater organophoephor& avec le
-------
888
mgtabolisme des graisses neutres in vivo. A des doses ou auoun
signe clinique d'intoxication ne peut encore Stre observe" t cer-
taines activites lipasiques tissulaires, subissent une reduction,
Cette diminution n'a toutefois pas la m§me importance que celle
subie par les cholinesteraaes, enzymes dont l'activit& reste le
meilleur temoin d'une exposition excessive aux eaters organophos-
phores.
ABSTRACT
The toxicological problem raised by the ever-increasing use
of organo-phoaphorus pesticides is the long-term effect ^f con-
tinued exposure to weak concentrations, for example in the form
of residues in food. Apart from inhibition of the cholinestep-
ase8f the inactivation of numerous hydrolases can be attributed
to organo-phosphorus esters. This is especially the case with
certain important enzymes involved in lipidic metabolism.
Various glyceridase activities were observed in vitro in
three rat tissues (heart, intestine and adipose tissue), using
organo-phoephorus esters. In order to see whether the inhibi-
tion of these enzyme activites which had been observed in vitro
would be reproduced in viyo^ and, at the same time, to examine
the possible consequences, an organo-phoephorus ester was ad~
ministered to certain animals(in some cases for three months,
others for one year), mixed in with their food (normal or fat-
enriched). After they had been killed, various enzyme activi-
ties (Upases and cholinesterases) were determined; various
quantitative analyses of neutral fat constituents (fatty
glycerol, cholesterol) were also carried out.
-------
839
This preliminary work showed clearly that an organo-phospho-
rus eater does in fact interfere with the metabolism of neutral
fata in the living animal. There ie a reduction in certain ac-
tivities of lipaae tissues even with doses which do not give rise
to any clinical signs of poisoning. This reduction in activity,
however, is by no means as important as that suffered by the
cholinesterasest and it is the activity of these enzymes which is
the best indicator of excessive exposure to organo-phosphorus
esters.
-------
890
1. Introduc jdon.
L1action des esters organophosphores sur les esterases agissant prefe-
rentiellement sur les esters aliphatiques simples ou les triglycerides a
courtes chaines d'acides gras a fait 1'objet de nombreuses etudes. Au
contraire, leur action sur les esterases hydrolysant les glycerides a
longues chaines d'acides gras (en d'autres termes les lipases) est encore
peu connue : seule la lipase pancreatique a regu quelque attention.
Dans un premier temps, nos travaux ont eu pour but la caracteriaation
des activites enzymatiques responsables de 1'hydrolyse des graisses neutres
dans les trois tissus du rat (coeur, intestin et tissu adipeux). L'utili-
sation des esters organophosphores nous a permis de mettre en evidence dans
ces trois tissus des activites mono et diglyceridases. Les resultats de
ces travaux ont ete publics [l-^J.
Nous nous sommes orientes dans la suite vers I1etude des activites
triglyceridases de ces tissus. Nous avons constate que les deux activites
responsables de l'hyd"olyse des triglycerides, a savoir la lipase hormono-
sensible (HSL) et la lipoproteine lipase (LPL), pouvaient toutes deux etre
inhibees aussi bien in vitro que in vivo par les esters organophosphores
(resultats non publics). II etait done interessant de verifier 1'effet
sur le metabolisme lipidique d'une exposition continue a de faibles concen-
trations de pesticides organophosphores.
2. Partie experimentale.
Cinquante jours apres la naissance,des rats femelles (souche Sprague-
Dawley) ont ete sounds par groupes de dix pendant un an a quatre types
d1alimentation dont la base est une farine classique : la farine DOS de la
firme U.A.R. {Villemoisson sur Orge, France).
Le regime A est constitue de farine enrichie en matieres grasses par
addition de 20 % d'huile de mais ; le regime B contient en plus 10 ppm de
I1ester organophosphore Triamiphos (T = 0) ou 5~aniino-(bis dimethylamido)
phosphoryl 3~phenyl-1,2,U-triazole ; le regime C est de la farine sans
aucune addition et enfin le regime D, la farine additionnee de 10 ppm de
T = 0. Chaque semaine les animaux ont ete peses et chaque mois du sang a
et6 preleve pour la determination de 1'activite cholinesterasique erythro-
cytaire de fagon a controler 1'absorption de 1'ester organophosphore. Les
figures 1 et 2 montrent les courbes de poids corporel et 1'evolution de i»
activite cholinesterasique des globules rouges durant le traitement. L"
administration de 10 ppm de Triamiphos permet de maintenir un taux d'inhi-
-------
891
400
^300
d
Q
s
"•
§200
CL
REGIME GRAS
*
1
§
§
u
100-
S
50S||
I
U
10
20
30 40
TEMPS (semaines)
Fig. 1 Courbe de poids et activite cholinesterase erythrocytaire des
rats temoins et traites au Triamiphos (regime normal).
PJxposes (— —; \^\] ) ; controles (- - ; i ; ).
400
-300
i '
o
I200
REGIME NORMAL
1
10
20
1
:
,
'
30 40
TEMPS (semaines)
50
X
--
ui
u)
g
U:
Ifl
•
i •
Fig. 2 Courbe de poids et activite cholinesterase erythrocytaire des
rats temoins et traites au Triamiphos (regime gras).
Exposes ( ; i I ) ; controles ( - ; , t } .
-------
892
bition de la cholinesterase du sang total compris entre i»0 et 50 % (par
rapport a 1'activite cholinesterase des aniraaux temoins correspondents,
conside"ree comme 100 %}.
Aucune difference dans 1'augmentation de poids n'est observee entre
le groups traite et celui non traite au pesticide, lorsque le regime est a
base de farine normale. Par centre, lorsque de 1'huile de mais est ajoutee
les animaux soumis au triamiphos ont un poids nettement superieur a celui
des animaux temoins des la 10eme semaine. Cette difference se maintient
pendant toute la periode d1observation et ce malgre I1augmentation de poids
constate'e a partir de la SOeme semaine chez les animaux temoins, soumis au
regime riche en graisse (A) par rapport aux temoins soumis au regime normal
(C).
A la fin de la periode d'exposition, les animaux ont ete sacrifie"s
et las mesures suivantes effectuees :
- activite"s cholinesterases dans les homogenats de coeur, intestin et
cerveau et dans le se"rum et le sang total,
- activite"s mono-, di- et triglyceridaaes (HSL et LPL) dano les homogenate
de coeur, intestin et tissu adipeux,
- contenu en acides gras, glycerol et cholesterol totaux des homogenats
de coeur et d'aorte et concentration en acides gras libres du serum.
L'examen de ces resultats, permet de degager les tendances suivantes:
- apres un an de regime riche en graisses ou non, certaines activites
lipases ont subi une reduction de 1'ordre de 10 a 20 % sous 1'influence
de 10 ppm de triamiphos (la difference n'est toutefois statistiquement
significative avec les deux regimes, qu'en ce qui concerne la monogly-
c^ridase du coeur),
- les activites cholinesterases subissent de plus importantes reductions et
cheque fois significatives au seuil de 5 % ' ± **0 % pour le coeur et le
sang total, + 25 % pour 1'intestin, + 75 % pour le s^rum et J 10 f seule-
" ment pour le cerveau. Cette dernie're inhibition tr§s iSgeTe, s'explique
par le manque de permeabilite" de la barrifere he'mato-ence'phalique au meta-
bolite actif du triamiphos [$]•
- en ce qui concerne les dosages d'acides gras, glycerol et cholesterol,
les constatations suivantes peuvent etre faites (tableau 1) :
a. dans le se'rura des animaux soumis au regime normal contenant 10 ppm de
triamiphos, la concentration en acides gras libres est reduite signifi-
cativement de 30 %. Get effet est peut etre la resultante d'une
-------
893
TABLEAU I : Dosage des constituents des graisses apres
un an de traitement au triamiphos (10 ppm)
chez le rat.
Substance dose'e
Regime normal
(n - 10)
Regime gras
(n = 7)
Acidea gras litres
du serum
Acides gras totaux
dans 1'aorte
Glycerol total
dans I1aorte
Cholesterol total
dans I1aorte
69* +
65
122
110
100
91
175
* P<0.05
+ Re'sultats exprime"s en % du groupe te"moin.
reduction de la lipolyse (par inhibition de la HSL) chez les animaux
traitSs par le pesticide. La concentration en acides gras libres du serum
ne change cependant pas chez les animaux sounds au regime gras et recevant
du triamiphos.
b. dans les homogenats d'aorte, les concentrations en acides gras et
glyce'rol ne changent pratiquement pas chez lea animaux soumiB a 1'ester
organophosphore" et au regime gras alors qu'elle tend § diminuer sans
toutefois atteindre le seuil de signification chez les aniraaux soumis
au regime normal et au pesticide : en ce qui concerne les dosages de
cholesterol dans 1'aorte, on observe, suite a 1'administration du
tri-amiphos, une augmentation de concentration de 22 % (P<0,01) chez
les animaux soumis au regime normal et de 75 % (P<0,001) chez ceux
soumis au regime gras.
3. Conclusion.
Ces travaux prSliminaires ont mis en Evidence 1'interference d'un
,prf...
pesticide organophosphore avec le mStaboliame des lipides alors qu'aucun
aigne clinique d1intoxication ne peut encore etre observS. ConcrStement,
-------
894
cette interference du triamiphos se traduit dans notre etude par :
- un gain de poids plus important des animaux sounds au regime gras,
- la reduction des activates glyceridases de differents tissus du rat
(reduction significative dans le cas de la monoglyceridase du coeur).
- des differences de teneur en constituents des graisses dans le serum et
les homogenats de coeur et d'aorte, et specialement dans ce dernier
tissu, une augmentation nette de la concentration en cholesterol.
Ces observations suggerent que I1interference possible des pesticides
organophosphores avec le metabolisme lipidique merite de retenir I1atten-
tion. De telles investigations devraient d'abord etre repetees avec
d'autres pesticides organophosphores a:'in de verifier si les reponses
biologiques observees suite a 1*administration prolongee de triamiphos
sont conmunes a d'autres representants de ce groupe de pesticides.
Ce travail a etc finance par le Fonds de la Recherche Scientifique Medi-
cale (projet 1198) et le Fonds de Developpement Scientifique (UCL).
J.P. Buchet a beneficie d'un mandat de Charge de Recherches du Fonds
National de la Recherche Scientifique.
References
1 BUCHET, J.P. et LAUWERYS, R., "Inhibition of rat heart diolein hydrolase
and brain acetylcholinesterase by organophosphate esters in vitro"«
Biochim. Biophys. Acta. 218, 369-371 (1970).
2 LAUWERYS, B. et BUCHET J.P., "Inhibition of diglyceridase and cholines-
terase activity in rats by organophosphorus and carbamate esters", Tox.
Appl. Fharmacol. . 19, 1*06 (1971).
3 BUCHET, J.P. et LAUWERYS, R., "Characterization of a diglyceride lipase
in rat heart and intestine", Life Sciences, 10, 371-3J6 (1971).
U BUCHET, J.P., ROELS, H. et LAUWERYS, R., "Further characterization of
mono and diglyceride lipases in rat tissues", Life Sciences, l!*, 371-
385 (1971*).
5 LAUWERYS,R. et BUCHET, J.P., "Studies on the mechanism of toxicity of
the organophosphorus pesticide triamiphos", European J. PharmacQl. 16,
361-366 (1971).
-------
895
DISTRIBUTION AND METABOLISM OF POLYCHLOROBIPHENYLS
M, BERLIN. J, C, GAGE/ S, HOLM
Department of Environmental Health, University of Lund, Sweden
ABSTRACT
The relation between the structure of the polyohlorobiphen-
yls (PCBs) and their susceptibility to metabolic degradation has
been studied in the mouse. The excretion and retention in fat
have been investigated with six pure compounds: 3, 5, 3 ',5'-tetra-
chlorobiphenyl (I), 2,5,3 ',4 '-tetraahlorobiphenyl (II), 2,5,2',
4 ',5 ' -pentachlorobiphenyl (III), 2,3 ,4,2 ' ,4 ' ,6 ' -hexachlorobiphen-
yl (IV), 2,4,5,2',4',S'-hexachlorobiphenyl (V) and 2, 3, 4, 2 ', 4 ',
5 '-hexaahlorobiphenyl (VI). Compounds III, IV and V were ob-
14
tained labelled with C, the excretion of these three was meas-
ured over 21 days, and the retention of all six compounds in fat
was measured 7 and 21 days after dosing.
The results obtained with III, IV and V conform with the
current view that two vicinal carbon atoms are involved in arom-
atic hydroxylation; III and IV have such an unsubstituted pair
and are not retained in fat and are vapidly excreted, while V,
which does not have the unsubstituted pair of carbon atoms, is
retained in fat and excreted only very slowly. Compound VI,
however, disappears very slowly from fat although it contains a
pair of unsubstituted carbon atoms in the 2,3 position, and it
is suggested that hydroxylation at this position is blocked by
o_-chlorine substitution on the other ring, but only if the two
rings are approximately co-planar. With three o_-chlorine
atituentB in the molecule, as in IV, the two rings cannot be
co-planar, and hydroxylation at the 2,3 position ie possible
-------
896
The Zt4 position, as in II and III, is available for hydpoxy-
lation even with o_-Qhlorines on the other ping. The tetrachloro
compound I does not contain two adjacent unsubstituted carbon
atoms, and its retention in fat is intermediate between the
Values obtained with PCBs that are slowly and rapidly excreted.
It seems likely that the 3,S-dichlorophenyl ring can undergo a
slower metabolism by a mechanism somewhat different than occurs
with II, III and IV.
-------
897
Over 70 different components containing up to 10 chlorine atoms
have been detected in the commercial mixtures of polychlorobiphenyls
(PCBs). Evidence in the literature is conflicting on the relative rates
of biodegradation of different PCBs, but it seems likely that the lower
chlorinated components, which form a considerable part of the PCBs>
liberated into the environment, are preferentially degraded in the
lower stages of food webs. Jensen and Sundstrom [l] have not
identified PCBs with fewer than five chlorine atoms in human fat.
Together with other laboratories in Sweden, we are engaged in a
programme to assess the risks to health from those PCBs that are
absorbed and retained by man; this programme is supported by the
National Swedish Environment Protection Board. We have already
presented our preliminary results on the distribution and retention in
the mouse of 2, 5, Z',41, 5'-pentachlorobiphenyl (Berlin et al. [2]}; a
PCB which occurs in traces in human fat. This compound is rapidly
taken up by the tissues after oral or intravenous administration,
mainly by the liver and brown fat, and then it migrates to the general
body fat where it reaches a maximum about 24 hr after dosing, at a
time when the concentration in other tissues is falling rapidly. A
localized higher concentration was maintained in the nasal sinuses and
bronchi, and 32 days after the dose the lungs were the most clearly
defined organ in a whole-body autoradiogram, with radioactivity located
in the bronchial epithelium. Radioactivity is excreted in faeces with a
half-time of about 6 days, and we have subsequently shown that the
compound excreted is mainly a free and conjugated hydroxy derivative
of the PCB.
At a meeting in Stockholm where these preliminary results were
discussed, we suggested that the biodegradation of PCBs in the
environment might not be influenced primarily by the degree of
chlorination, but that it was dependent on the presence of two vicinal
unsubstituted carbon atoms in the molecule, a condition more likely to
be satisfied in low than in high chlorinated PCBs. This was based on an
earlier suggested mechanism for the hydroxylation of chlorobenzenes
(Jondorf et al. [3j ), and is in line with modern views on aromatic
hydroxylation (jerina [4j ). We have now tested this hypothesis by
studying the retention and excretion by the mouse of six pure PCBs
(Fig 1), supplied by Decent C.A. Wachtmeiater of the Wallenberg
-------
898
Ci
Cl
Cl
in
Cl
Cl
Cl
Chemical structure of PCBs used in the experiments.
Laboratory, University of Stockholm. Compounds III, IV and V were
14
obtained labelled with C.
The six compounds were administered orally in the lipid phase of
an aqueous emulsion to groups of 3-6 mice. With the three radioactive
compounds the excretion of radioactivity over a 21-day period, and
the tissue retention 1, 7 and 21 days after dosing were determined as
described in our earlier publication. In addition, all of the compounds
were determined in fat 7 and 21 days after dosing by the analytical
method of Jensen et al. [5J .
Fig 2 shows the faecal excretion of the three radioactive PCBs III,
IV and V, expressed as a percentage of the dose; less than 1% of the
dose is excreted urine. Compounds III and IV, both of which contain a
pair of unsubstituted.carbon atoms, are excreted with half-times of
about 6 days and 1 day respectively, while compound V, which does
not contain the pair of carbon atoms, is very slowly excreted. As
expected, the differences between the rates of excretion are inversely
related to the retention of radioactivity in fat and skin, the main body
reservoirs of PCB (Fig 3). The very rapid excretion of IV is not due
-------
899
1OO
•o
«
o
X
o
k
•
5 10 15
Days after dosing
20
25
Fig. 2. Excretion of radioactivity in faeces
Dose
165
13
fat skin liver
31
SO
25
0
SO
25
O
150
1OO
SO
7 days
21 days
&ea
vertical lines Indicate range
Fig. 3. Retention of radioactivity In tissues
calculated as PC B equivalent
-------
900
to incomplete absorption from the gut, as 1 day after dosing the
concentration in liver per/ug dose was found to be more than double
that of the other two, and Fig 3 shows that it remained higher after 7
days. The concentrations of PCB in fat at 7 and 21 days after dosing
are shown in Fig 4. The good agreement between the chemical and
radioactivity measurements with compounds III, IV and V indicates
that all of the radioactivity in fat was present as unchanged PCB. The
results with these three compounds support our original hypothesis,
but the prolonged retention of hexachlorobiphenyl VI, which contains
two unsubstituted vicinal carbon atoms, indicates that this is not the
only structural requirement for the metabolism of the PCBs.
The recent investigation of Jensen and Sundstrom [l] into the
retention of PCBs in human fat suggests an explanation of the failure
of compound VI to be metabolized and excreted. These authors found
that few PCBs with three or four o_-chlorine substituents were present
in fat, but PCBs with one or two £-chlorines were abundant, and they
suggested that £-chlorine substitution may influence metabolic
degradation. The structural difference between the two
hexachlorobiphenyls IV and VI is IV, which is rapidly metabolized,
Dose 25 25 165 13 31
31
O
•o
150
100
O 50
o>
c
I
7 day*
21 days
Fig. 4. Retention of PCB In fat
D0 U
, ,
1
J
\
\
\
s
fy
^
'<,
^
ft
\
\
tf
't
>
2
;
>
. E
-------
901
has three o-chlorines, while VI, which is not metabolized, has only
two. With two £-chlorines the biphenyl molecule can assume a
co-planar configuration while with three £-chlorines it cannot, and it
seems likely that £-chlorine substitution can inhibit hydroxylation in
the 2, 3 position of the other ring, but only if the two rings are
co-planar.
Jensen and Sundstrom found traces of III in human fat, and rather
more of 3,4, 2', 3', 6'-pentachlorobiphenyl, and they concluded that
two £-chlorines could depress hydroxylation in the 3,4 position. Our
investigation has indicated that III is fairly rapidly metabolized in the
mouse, so the o-chlorine substituents have little or no influence on
this pair of carbon atoms. The concentration in fat is, however, a
function of not only the rate of absorption but also of the rate of
intake, and it is possible that these two pentachlorobiphenyls constitute
a considerable proportion of the PCBs in human diet, although positive
evidence of this is not available.
The low retention of the tetrachlorobiphenyl II in fat is to be
expected from the presence of a 3,4 pair of unsubstituted carbon atoms,
but I, which contains no such pair, shows a retention in fat that is
intermediate between that of II and of the slowly metabolized
hexachlorobiphenyls V and VI. Jondorf et al. fsj have shown that
1, 3, 5-trichlorobenzene gives a small proportion of a hydroxy
derivative, and it is possible that the 3, 5-dichlorophenyl group does
not undergo the rapid metabolism associated with a vicinal
unsubstituted pair of carbon atoms, but that degradation proceeds by
an alternative slower mechanism.
REFERENCES
1 JENSEN, S. , SUNDSTROM, G., "Structures and levels of most
chlorobiphenyls in two technical PCB products and in human
adipose tissue", Ambio 3 (1974), in press.
2 BERLIN. M. , GAGE, J.C., HOLM, S., "The metabolism and
distribution of 2, 4, 5, 2', S'-pentachlorobiphenyl in the mouse",
National Swedish Environ Protection Board Publ 4E. 101-108,
(1973).
-------
902
JONDORF, W.R., PARKE, D.V., WILLIAMS, R.T., "Studies
in Detoxication. The metabolism of halogenobenzenes. 1, 2, 3-,
1,2,4- and 1, 3, 5-trichlorobenzenes", Biochem. J. 61,
512-521 (1955).
JERINA, D. M. , "Hydroxylation of aromatics, chemical models
for the biological processes", Chern. Technol. 3(2), 120-127
(1973).
JENSEN, S., JOHNELS, A.G., OLSSON, M., OTTERLIND,
G. , "DDT and PCB in herring and cod from the Baltic, Kattegat
and Skagerak", Ambio Special Report 1, 71-85 (1972).
DISCUSSION
DANIEL (U.K.)
1. Have you determined the level of glutathione in the liner
following the administration of the various polychlorobiphenyls?
2. Does the degree of ortho-substitution affect the difference
spectrum given with microsomal suspensions.
HOLM (Sweden)
The present investigation has been limited to a study of
the relation between the structure of the PCBs and their excretion
and retention in fat. It is hoped that it may be possible to
extend the investigation to include a more detailed study of the
mechanism of biodegradation. The point raised by Dr. Daniel
will be considered at that time.
-------
903
RELATION OF THE PHYSICAL/CHEMICAL STATE OF A
PLASTIC IZER, DI-C2-ETHYLHEXYDPHTHALATE
(DEHP) TO ITS BIOLOGICAL DISPOSITION AND ACTION
R, J, RUBIN AND C, 0, SCHULZ
Department of Environmental Medicine, Johns Hopkins School of
Hygiene and Public Health, Baltimore, Maryland, USA
ABSTRACT
DEHP, a highly water-insoluble plastiaizer which has been
shown to be widely distributed in the environment as well as to
be a contaminant of human blood stored in vinyl plastic bags, was
prepared in 3 different vehicles. Each preparation represented
a different type and degree of oil-in-water dispersion. These
were respectively: (2) a sonicated, aqueous emulsion which was
milky white and opaque, (2) a detergent-dispersed suspension in
S0% DMSO, 5% Tween 80 in saline which was opalescent, and (3) a
detergent-solubilized preparation in 25% DMSO, 10% Tween 80 in
saline which was completely transparent and, microscopically,
exhibited no droplets. DEHP (250mg/kg iv) in each of these S
preparations, had a markedly different effect on in vivo reticulo-
endothelial (RE) function in rats, implicating the physical state
of the DEHP in the HE effect. There appeared to be an optimal
droplet size for maximal inhibition of RE function with (3) having
no significant effect. The kinetics of disappearance from blood
and distribution were also markedly different for DEHP in (1) and
(S). In (1), it disappeared biexponentially and accumulated pri-
marily unchanged in the liver while in (3) it disappeared mono-
eteponentially and accumulated primarily in the eviscerated car-
cass. DEHP, solubilized in Tween 80, resulted in a respiratory
distress syndrome and lethality due to an acute alveolar inflam-
matory response which was not seen with either compound alone.
(Supported by grants ES34, ES 454 and ES 44887 from NIEHS and
contracts NAS-S-22071 from NASA and 72-29903 from NHLI).
-------
904
1. INTRODUCTION. The annual American production of phthalate
ester plasticizers is approximately one billion pounds [1].
With recent evidence of its widespread environmental distribu-
tion and contamination of human blood stored in plastic bags,
considerable interest has centered on its potential toxicologic
hazard [2], Because DEHP, the most widely used phthalate, is a
water-insoluble oil, we have been interested in its disposition
and biological effect when administered in various vehicles
that result in different physical states. Three different form-
ulations have been studied: (1) a sonicated emulsion in 3% gum
acacia (DEHP=50 mg/ml). This preparation is milky white and
completely opaque. Under phase contrast microscopy it
exhibits 19xl06 droplets/ml with a diameter of 1.9-2.6y,
(2) a detergent-dispersed suspension in a vehicle of 50% DMSO
and 5% Tween 80 in saline (DEHP=100 rag/ml). This preparation
is opalescent and contains 5.4xl06 droplets/ml of diameter
10.5-15P- (3) a detergent-solubilized preparation in a vehicle
of 25% DMSO and 10% Tween 80 in saline (DEHP=50 mg/ml). This
preparation is completely transparent and under phase contrast
microscopy exhibits no visible droplets. Extensive Brownian
movement suggests subraicron solubilized micelles of DEHP.
Dilution of these 3 preparations with rat plasma to an extent
that mimics their in vivo dilution results in the settling out
of additional oil droplets with the first two preparations
but has no effect on the solubility of DEHP in the third.
2. RESULTS. In Table 1 are shown the effects of the three
different formulations of DEHP on in vivo reticuloendothelial
function in rats. The dose-response curve for formulation (1)
is shown for doses of DEHP between 125-500 mg/kg. There
is no significant alteration at 125 mg/kg with the maximum
inhibitory effect seen at 250 mg/kg. At this latter dose the
-------
905
detergent-dispersed preparation (formulation (2)) produces an
approximately 2.5 fold greater effect while the detergent-
solubilized preparation (formulation (3)) results in no
significant alteration. These results clearly implicate the
particle size and number in the RE effect and indicate no
direct inhibitory effect of DEHP.
TABLE 1.
EFFECT OF DEHP ON RETICULOENDOTHELIAL FUNCTION IN RATS
% CHANGE IN CLEARANCE HALF-TIME
DOSE FORMULATION
mg/kg iv (1) (2) (3)
125 - 9 ± 5
250 +75 ± 5* +210 ± 10 +10 ± 5
*
500 +77 ±5
Twenty-four hours after the administration of the indicated
doses of DEHP or appropriate vehicle for each formulation,
a test dose of carbon particles was injected i.v. and the
clearance of the carbon from the blood was followed. The
carbon clearance was monoexponential. Half-time was
calculated from the rate of clearance and the data are
shown as the mean % change in clearance half-time from the
respective vehicle-injected controls ± S.E.M. Increases
in clearance half-time represent paralysis of RE function.
*
- p<.001
-------
906
The clearance rates of both emulsified and solubilized
DEHP are shown in Table 2. The emulsified DEHP disappears
biexponentially with rate constants for the initial rapid a
phase and slower 3 phase of .200 and .021 min , respectively.
In addition, approximately 90% of the injected dose disappears
from the blood during the ot phase. The solubilized DEHP
disappears monoexponentially with a rate constant of .036
min , which is not significantly different from that of the
0 phase for the emulsified DEHP. No rapid distribution phase
is seen.
TABLE 2.
KINETICS OF DEHP DISAPPEARANCE FROM BLOOD
FORMULATION FIRST-ORDER RATE CONSTANT (min" )_(x±SEM)_
a phase B phase
Emulsion in
4% BSA .200 ± .082 .021 ± .017
Detergent-
solubilized .036 ± .055
C-labelled DEHP at a dose of 200 mg/kg was injected i.v.
in the indicated formulation. At 2, 5, 15, 30, 45, and 60
minutes, blood samples were taken and the amount of unchanged
DEHP remaining in the blood was determined. After 1 hour
approximately 5% of the initial concentration of the
emulsified DEHP and 20% of the solubilized DEHP remained in
the blood. In these experiments DEHP was emulsified in 4%
bovine serum albumin (BSA). Visually and microscopically
this preparation was similar to the one in 3% gum acacia.
Preliminary experiments indicate that the in yiyg rates of
disappearance from blood are also similar.
-------
907
In Table 3 is shown the distribution of emulsified and
solubilized DEHP at 1 and 24 hours after i.v. injection. It
can be seen that the liver contains 48.4% of the emulsified
dose as unchanged DEHP after 1 hour; after 24 hours this
organ still retains 9.6% of the dose. In contrast, at 1 and
24 hours the liver contains only 11.3 and 0.2% of the solu-
bilized dose, respectively. Likewise, significant amounts of
the emulsified DEHP are found in the spleen at 1 and 24 hours
while only small or trace amounts of the solubilized DEHP
are recovered in that organ. In contrast to the preferential
uptake of the emulsified DEHP into liver and spleen, the
solubilized DEHP is recovered primarily in the eviscerated
carcass (i.e. skeletal muscle and bone). Note the lack of a
significant difference in the distribution of the two forms
of DEHP into lung. In spite of its lack of a major accumula-
tion in the liver, the solubilized DEHP is readily metabolized
to water-soluble metabolites. In fact, after 1 hour there is a
slightly but significantly greater accumulation of metabolites
in the liver, and after 24 hours a significantly greater amount
of the solubilized DEHP is recovered in the urine and feces
as metabolites.
During these experiments we noted that rats given the
detergent-solubilized DEHP die in respiratory distress at
doses of DEHP which have no overt effects when administered
as aqueous emulsions. The respiratory distress begins almost
immediately and death occurs within 90 minutes. At autopsy
the lungs are grossly enlarged and covered with dark, hemorr-
hagic patches. The DMSO, Tween 80-containing vehicle alone
does not produce the pulmonary damage and subsequently we
found that DMSO could be left out of the preparation without
-------
TABLE 3.
TISSUE DISTRIBUTION OF 2 DOSAGE FORMS OF DEHP
(% of injected dose: 200 mg/kg iv)
EMULSIFIED
SOLUBILIZED
% recovered as DEHP (±SE)
LIVER
CARCASS
SPLEEN
LUNG
1 hr.
48.4+5.2
12.1±3.1
6.1H.O
1.2±0.1
24 hrs.
9.6±1.4
2.910,2
1.810.4
0.3±0.02
% recovered as H.O-soluble
LIVER
CARCASS
EXCRETA :
1 hr.
1.7±0.3
1.610.6
4.3+1.6
24 hrs.
1.2+0.3
0.210.1
53.212.8
1 hr.
**
11.3 +0.5
33.5**+1.7
0.6* +0.08
1.3 10.2
metabolites
1 hr.
3.0* ±0.3
10.9**±1.8
6.2 ±0.5
24 hrs.
0.2 ±0.05
**
12.4 ±4.0
**
TRACE
vO
0.2 ±0.05 »
(±SE)
24 hrs.
0.5 ±0.05
0.6 10.2
66.9* ±4.3
Intest. cont,
Feces
Urine
**
p<0.05 or p<0.01 when compared to corresponding emulsified distribution
-------
909
altering the respiratory syndrome and lethality. Table 4 shows
the effect of Tween 80-solubilized DEHP on lung weight/body
weight ratios and lethality in rats. The varying doses of
TABLE 4.
LETHALITY OF TWEEN 80-SOLUBILIZED DEHP AND
EFFECT ON LUNG WEIGHT IN RATS
LETHALITY WET_LUNG WT/BODY WT
No. Dead/No. Treated (x ± SEM)xlQ3
DEHP vehicle DEHP vehicle DEHP #
Dose injected treated injected treated
mg/kg controls controls
200 0/10 0/10 5.02±0.15* 5.85±0.17+
250 0/8 4/17 4.78±0.14* 6.18±0.36+
300 0/13 10/15 4.7510.12* 7.1710.62*
*
Not significantly different from non-injected controls
(4.6910.16)
**
Data reported only for animals surviving for 90 minutes
Statistically different from corresponding vehicle-injected
controls (p<.01)
DEHP were achieved by injecting various volumes of the same
concentration of DEHP in 13.3% Tween 80. Each dose level was
controlled by a group of rats receiving an equal volume of
vehicle alone. The vehicle alone produces no significant
alterations in lung weight/body weight ratios when compared
to non-injected controls. In addition, no deaths are seen
with the vehicle-injected controls. However, a clear dose-
response relationship can be seen for lung weight/body weight
-------
910
ratios and lethality as a function of DEHP dose. The acute
LD5Q for the DEHPrTween 80 solution lies between 250-300 mg/kg.
At 150 mg/kg there are no overt symptoms and the lung weights
are not significantly different from controls. We have further
observed that the administration of 300 mg/kg of corn oil or
another closely related plasticizer, di(2-ethylhexyl) sebacate
solubilized in Tween 80 produces no lung alterations nor
deaths. Thus, the results shown in Table 4 must be due to some
specific interaction between the micellar DEHP and Tween 80.
Histologically, vehicle-injected lungs are indistinguishable
from non-injected control lungs. The pulmonary lesion seen
with solubilized-DEHp is due to an acute alveolar inflammation
and is characterized by a dramatic thickening of the intra-
alveolar septa arising from the migration of polymorphonuclear
leukocytes from the capillaries. As the acute pathology
progresses, other blood elements can be seen in the alveolar
walls; occassionally, migration across the wall into the air
spaces themselves can also be seen. Time course studies
indicate that the pathological lesion is fully developed with-
in 15 minutes after injection and, in animals that survive,
can be seen for at least 50 hours. In addition, the lesion
can also be seen histologically following doses of DEHP as
low as 50 mg/kg.
3. CONCLUSION. These experiments demonstrate that alteration
of the physical state of the dosage form of DEHP leads to
differences in biological activity, kinetics of disappearance
from the blood, tissue distribution, and, perhaps most import-
antly, unmasks a type of pathology not previously seen. These
results emphasize the necessity for knowledge of the physical
state of DEHP in evaluating its toxicologic potential and
signals a concern for the interaction with Tween surfactants
that are so readily available in the human diet.
-------
911
REFERENCES
1. Graham, P.R. "Phthalate Ester Plasticizers - Why and
How They Are Used". Env. Health Perspectives 3_:3
(1973). DHEW Publication No. (NIH) 73-218.
2. conference on Phthalic Acid Esters. Env. Health
Perspectives Vol. 3 (1973). DHEW Publication
No. (NIH) 73-218.
DISCUSSION
De BROIN (Netherlands)
Two questions:
1. Type of metabolic change of DHEP and structure of metabolites?
2. May DEHP be categorized as a microsomal enzyme inhibitor in
view of its effects upon barbital sedative action?
RUBIN (U.S.A.)
1. First a mono-deesterification of the DEHP to form the monoester,
MEHP. This is followed by oxidation of the terminal carbons of the
remaining side-chain to form the alcohol, ketone and terminal carboxyl
group. This is followed by 3-oxidation that results in a shortening
the sidechain by 2 carbons.
2. Although DEHP prolongs hexabarbital sleeping time, our studies
indicate that it does not do so by inhibiting the microsomal meta-
bolism. It apparently affects the distribution of the hexabarbital
to the brain or perhaps even brain sensitivity to the bariturate.
BUSH (U.S.A.)
Did you measure passage of your emulsions and mycellular
dispersions through the Gl tract?
-------
912
RUBIN (U.S.A.)
Following the oral administration of DEHP in corn oil, we
have observed the almost quantitative excretion of the DEHP as
water soluble metabolites in the urine and feces. The presence
of these metabolites in urine indicates the passage of either
the DEHP or its metabolites through the systemic circulation.
However, we have not been able to find any evidence of DEHP or
its metabolites in the blood following oral administration
(single intragastric dose). We interpret these results to mean
that the rate of GI absorption is slow while metabolic conversion
and urinary excretion of metabolites are rapid. This would tend
to minimize the accumulation of either DEHP or its metabolites
in the systemic circulation.
-------
913
THE EFFECT OF MERCAPTODEXTRAN AND N-ACETYLHOMO-
CYSTEINE ON THE EXCRETION OF MERCURY IN MICE
EXPOSED TO METHYL MERCURIC CHLORIDE
J, AASETH+ AND T, NORSETH++
+ University of Oslo, Rikshospitalet, Oslo, Norway
++ Institute of Occupational Health, Oslo, Norway
ABSTRACT
In order to prevent the reabsorption of biliary excreted
mercury in methyl mercuric chloride exposed mice, the animals
were treated- with a macromolecular polythiol (Meroaptodextran)
synthesized by thiolating a dextran compound using N-aoetylhomo-
oyteine thiolactone.
The polythiol given in food at a concentration of 5% reduced
the average biological half time of mercury from 11.6 days to 5.9
days. Faecal excretion of mercury compounds was not changed
during 10 dayst but urinary excretion increased by a factor of S.
The effect of the maoromoleoule seems to be related to N-
acetylhomooyeteine being released in the gastrointestinal tract.
N-aoetylhomocyeteine thiolaotone had the same effect as the macro-
molecule. Corresponding results could be obtained by intravenous
injection of N-acetylhomooysteine. The mechanism seems to inc-
lude the formation in the gastrointestinal tract and in the blood
of a N-aoetylhomooyeteine-methyl-merourio complex. This complex
is easily absorbed when formed in the gastrointestinal traott and
it is rapidly excreted in the urine.
-------
914
1. Introduction.
Biliary excretion of methyl mercuric salts is higher than
faecal excretion indicating intestinal reabsorption (Norseth,
Clarkson fl] ). A sulphydryl substituated poly vinyl has been
demonstrated to increase the elimination probably by complexing
mercury in the gastrointestinal tract (Clarkson, Small, Norseth
[ Zj). The aim of the present investigation is to study the effect
of mercaptodextran, a thiolated dextran of high molecular weight
(Jellum, Aaseth, Eldjarn[3]), on mercury excretion and retention
after exposure of mice to methyl mercuric chloride.
2. The effect of Mercaptodextran on excretion of mercury.
Female mice weighing about 20 g were given a single
intravenous injection of 5 /umol Hg/kg as radiolabelled methyl
mercuric chloride. Mercaptodextran (SH-500) was synthesized from
dextran with average molecular weight of 500,000 (Eldjarn, Jellum
[4]). Ten mice were given a diet containing 5% SH-500, and
10 mice served as controls. The animals were started on the
diet to be tested 48 hours prior to the injection of methyl mercuric
chloride. Radioactivity was determined every other day in the
whole animal using a whole body counter. The semilogarithmic
graphs of the body burden of mercury in control and treated animals
is seen to be approximately linear over the time period of obser-
vation. Accordingly a constant fraction of the body burden was
excreted per time unit, and the biological half-times ( t ± ) of
2
mercury can be calculated according to the formula
B_ : B . e " 7"t where >- = In2/ti
to 2
B = initial body burden of mercury
B = body burden of mercury after
a time period t
Mercaptodextran reduced the biological half-time of mercury after
exposure to methyl mercuric chloride from 11 days in control
animals to 5.9 days in the treated animals ( Fig. 1).
Thus Mercaptodextran decreased the biological half-time of
mercury by a factor of two, which equalized the reduction previously
shown for a polyvinyl resin (Clarkson, Small, Norseth I 2)).
During the treatment with Mercaptodextran radioactivity in
-------
915
O)
J£
O>
O
1
3
i
2
i i
4
i
i i
6
i
8
1
1
10
Days
Fig. 1
Retention of mercury in 2 groups of 10 mice at different
time intervals after a single intravenous injection of
methyl mercuric chloride. Triangles indicate mean
values in the control group, and squares indicate values
in the group treated with food containing 5% mercapto-
dextran (SH-500). Standard deviation is given in the
figure.
-------
916
faeces and urine was determined daily. The total amount of
mercury excreted in faeces and urine during 10 days is shown in
Fig. 2. Faecal excretion of mercury unexpectedly did not change
as a result of treatment with SH-500. Mercaptodextran, however,
increased the urinary output of mercury by a factor of 5. About
40% of the dose given was recovered in the urine of experimental
animals versus about 8% in the urine of controls.
FAECES
40 r
S
o
_ 20
5
£
SH-500 Cont.
URINE
8
•g
£
20
SH-500 Cont.
Fig. -2 Mercury excretion for 10 days in faeces and urine from
the same groups of mice after a single intravenous
injection of methyl mercuric chloride.
The bars denoted SH-500 and cont. represent mean
excretion from mice treated orally with SH-500 and from
the controls, respectively. Experimental range is
indicated.
-------
917
3. The effect of N-acetylhomocysteine and its thiolactone on
excretion of mercury.
As the synthesis of Mercaptodextran involves the use of
N-acetylhomocysteine thiolactone, the latter compound was mixed
in the diet of a mouse group in a concentration of 10/umol/g food.
The thiolactone appeared to have an effect almost similar to that
of Mercaptodextran.
The similar effect of Mercaptodextran and N-acetylhomo-
cysteine thiolactone indicates that the active agent in both cases
is N-acetylhomocysteine. The thiolactone is easily hydrolyzed,
and the macromolecule SH-500 ( = N-acetylhomocysteinylaminoethyl-
dextran) may well release N-acetylhomocysteine in the gastro-
intestinal tract by enzymatic cleavage.
In order to identify the active agent, groups of mice were
given intravenous injection of 500/umol/kg of SH-20 (mercapto-
dextran, average molecular weight 20.000), N-acetylhomocysteine
thiolactone or N-acetylhomocysteine immediately after the mercury
injection, 6 mice served as controls. Urinary excretion of
mercury was increased by a factor of about 5 when N-acetylhomo-
cysteine was given intravenously (Fig. 3). Treatment with SH-20
or N-acetylhomocysteine thiolactone was less efficient.
The latter experiment supports the hypothesis that N-acetyl-
homocysteine released in the gastrointestinal tract is responsible
for the effect of Mercaptodextran. Thus, in the gut a complex
may be formed v/hich is different from that normally excreted in
the bile. This compound, probably a methyl mercuric-N-acetyl-
homocysteine complex is, in the same way as the biliary complex,
easily reabsorbed, but is rapidly excreted in the urine.
Provided that only the biliary excreted mercury in the gut
is available for complexation, the highest estimated effect is a
reduction of the biological half-time of mercury by a factor of 4.
By increasing the dose of N-acetylhomocysteine thiolactone to 10
and 30/umol/kg per day given in the food the biological half-time
was reduced to 1.3 and 1.1 days respectively, versus 8.1 days
in the controls. This reduction in the biological half-time by a
factor of about 7 is incompatible with the hypothesis of complex-
ation only of biliary excreted mercury in the gut. The formation
-------
918
10 r
FAECES
41
8
SH-20 NAHT NAH Cont.
URINE
10 r
01
13
•*-
O
"c
I
rfl
\
SH-20 NAHT
NAH
Cont.
Fig. 3 Mercury excretion for 2 days in faeces and urine from
mice after a single intravenous injection of methyl
mercuric chloride. The bars denoted SH-20, NAHT, and
NAH represent mean excretions from 12, 6, 8, and
6 mice, treated intravenously with 500/umol/kg of
mercaptodextran (SH-20), N-acetylhomocyateine thiolactone
or N-acetylhomocysteine, and the column denoted cont.
represents excretion from 6 control animals. The bars
showing urinary excretion indicate values for the first
and second day. Experimental range is indicated for
each group.
-------
919
of a rapidly excreted complex, possibly between methyl mercury
and N-acetylhomocysteine, accordingly is thought to occur in the
blood as well as in the gut.
References
NORSETH, T. , CLARKSON, T.W., "Intestinal transport of
203Hg-labelled methyl mercury chloride: Role of biotransfor-
mation in rats", Arch. Environ. Health, p. 568 (1971).
CLARKSON, T.W., SMALL, H. , NORSETH, T. , "Excretion
and absorption of methyl mercury after polythiol resin
treatment", Arch. Environ. Health, 26, p. 173 (1973).
JELLUM, E. , AASETH, J. , ELDJARN, L. , "Mercaptodextran,
a metal chelating and di sulphide-reducing polythiol of high
molecular weight", Biochem. Pharmacol. , 22, p. 1179(1973).
ELDJARN, L. , JELLUM, E. , "Organomercurial-poly-
saccharide, a chromatographic material for the separation
and isplation of SH-proteins", Acta Chem. Scand. , 17,
p. 2610 (1963).
-------
921
POLLUTION DES ALIMENTS PAR LES HYDROCARBURES
PARAFFINIQUES: ETUDE DU DEVENIR DE CES SUBSTANCES
CHEZ LES ANIMAUX SUPERIEURS
J, TULLIEZ
Laboratoire de Recherches sur les Additifs alimentaires, INRA-
CNRZ, Jouy-en-Josas, France
RESUME
L1etude du metaboliame dea hydrooarburee aliphatiquee chest
lee mammifevee preaente un interSt certain, a la lecture de plu~
aieure travaux reoente, faiaant etat de I'augmentation trds nette
depuie 1940 tdee cae de lipidoae folliculaire de la rate et de
I'accumulation d'hydrocarburea dane lea nodulea lymphatiquea ches
I'homme, Dee hydrooarburea aliphatiquea ont egalement ete isoles
du tieeu arteriel et dee plaques ath6vomateit.eea ohez I'homme.
Dee ISaione, aolSroaee et degenSreaoenoea ont ete provoqueea ohea
le rat et le lapin apres ingeation prolongSe de doses importantee
d'huilee mineralee. Lea hydrocarburea ieolSe dea tiaaua humaina
ont pu Stre identified oomme provenant de I'ingeation d'huilee
minSvalea qui aeulea renferment dea cyoloparaffineaf en revanche,
lea n-alcanea peuvent provenir egalement dea alimentst lea vege-
taux renfermant & l'6tat naturel de 0,001 d, 0,1% de n-alaanea
lourda (Cl? a °31).
E« dehora de tout usage thevapeutique ou di&tetique, la
quantite annuelle d'hydrocarburea ingirea par individu a ete
evalu&e d. 400g aux Etate-Unie. La FDA a evalue a SOg/an/individu.
la quantitS d'huilee miner ale 8 ingereea. L'origine de oea der-
nidrea ae aitue au niveau dea traitementa teohnologiquea alimen-
tairea (enrobage dea fruita, revStement intdrieur dea emballagea
alimentairea en carton, agent de dfmoulage ...).
-------
922
Le d&veloppement recent de la production de levures d'alcanea
destinies & I 'alimentation animale et plus tard sans doute a Z
mentation humaine pose un probleme analogue. Ces levures^
elles sont cultivees BUT gas-oil* oontiennent des residua d'hydro-
carbures (ayalo et iso-paraf fines) dont le taux maximum autorisg
est de Ot08%. Des sources nouvelles de protSines , telles les
algues spirulines, aontiennent des quantites non negligeables
d'hydrocarbures (0,15% d' hep tade" cane ) .
Les travaux suivants sont
- Une etude de I' absorption des diffSrentes paraffines et
des quantit£s rSaiduelles non metabolise' es3 fixees dans la oar-
casse de I'animal.
- Une experience destinee d. suivre I 'accumulation de I'hepta-
deoane dans les carcasses et lea diffSrents tissus de rats rece~
vant un rSgime oil les spirulines constituent la source unique
prot&ines .
- Une etude plus fine du stockage realisee, &galement aur
ratst (J la suite de I'ingestion d'un regime alimentaire renfermant
une dose elevee de dodecyleyclohexane. Un niveau de saturation
des tissus en ce produit ayant ete atteint, I 'Evolution de la
quantit^ stockee a ete etudiee ohez des rats eoumis d un
amaigrissant.
ABSTRACT
Several recent studies noting the very distinct rise since
1940 in the incidence in humans of follicular lipidosis of the
spleen and hydrocarbon accumulation in the lymph nodules are of
considerable interest in the study of the metabolism of
-------
923
hydrocarbons in mammals. Aliphatic hydrocarbons have also "been
isolated in human arterial tissue and atheromatous plaques. Pro-
longed ingeetion by rats and rabbits of considerable quantities
of mineral oils produced lesions, scleroses and degenerative ef-
fects. It has been possible to identify the source of hydrocar-
bons isolated in human tissues as the ingestion of mineral oils,
which are the only source of cycloparaffins. On the other hand,
•n-alkanes can also come from food, since plants contain in their
C C
natural state between 0.001% and 0.1% ( 17 to 31) of heavy n-alkanet
The annual hydrocarbon intake per person, apart from that ta-
ken for therapeutic or dietetic purposes, has been estimated in the
United States as 400g. The FDA has estimated the intake of mineral
oils per person per year as 50gt the source of which is to be found
in technological food processing (wrappings of fruit, inner lining
of cardboard food containers, non-stick coatings, etc.).
A similar problem is created by the recent development of
alkane yeast production for animal feeding and later, doubtless,
for human food. When these yeasts are cultured on fuel oil, they
contain hydrocarbon residues (cyclo- and iso-paraffins), the maxi-
mum permissible proportion of which is 0.08%. New sources of
proteins, such as spiruline algae, contain not inconsiderable
amounts of hydrocarbons (0.15% heptadecane).
The following studies are presented:
- A comparative study of the absorption of different paraf-
fins and non-metabolized residues in the animal carcass.
- An experiment to trace the accumulation of heptadecane in
the carcasses and different tissues of rats given a diet «n which
spirulines constitute the sole source of proteins.
- A closer study of the storage in rats given a diet with a
large dodecylayolohexane content. When the saturation of the tis-
sues with this substance reached a certain level, the development
of the quantity stored was studied in rats subjected to a reducing
diet.
-------
924
Introduction
Les hydrocarburas aliphatiques ont ate1 mis en evidence chaz las memmi-
feres 11 y a deja longtemps, mais la role biologlque de ces substances,
souvent conside'rees comme inertes, n'sst pas clair, Ds nombreuses etudes
effectuees sur te Rat, IB Poulet, IBS ruminants at les poissons ont permis
de deceler das n- at das cyclo-alcanas ainsi qua das paraffines ramifie'as
au nivsau de divers organes et tissus, at en particulier dans la foie, la
rate at 1'ancephale. Las paraffines sont figalement prasantes chez 1'Homme
ou leur existsnce a 6ta signalee dans la rate, IB foie, las ganglions lym-
phatiquas m^sente'riques, las ganglions du systema porta hfipatique, las tis-
sus da 1'ertere femorala at dans IBS maninges.
L1absorption des hydrocarburas aliphatiquas 93t maintenant bien admi-
se, aussi attribue-t-on laur presence chaz les animaux superieurs a una
origina alimentaira (vegfitaux en particuliar) et, en ce qui concerns las
cycloparaffines, aux contaminations des aliments par les huiles minerales.
Les quantities ingerees par 1'Homma sont en augmentation constants,, compte
tenu d'une part de 1'usage des huiles da paraffins en technologie alimen-
taire lenrobage das fruits, ravfitement interieur das emballap;es alimantai-
res en carton, agent da demoulage), 3 des fine thSrapeutiques ou dans des
regimes basse-calorie, et d'autre part, das hydrocarbures accumulfis chez
diverses especes animales dont nous consommons les tissus,
Le dSveloppement recant de la production de levures d*alcanas desti-
nies a 1'alimentation animale et plus tard sans doute a I1alimentation
humaina, pose un problems analogue. Ces levures, lorsqu'alles sont culti-
v6es sur gas-oil, contiennent des rfisidus d'hydrocarbures (cyclo at iso-
paraffines) dont la taux maximum autorisfi est de 0.08 \, Enfln, des sources
nouvelles de protsines, tellss les algues apirulines, contiennent des quan-
titis non nSgligeables d'hydrocarhures [0,15 % d'heptadecane).
L'ingestion d'origina allmantalra a et6 Svaluee a 400 g/tfite/an aux
U.S.A.,quantit6 a laquelle 11 faut ajouter 50 g d'hulles minfirales. On peut
se demander quelles sont les repercussions de telles quantitds ingfirees sur
I'organiame humain. En affet, BOITNOTT et MARGOLIS ont fitabli une corrfila-
tlon antre la quantlt£ d'inclusions sous forme de gouttalattes dans IB foie
volre mime la degonerescence das cellules he"patiquas et la quantit§ meaurfie
d'hydrocarbures dans ce tissu t d'autre part, lls ont mis en Evidence une
relation antre Is nombre da cas de lipidose folliculaire de la rate et
I'utillsation ou la consommation crolssante d'huiles minerales depuls 1940.
-------
925
LIBER et ROSE. etudiant les cas de lipidose folliculaJre. ont constatS qua
les rates da sujets attaints etaient plus riches en Isomeres ramifies tan-
dis qua les rates normales contanaiant sssentiellement das n-alcanes. CAIN
a montre qua les meninges de sujets attaints de meningiomes ranferment das
quantities de paraffines plus importantes qua eelles de sujets sains. Une
constatation identique a et6 faite au niveau de 1'aorte de lapins souf-
frant d'atheroacleross. De plus, das lesions, scleroses et d£generescances
ont etd provoquees chez le Rat et la Lapln apres ingestion prolongee de
doses irnportantes d'huiles minerales. Enfin.l 'utilisation accrue de pesti-
cides ou de produits aglssant en synergie avec les pesticides, peut fitre
a 1'origins de 1'augmentation des paraffines tissulaires chez les mammifa-
res, du fait de leur role inhibiteur des processus de detoxification micro-
somale.
II Qxiste quelques travaux concernant 1'absorption d'huiles minerales
et des differentes classes d'alcanes chez le Rat et les ruminants. Les taux
de retention apparente (ingere molns excrete) ont 6tS determines pour dif-
ferentes longueurs de chalne et les voles d'absorption stabiles.
Nous rapportons lei les travaux concernant 1'absorption, la retention
et 1'accumulation chez le Rat de divers hydrocarbures paraffiniques.
I. Bilan metabolique at retention
Les experiences ont port6 sur :
Heptadecane
Elcosane
- Cinq n-parafflnes Hfineicoaans
Tetracosane
Dot rlacontane
- Deux cyclo-paraffines Dodecylcyclohexane
Heptadecylcyclohexane
- Deux paraffineB ramifieas 2,2,4,4,6.8.6. heptamethylnonane
2,6,10,14 tetramethylpentadecane
(Priatana)
Pour cheque hydrocarbure etudie, huit rats femelies de 150 g environ
recoivent individuellement au jour 0 une dose de 15 mg du produit consi-
dera, incluse dano leur nourrlture en solution dans 1'hulls d'arachide
entrant dans la ration. Urines et faces de trois rate sent collectea pen-
dant dix jours tandls que pour 1'etude de la retention, les animaux sont
abattua respectivament 1.2.3,5.7,10,15 at 21 Jours apres 1'administration.
-------
926
Les conclusions de ces premieres etudes sont IBS suivantes :
Las hydro carburea envisages n'ont donne lieu a aucuns Elimination uri-
naire. En revanche, il exists uns elimination fecale qui donna una idee du
nivaau de retention apparente. Ces resultats na parmettent pas da degager
de ligne generals applicable S 1 'ensemble des paraffines et ce d'autant
plus qu'il existe une assez grande variability individualle. Toutafois. il
ressort qua la retention apparente est treg elevee, de 1'ordre de 90 a
95 p. 100, tant qua le nombre d'atomea decarbone raste inferieur a 24.
En ce qui concerne la retention du produit inchange au nlveau de la
carcassa, las resultats obtenus permettent de divisar les paraffines en
deux categories :
- d'une part, les isoparaf fines qui semblant avoir un comportement parti-
culier : bien que donnnnt lieu a una retention apparente importanta, elles
ne sont a 1'origina qua da quantites residuslles non metabolises tres fai-
bles, voire nulles ;
- d'autre part, n-paraf fines et cyclo-paraf fines paraissent avoir un com-
portement identique, saul la nombre d'atomes de carbone intarvenant. Da
plus, il sembla qu'il existe une discontinuity au nivsau C_0-C i an deca.
les retentions sont du mama ordre de grandeur et la niveau, 15 jours apres
1'ingestion, se situe autour de 8 p. 100 da la dose ingeree i au-dela da
cette limite, la retention decrolt Jusqu'a davenir nulla(cas riu dotriacon-
tane).
Les resultats concernant la retention nous amenent a constater qu'a-
pres une diminution assez rapida durant les trois premiers Jours, les quan-
tites residuelles re ten UBS dans la carcasso entiera attaignent un palier
entre la 5eme et le 21eme Jour, compte tsnu de 1 a variability individualle.
Caci nous a conduit naturellement a envisager una accumulation chaz 1 'ani-
mal a la suite d'una administration prolongea,
II. Accumulation at localisatian tissulairB
consistent 3 repatar 1' administration da
15 mg d1 heptadacana et de dodacylcyclohexana durant 7 Jours consecutifs a
conduit a das resultats comparables, a savoir 1' accumulation dans la car-
casse d 'environ 7 p.ldO de la dose ingaree, signifiant que durant catte
periods 1' accumulation est lineaira. On observe una fixation preffirentiel-
le au niveau des graissas da reserve (300 ppm) at du muscla (35 ppm) . Pour
ce qui est des organes preleves, las teneurs se situant entre 3 et 20 ppm
pour foie, coBur, poumon, rate ou rein.
a B^B realises dans le
-------
927
cadre d'un ensemblG d'6tudes concernant la toxicologis das alguas spiruli-
nas en collaboration avac le Lahoretoire da Toxicologis de la Facultfi da
Pharmacie. II s'ast avere qua ces alguas renfermaiant 0.2 p.1DO d'hydro-
carburas dont las deux tiers sont constitues par da I'haptadecane. La re-
tantion da cette paraffins a ete etudi§a chaz la Rat racavant un regime
contenant 25 p.100 d'algues spirulines. La retention dans 10s carcasses a
ete rnasuree tous IBS moi3 pendant 12 mois at a la fin da cetta experience
las tanaurs dans las divers organes ant et6 datarminees.
Las resultats sont las suivants :
- La quantity residuelle d'haptadecana dans les carcasses plafonnedes
le 4emo mois ; alia attaint en moyenno 17 mg chez la femelie at 33 mg chez
le male, ca qui correspond quel que solt le sexe a environ 65 ppm par rap-
port au poids vif S 1'abattage.
- Au niveau dss organes at tissus, les teneurs las plus elevees sont
indiquees ci-dessous : ,
-------
928
- Les chiffres correspondent aux quantites residualIBS dans IBS car-
casses nous invitent 5 conclurs 5 une difference de comportement des deux
produits 6tudies :
. pour le dodecylcyclohexane, il semble qua 1'on attelgna un maximum voisin
da 130 mg des le 2ema moia i
. en revanche, les 370 mg d'aicosane accumula's ne peuvent 6tre consideres
comma un maximum meme si la teneur dans le tissu adipeux semble s'Stre
stabilises.
- Un point commun est constitue par le fait que 1'accumulation s'est
effectuee de fagon quasi linealre et que les teneura finales observees
tant au nlveau du muscle que du tissu adipeux sont voislnes pour les deux
hydrocarbures.
- II est difficile de comparer les resultats o tit en us lors de 1'amai-
grissement du fait des differences relevees dans les quantites stockees et
dans les poids des animaux en fin d*accumulation, mais nous pouvons noter
qu'une utilisation partialle des reserves lipidiquaa entrains une mobili-
sation d'abord lente des hydrocarbures stockes SB traduisant par un accrois-
sement de la teneur dansle tissu adipeux. Puls lorsque toutes les reserves
ont ete utilisees, seule subsists una faible quantitfi residualle correspon-
dant sans doute a cells flxee au nlveau das lipides structuraux.
- Soulignons encore le fait que dans le cas du dodecylcyclohexane
seule la moitie1 de la quantit6 stockee est metabolisee en 5 mois lorsqu'un
regime normal est distribue ad libitum aux animaux.
- En ce qui concerns les quantitds residuelles obaervSes au niveau
des divers organes, elles sont tres volslnes an fin d'accumulation pour
les deux hydrocarburas. Nous an retrouvons environ 130 ug dans las poumona
et les reins alors que le cerveau. la rate et le cosur n'en contiennent
que de 10 a 30 ug. En revanche, pour les rats amaigris comma pour ceux qui
recevalent ad libitum une alimentation normals, les organes n'en contien-
nent que de 3 a 15 ug.
Conclusion
Les hydrocarbures envisages sont tres largement absorbes tant que le
nombre d'atomea de carbons n'est pas trop elevei en fonction de leur nature,
ils donnent lieu a une accumulation plus ou molns importante dans les car-
casses et dans tous les cas, le tissu adipeux est la lieu de fixation pr6-
ferentiel. Enfin, lorsqu'on cease I1administration, la quantitd stockee eat
mobilises lentement meme si les animaux sont soumls i un regime lipidopriva.
-------
929
DISCUSSION
MERIAN (Suisse)
Vous avez 6tudi6 les me'canismes metaboliques apr&s adminis-
tration orale des hydrocarbures. Puisqu'on trouve environ 0,5
a 1 mg d1hydrocarbures par m dans 1'atmosphere des rues, 1'i-
nhalation joue aussi un rOle important. 80% des hydrocarbures
dans 1'air sont des paraffines 2O% des aromates. Est-ce que
vous voulez faire des commentaires en relation a cette inhalation
qui est principalement une inhalation d*essence.
TULLIEZ (France)
Je dirai tout d'abord que le chiffre de 80% de paraffines
parmi les hydrocarbures presents dans 1'atmosphere me surprend
quelque peu. Toutefois, il est certain que cette presence per-
manente de paraffines dans 1'air devait susciter 1'etude de leur
absorption par la vole pulmonaire et ce d'autant plus que mfime
lors de 1'administration per os de n-alcanes ou cycloalcanes a
chalne longue, le poumon est 1'un des sites de fixation prSffiren-
tielle (nettement apres le tissu adipeux et le muscle cependant);
or il a 6t6 d6crit des cas de pneumopathies huileuses et d'olSo-
granulome pulmonaire chez 1'Homme.
-------
931
DISTRIBUTION IN THE BODY OF THE FLUORIDE
INTRODUCED FROM THE DIET IN HIGH DOSES
AND ITS PLACENTA TRANSFER
G, SCASSELLATI SFORZOLINI AND A, SAVING
Institute of Hygiene, Medical Faculty, University of Perugia,
Italy
ABSTRACT
Rabbits were fed on a controlled diet containing different
amounts of fluoride (Ippm and 60ppm of F in the water).
After a certain period of this treatment the rabbits were
mated andt after the birth of the baby rabbits^ were "killed to-
gether with the new-borns. The following parameters were con-
trolled during the course of the experiment which lasted 100 days.
a) daily excretion of the fluoride with the urine;
b) daily elimination of the fluoride with the faeces;
c) dosage of the fluoride in the blood;
d) dosage of the fluoride in the bonesf teetht muscles and
principal soft tissues of the rabbits at the end of the
experiment;
e) dosage of the fluoride in the new-born rabbits and in
the placentas.
The data was compared with the results obtained by similar
researches on non-pregnant rabbits (controls).
From all researches carried out the Authors conclude that:
-------
932
- Fluoride -introduced from the diet ia partly eliminated
with the faeces and partly absorbed; a very small part of the
absorbed fluoride is found in the blood, while a large amount,
proportional to that introduced, is excreted with the urine.
- The hard tissues (teeth and bones) fix notable amounts oj
fluoride in proportion to the quantity introduced, while in the
soft tissues the amount of fluoride is always small.
t
- During the pregnancy the rabbits, especially those treated
with high concentrations of fluoride, eliminate a higher quantity
of the halogen with the urine than the control rabbits, while a
smaller quantity is fixed in the hard and soft tissues.
- The placenta allows the transfer of the fluoride to the
foetus in an almost constant quantity; in fact the quantity of
fluoride found in the new-born rabbits is always the same, inde-
pendent of their weight and the quantity of fluoride taken by
the mother. The placenta itself in part fixes the fluoride,
especially when the amount introduced is very high.
In rabbits fed on diets containing different amounts of
fluoride, during the pregnancy, therefore, a part of the halogen
is subtracted from the incorporation into maternal tissues,
passing through the placenta to the foetus; the urine excretion
and the placenta are the regulating organs of an excessive pas-
sage of the fluoride to the foetus. This is also demonstrated
by the rarity of mottled enamel of deciduous teeth in endemic
fluorosis areas.
-------
933
1* Introduction
It is well known that the biological action of fluoride is
different depending on its concentration; in fact while it has been
shown that an excess of fluoride introduced into the body may represent
a danger owing to its high toxicity, it is also certain that an
insufficient introduction of this element affects the mineralisation
process of the biological hard tissues (bone and teeth) and influence
the formation of dental caries.
Sereral papers have been published directed to study the
distribution in the body of the fluoride introduced in optimum doses by
the digestire tract and its passage from the mother to the- foetus, in
order to contribute to the study of dental carlea prophylaxis by the
fluoride.
Less numerous instead are the researches directed to investigate
the placenta transfer when the fluoride is introduced in high doses.
Smith and Smith [11, Uc Clure[2], Marci[31, have emphasized the
rarity of mottled enamel in deciduous teeth in endemic fluorosis areas
and have connected this fact to the limited permeability of the human
placenta to a certain amount of fluoride (Gardner et al.[43, Feltman
and KoselCS], Ziegler [6], Ericsson and Ullberg [7], Auermann et al.[8l,
Gedalia et al. [0], dedalin et al.[10], Blayney and Hill [11] ,Ericsson
and nammarstrSo [12], Gedalia et al.USl, Gedalia [14]) and also of the
placenta of laboratory animals, such as rabbits and rats (Hurray [15],
Ifaplesden et al.[16l).
3. Experimental
To study the distribution of the fluoride in the body and its
placenta transfer we used rabbits, because the placenta of these
animals is classified under group IV of Grosser** classification, that
is haemooorial as human placenta (Stefanelli [17]).
The research was carried out on 20 primiparous rabbits aged 4
months and weighing about 2 Kg, kept in metabolic cages throughout the
treatment period, in order to collect daily separately the faeces and
urine.
To all the animals food and water containing two different amounts
of fluoride were administered in order to study the different
distribution in the body of the halogen introduced from the diet in
optimum and in high doses, normally considered toxic*
The 20 rabbits were diTided into two groupsi the first group was
kept as a control and therefore not mated, while the second group was
mated.
For further details of this research, we refer to our works
published in detail (Seppilli et al.[18], Scassellatl Sforzolini et
-------
934
2,1 DistrijwUjmj)f the f luoride in^ the body of control rabbit a
(Group I)
Daring the first tiro weeks of the experiment the 10 rabbits were
fed daily on 100 g of food (containing 20.203 ppm P~) and non-
fluoridated water and in the daily faecea and urine the fluoride
present was determined (Tab. I). The rabbits were successively
subdivided into two sub-groups a) and b)t the rabbits of the sub-group
I-a) were fed daily on 100 g of food and water containing optimum doses
of fluoride (l ppm of F~)j the rabbits of sub-group I-b) were fed daily
on 100 g of food and water containing high doses of fluoride (60 ppm of
r).
In the first two weeks of this second phase of the experiment in
the faeces and urine the fluoride was determined daily (Tab* II), while
for the rest of the experimental period which lasted in all 100 days,
the fluoride was measured once a week only in the urine (Fig. 1). In
the sane rabbits the fluoride present in the blood was also determined
(Tab. III).
At the end of the experimental period, all the rabbits were killed
and in the h\ard and soft tissues the fluoride present was measured
(Tab. IV and Fig. 2).
From the results given in the tables and in the figures we can
emphasize that!
l) The amount of fluoride eliminated with the faeces is connected with
the means of its administration; in fact the highest percentage of
the halogen is eliminated when it is introduced only through the
food.
8) The urinary excretion of the fluoride in both the sub-groups remains
relatively constant during the whole period of treatment and the
amount found is proportional to the quantities introduced.
3) The quantities of the: fluoride found in the blood are constant in
time and always very low, even if in proportion to the quantities
introduced.
4) The tissues wich most concentrate the fluoride are the hard tissues
(teeth and bones), while in the soft tissues the amounts found are
always small} the amount of fluoride fixed by the various tissues
increas'es with the increase of the amount introduced.
8.2 Distribution in the body of the fluoride during the pregnancy MJ
its placenta transfer (Rabbits Group II).
As in the experiments on the control rabbits, this group was
subdivided into two sub-groups! to every rabbit of each sub-group II-a)
-------
935
and Il-b) was given food and fluoridated water, containing two
different amounts of fluorides 1 ppm of F~ and 60 ppm of F~, following
the same proceedure described for the control rabbits) the onlj
difference was that the treatment with food and non-fluoridated water
lasted only one week.
After 9 weeks from the beginning of the experiment, all the
rabbits were mated. For these rabbits also* as in the controls, the
treatment lasted a total of 100 days, during which time the urinary
excretion was determined once a week (Fig. 3).
At the end of the experiment, both the rabbits and the new-boras
(these last immediately after the birth) were killed and the fluoride
in the different tissues of the mothers, in the plecentas and in the.
new-boras "in toto" was determined (Tables VI, VII, VIII).
The results of this second group of researches hare been compared
with the results of the researches carried out on control rabbits
(first group) (Fig. 4).
From the results shown in the tables and in the figures we can
emphasize that!
l) During the pregnancy the amount of fluoride excreted with the urine
greatly increases in both the sub-groups and in particular in sub-
group Il-b), and than decreases immediately after the pregnancy to
levels more or less normal.
2) The weights of the new-boms and of the placentas of rabbits of the
same sub-group don't differ substantially, whereas the new-borns of
rabbits from sub-group Il-b) constantly weight less than the new-
borns of rabbits of sub-group Il-a).
3) In the hard and soft tissues of pregnant rabbits the amount of
fluoride present is always less than that found in the same tissues
of non-pregnant control rabbits.
4) In the new-borns, independent of their weight, fluoride is always
found in the same quantity for two sub-groups*
5) In the placentas fluoride is always present and in quantities
proportional to the amount introduced*
3. Conclusions
From the researches carried out we can concludes
- Fluoride introduced from the diet is partly eliminated with the
faeces and partly absorbed; a very small part of the absorbed
fluoride is found in the blood, while a large amount, proportional
to that introduced, is excreted with the urine.
-------
936
- The bard tissues (teeth and bones) fix notable amounts of fluoride
in proportion to the quantity introduced, while in the soft tissues
the amount of fluoride is always small*
- Daring the pregnancy the rabbits, especially those treated with high
concentrations of fluoride, eliminate a higher quantity of the
halogen with the urine than the control rabbits, while a smaller
quantity is fixed in the hard and soft tissues.
- The placenta allows the transfer to the fluoride to the foetus in an
almost constant quantity) in fact the quantity of fluoride found in
the new-born rabbits is always the same, independent of their weight
and the quantity of fluoride taken by the mother. The placenta itself
in part fixes the fluoride, especially when the amount introduced is
rery high.
In rabbits fed on diets containing different amounts of fluoride,
during the pregnancy, therefore, a part of the halogen is subtracted
from the incorporation into maternal tissues, passing through the
placenta to the foetus; the urine excretion and the placenta are the
regulating organs of an excessire passage of the fluoride to the
foetus. This is also demonstrated by the rarity of mottled enamel of
deciduous teeth in endemic fluorbsis areas.
-------
Table I
fig of F" eliminated daily with the faeces and urine of control rabbits fed on diet containing fluoride
and non-fluoridated water. Average from 10 rabbits with standard deviation (£_) and variation coefficient
(Cyg). Determinations carried out for two weeks: weekly averages with test of significance of the difference
between the two averages (£ and P).
Days from
beginning
of treatment
2nd
3rd
4t!i
5th
6th
7th
Average of
1st week
8th
10th
12th
14th
Average of
2nd week
1
I
Rabbits fed on 2.0203 nig of P~ daily
(only with the diet)
Faeces
/ig F~ eliminated daily
4 average fron 10 rabbits)
1226.4
1261.8
1151.7
1252.1
1230.5
1167.6
1215.0
1144.5
1175.7
1214.3
1190.2
1181.3
1.3124
>0.05
£_ °v£
53.5884 4.3695
58.5525 4.6404
55.5822 4.8261
59.4675 4.7494
54.9675 4.4670
56.4998 4.8385
52.7445 4.6085
47.4654 4.0372
52.7565 4.3446
48.7644 4.0971
I
Urine
jug PT excreted daily
(average from 10 rabbits)
330.3
304.6
328.9
290.4
340.3
298.8
315.6
320.2
297.4
312.6
316.5
311.8
0.3509
>0.05
.£_ Oy#
24.3363 7.3679
23.3445 7.6639
26.4354 8.0375
23.4153 8.0631
25.6131 7.5266
23.5635 7.8860
24.1833 7.5525
21.8184 7.3364
20.4822 6.5522
23.8839 7.5462
I
-------
Table II
/ig of 7" eli=ir.ated daily with the faeces and urina of control rabbits (group I) fed on diet containing fluoride and water containing different
mounts of fluoridei sub-group I-a) • 1 ppn of K~; sub-group I-b) • 60 ppn od F~. Average from 5 rabbits per sub-group with standard deviation
(ff_) and variation coefficient (£~g). Determinations carried out for two weeks: weekly averages with test of significance of the difference
between the two averages (.t and F).
!
3sys from
'searijming
of treatment
I
2nd
1 3rd
4th
| 5th
! 6th
i 7th
' Average of
1 1st wfcek
cth
10th
12th
14th
I Average of
2nd week-
is
2
Sub-group I-a)i
rabbits fed on 2.0553 ag of r daily (2.0203 mg with the diet and
0.035 n**with the water)
Faeces * Urine
fig F~ eliminated
daily (average
from 5 rabbits)
i
1230.6
1240.5
1231.2
1246.6
1215.8
1204.5
1228.2
1200.4
1170.8
1205.2
1217.3
1198.4
2.6695
>0.05
' S^* r excreted
2_ ' &£ { daily (average
» from 5 rabbits)
a
52.6496 4.2783 jj 363.5
54.2095 4.3699 " 352.4
52.7350 4. 2832 f 325.4
50.4291 4. 0453 ( 360.6
50.6659 4.1689J 371.5
54.0900 4.4906. 318.6
i ——————
S 352'°
p
5
50.1499 4.1777! 327.3
50.9904 4.3551 ! 394.O
50.6277 4. 2007 i 345.6
55.6316 4.5700J 326.4
!
\ 348.3
| 0.2050
• >0.05
£_ ' 5vi§
i
!
25.71 ?7I7. 0736
25.9347|7.3594
25.165417.7336
25.34 50J 6. 5592
25. 87371 6.9M6
25.45881 7.9908
1
1
1
26.3110'8.0388
25-1319,6.3786
25.67291 7.4285
25. 3311 [7.7607
t
t
1
*
j Cub-group I-b) i
1 rabbits fed on 4.0203 m« of 7~ daily (2.0203 mg with the diet and
2 mg with the water)
Faeces J Urir.e
jug F- elininated
daily (average
from 5 rabbits)
2045.0
2026.0
2100.5
2000.4
2020.8
2030.4
2037.2
i 2022.4
198O.6
2010.0
1990.2
2000.8
1.9147
>O.OJ
jpg F" excreted
0~ Cv£ ndaily (average
Sfroo 5 rabbits)
n
63.1485 3-0879 « 645.1
i
63.3597 3.1273 jj 755.2
65.9239 3-1384 3 602.4
66.1231 3.3054 ?. 704.6
65.8506 3.2586 3 621.3
66.0425 3.2526 5 746.2
•1
S 745,8
•i
65.6656 3.2469 J 781. 8
63.5744 3-2098 jj 750.0
63.9507 3.1816 I 791.6
63-9352 3.2125 j 800.6
J 781.0
„„ .„<„„ i
; 1.0345
| >0.05
5L cvj=
45.8?33 7.1141
47.7?44 6.32S7
52.5~94 6.5527
43.5?£6 6.1£"4
49.2923 6.CC17
46.03*5 6.1f95
4S."1336 6.2335
50.91-7 6.-7F90
49.6C-0 6.266C
45.9CC5 5.7332
VO
U)
Co
-------
Table III
pjn Of r-in th* Mood of eontj-ol rabbit* (group I) f«l on di.t containing fluoride and water containing different
tmnmt, of fluortdai aid-group 1-4) . 1 pp. of »-i aub-froup I-b) . 60 pt. of *-. Average fro» 5 rabbit! p.r eu"
with standard deviation (£_) and variation coefficient (£
Ktmrj froei
beginning
ef treataent
1
3
6
10
24
48
72
96
120
144
16ft
Average
Sub-group I-a)« "~
rabbi te fed
•« of I" In tli» hard «n I-a) - 1 mm of r~; aub-i-roBp I-b) . «0 ran of r. Average fro« 5 rabbita per sub-rroup .vit-i
atandard deviation (ff_) (oaleulated on f value* in total matorJi.1) and variation coalriciwit (Cy^).
OrrieU
Bore.
Teeth
Internal
thyroid
Kurcl*
rabbita fed on 2.0553 ag of r daily
(2.0203 w with the diet and O.035 •* with th* wat*r)
Average froa 5 rabbits
Dry
neijglt
(In g)
of total
ewterla]
113.1624
5.9380
40.301S
0.3999
448.05 6C
> of
aahea
over dry
•eight
39.4554
81.6100
4.8540
29.5460
3.3101
pod Of
r in the
aahea
697.4960
422.5$ 50
55.9730
n.d.
101.0240
p«a» of
r in
dry
Bttarial
347.0145
344.6804
2.7170
n.d.
3.3440
ng of r
in total
•aterlal
39.6690
2.0479
0.1095
n.d.
1.4983
«_ Cyjt
4.3559 11.0008
O.2O44
0.0157
-
9.9809
14.3378
, -
0.2320 ,15.4842
1
Sub-group 1— b) i
rabbits fed on 4.0203 Bg of F~ daily
{2.OTC3 »r with the diet end 2 eir with the writer)
Average from 5 rabbits
Dry
weirtit
(ing)
of total
aatarial
152.0224
4.7734
32.0635
0.4877
349.1742
•f. of
aahea
over dry
weight
45.4759
83.5546
5.2780
30.1360
4.6040
ppa of
T~ in the
aehea
1206.6740
60C.3140
119.4810
n.d.
279.9760
t>ia of
r in
dry
•aterlal
548.7461
673.7126
6.3062
n.d.
12.6°01
«« of P-
in total
•aterlal
83.4217
3.2159
0.2072
n.d.
4.5C09
i
1
1
£- | &r2*
1
9.53SF Jii.TJie
0.2510 '10.9145
|
C.C142 '16.913?'
_ 1 _
O.<"03 1 14. 6925!
i
(a) _ luaga, heart, liver, paneraaa. epleen, kUneva and «*nltala.
n.d.- aon detectable.
-------
Table V
mg ot TT fixed by the various cre«ns of the ocntrol rabbits («roup I) fefl for 100 days with diet containing fluoride
and water containing different amounts of fluoridei subgroup I-a) • 1 TWO of F~t sub-ftroup I-b) - 60 ppra of F".
Animal*
Sub-group I-a) I
rabbit» fed on 2.0553 mg of 7~
daily < 2. 020 3 mg with th* diet
and 0.035 ng with the water)
Sub-group I-b) t
rabbits fed on 4.0203 mg of F~
daily (2.0203 mg with the diet
ar.d t og with the water)
Total mg of F-
f ixed by the
various organs
examined
43.5247
91.3407
mg of 7~ found in th8 various organs of the rabbits
i of
Bones total 7~
fixed
39.8690 91.6009
83.4217 91.3303
i
•f. of
Teeth total f
fixed
2.0479 4.7051
3.2159 3.5208
internal *?
or^a fixed
0.1095 0.2516
0.2022 0.2214
1
. * of
Bueole total f"
fixed
1.4983 3*4424
4.5009 4.9276
t
Table VI
Weight of newborn rabbits and of the placentas of rabbits (group II) fed for 100 days with diet containing fluoride and water
containing different amounts of fluoride: sub-srou-a II-a) - 1 m>m of F~; sub-group Il-b) » 60 pro of F~.
Saf
rabbits
Ir £ 'S
ir «• 5
.. oo-..
1 C >-. -H
I-. 0 r- T! ,E
*-• *• '*'
C- I • "3 .C *
o i t;
j. c i. i E
V +> +» ^^
i .- i- -n jr t.
3 .2 o *•
n c V v • o
I- E E i t
O O -0 <—
ev r. C U
c c. 3 t.
-~ T- f^ «• c?
a —at
t S >>•"
t- O rt fj C
w •- X
c^^J4*
^ ** ** ?
1
2
3
4
5
Average
1
2
3
4
5
Average
Fresh wgt Tin g) of each
I
64.0
56.5
57.5
61.4
49.7
II
60.0
42.5
62.3
£2.8
70.4
III
59.0
66.5
63.5
70.3
45.1
IV
37.0
60.0
64.7
•»
46.8
V
-
55.0
-
, —
ta
50,0
71.3
39.8
54.2
48.7
52.0
49.8
33.2
44.3
39.8
43.2
37.9
30.4
48.7
45.9
-
66.4
44.3
47.4
-
-
-
-
39.0
-
Tresh wgt
(in K> of
all
newborno
220.0
280.5
228.0
194.5
212.0
227.0
146.1
225.4
147.7
233.6
134.4
177.4
Dry wjrt
(in g) of
all
newboms
46.5920
66.3400
52.4322
40.4851
47.0122
50.5723
28.0565
46.8349
30.79H
50.4469
26.2363
36.4732
£ ashes
dry wgt
12.8322
10.0976
13.8742
11.1245
13.4990
12.2855
10.8413
H.7510
13.8115
15.1015
12.0842
13.3179
?resh wjvt (in g)
i of all placentas
4.9
7.3
5.0
4.1
1 4.7
! 5.2
4.8
6.0
5.5
7.7
5.0
5.8
Placentas
Dry wgt (in g)
of all placentas
0.8243
0.9498
0.8444
0.7614
0.8021
0.8364
0.7825
0.9830
0.9700
1.2000
0.8205
0.9512
% ashes over
dry wgt
10.0736
11.2230
10.3242
9.0136
9.8764
10.3042
11.0078
12.0548
14.5964
13.5882
10.9878
18.4474
-------
Table VII
Big of f In the hard and soft tissues of pregnant rabbits (group II) fed for 100 days with diet containing fluoride and water containing
different asounts of fluoride: sub-group Il-a): - 1 ppo of T"; sub-group Il-b): - 60 ppm of 7~. Average from 5 rabbits per aub-proup with
star-lard deviation (o~ ) (calculated on F~ values in total material) and variation coefficient
/s— ..__ —
Grgs2is
Bones
Jeeth
Tr.terr.al
Thyroid
j-U»Cl«
ouo-group Il-a) :
rabbits fed on 2.0553 ng of f daily
(2.C2C1 v.g with the diet and 0.035 as with the water)
Average from 5 rabbits
Dry
weijrht
(ir. «)
of total
eeterial
125.4000
4.1612
35.2565
0.4301
379.7241
£ of
ashes
over dry
weight
41 .4947
SO. 6046
5.1060
25.7212
4.2984
pro of T
in the
ashes
753-8870
410.3700
52.0600
n.d.
91.1910
T>PB Of
IT in dry
material
312.B238
331.6034
2.6602
n.d.
3.9197
mg of F~
in total
material
39.2281
1.3865
0.0939
n.d.
1.4884
-
— £**
4.3547 11.1010
0.1418 10.2316
0.03^4 14.5215
_
0.2355 15.8244
Rub— croup Il-b) I
rabbits fed on 4.0203 mg of F~ daly
(2.0703 OK with the diet and 2 n«t with the water)
Average from 5 rabbits
Dry
i weifht
(in K)
of total
material
120.6348
5.6O44
38.7018
0.3863
548.4061
* of
ashes
over dry
weight
44.3772
80.0978
5.1920
28.3154
4.8925
ppm of F~
in the
ashes
1075.1500
716.9420
80.1800
n.d.
192.6900
ppffl Of
F" in dry
material
477.1227
576.1901
4.1626
n.d.
9.4273
ag of P~
in total
material
57.5576
3.2292
0.1611
n.d.
5.1700
i
£- , HvS
(
6.6043 , 11.P217
0.3555 ' 11.P065
i
8.025-7 • 15.9765
— : _
0.776C ' 15.0105
1
f
(*) - Lungs, heart, liver, pancreas, spleen, kidneys and genitals.
n.d.- nor. detectable.
Table VIII
Be Of f in the newborn rabbits and in the placentas of rabbits (group II) fed for 100 days with diet containing fluoride and water containing
different erounts of fluoride: sub-group Il-a) - 1 ppm of F~j sub-group Il-b) » 60 pan of P~. Average from 5 rabbits per sub-group with
deviation (o_) (calculated on the values of 7" in the total material) and variation coefficient
Material
:reirboms
Placentas
Sub-group li-a)s
rabbits fed on 2.0553 mg of T daily
(2.0203 a* with the diet and 0.035 m« with the water)
Average from 5 rabbits
Dry *; cf
neifht [ashes
(in 5) lover dry
of total Wight
aat«rial •
50. 572 J
O.B3«
12.2655
10.3042
ptm of 7"
in the
ashes
32.9960
257.5410
ppm of T~
in dry
material
4.0557
26.5423
jag of y~
in total
material
0.2051
0.0222
£. £v^
3.2174 15.6870
0.3267 '14.7180
1
l
Sub-group II-b)i
rabbits fed on 4.0203 mg of P~ daily
(2.0203 RK with the diet and 2 mK with tha water)
Average from 5 rabbits
Dry
weight
(in E)
of total
material
35.4732
0.9512
t of
ashes
over dry
weight
13.3179
1 2.4474
P3B Of F"
in the
ashes
43.2944
1072.6350
ppo of T
in dry
material
5.7659
133.5155
eg of T~
in total
material
0.2103
0.1270
SL £v5
3.1873 15.1558
2.0641 16.2531
-------
— 900-
o
•B
£s
t I5O-
J^»
|
I
— ' A /
X\ f" ^v "/'
/ \ /
\ / \ /
X N / V '
X
.^ —— — ( overage 82U ) rabbits
tub-group I-bl
3
350
300-
250-
(average 322.6) rabbits
sub-group I-a!
vo
*>
K>
T"
2
T
3
T
4
"I
6
1 - 1 - 1 - 1 - 1-
8 9 10 11 12
we«k» of treatrnenl
Fig. 1. F~ excreted with the urine of control rabbits (group I) fed on diet containing
20.203 PP» of F~ and water containing different amounts of F~: sub-group I-a)
= 1 ppm of F~. sub-group I-b) = 60 ppm of F".
-------
943
400-,
350-
300-
250-
200-
150-
100-
50-
Fig. 2.
Rabbit* tub-group l°-a)
Rabbits tub-group l°-b)
Distribution of the fluoride in control rabbits (group I)
fed for 100 days on diet containing 20.203 PP» of T~ and
water containing different amounts of F~: sub-group I-a)
s i ppm of F~j sub-group I-b) «= 60 ppn of F~.
-------
Rabbits
sub-group Q-b
Rabbits
sub-group [la
•
.
1st ten days 2nd ten days 3rd ten days last
. week of
treatment
o treatment with the diet and non - fluoridated water
Fig. J>. T~ excreted with the urihe of pregnant rabbits (group II) fed on diet containing
20.205 PP» of F~ and water containing different amounts of F~: sub-group Il-a)
= 1 ppin of F ;sub-group Il-b) = 60 ppm of F .
-
-
-
-------
945
Rabbits group I
- Rabbits group []
A = F" introduced in 100 days of treatment
F~ eliminated in 100 days with the urine
F" eliminated in 100 days with the faeces
F" found in various organs and tissues
O F" found in the placentas and in the new-born
rabbits
10 mg of F*
A B C D
Rabbits tub-groups La land Q a)
1
ll
I
A B CD
Rabbits sub groups I bl and lib)
Fig.
Distribution of the fluoride in control rabbits (group I)
and in pregnant rabbits (group II) fed for 100 days on diel
containing 20.203 ppn of F~ and water containing different
amounts of F~ : sub-groups I-a) and Il-a) = 1 ppm of F~;
sub-groups I-b) and Il-b) = 60 ppm of F~ .
-------
946
References
1 SMITH, M.C., SMITH, H.V., "Mottled enanel of deciduous
teeth", Science. 81, 77 (1935).
2 Me CLURE, F.J., "Flnoridation of drinking water and control
of dental caries", p. 197, Bethesda, Md., National Institute
of Dental Research (1962).
3 UARCI, F., "Incidenza della carle, della fluorosi e contenu-
to di fluoro nello smalto nei soggetti di et& scolare di ana
zona fluorotica (Campagnano Romano) a distanza di 6 anni dal_
la sostituzione delle acque", Paradontol. e Stomatol. 5,
456 (1967).
4 GARDNER, D., SMITH, F.A., HODGE,H.C., OVERTON, D.E., FELTMAN,
R., "Fluoride content of placental tissues as related to
fluoride content of drinking water", Science, 115, 208 (1952).
5 FELTMAN, R., KOSEL, G., "Prenatal ingestlon of fluorides and
their transfer to the fetus", Science. 122, 560 (1955).
6 ZUEGLER, E., (1956), clt. by Gedalia, 1970.
7 ERICSSON, T., ULLBEAG, S., "Etude autoradiographique de la
repartition du radiofluor chez la souris et le rat", J.Dentaire
Beige. 4, 237 (1959).
8 AUEJoiANN, E., BOIUIIS, W., GROSSER, H., "Uber den Uetabolismus
des fluors im Neugeborenen-Stadiun". Advances in Fluorine
Research and Dental Caries Prevention, 2, 215 (1963).
9 GEDALIA, I., BRZEZINSKI, A., PORTUGUESE, N., BERCOVICI, B.,
"The Fluoride Content of Teeth and Bones of Human Fetuses",
Arch.Oral.Bio1.. 9, 331 (1964).
10 GEDALIA, I,, BRZEZINSKI, A., ZUKERUAN, H., MAYERSDORF, A.,
"Placental Transfer of Fluoride in the Human Fetus at Low
and High F-Intake", J.Dent.Res.. 43, 669 (1964).
11 BLAINEY, J.R., BILL, I.N., "Evanston dental caries study.
XXIV. Prenatal fluorides-value of waterborne fluorides during
pregnancy", J.Amer,Dent.Assoc.. 69, 291 (1964).
.12 ERICSSON, Y., HAUMARSTRtiH, L., "Mouse placental transfer o£
P._ in comparison with Ca. *, Aota Odont.Scand.. 22. 523
(1864). 45
13 GEDALIA, I.*, ZUKERUAN, H., LEVENTBAL, H., "Fluoride content
of teeth and bones of human fetusesi in areas with about I
ppm of fluoride in drinking water", J.Aner.Dent.Assoc., 71,
1121 (1965).
-------
947
14 GEDALIA, I., "Distribution in placenta and foetus",
Fluorides and Human Health, W.H.O., Monograph Series
n, 69, p. 128, Genera 1970*
15 MURRAY, 11.11., "Maternal transference of fluorine",
J.PhTSiol. 87, 388 (1936).
16 MAPLESDEN, D.C., 110TZOK, I., OLIVER, W.T., BRANION, H.D.,
"Flacental Transfer of Fluorine to the Fetus in Rats and
Rabbits", J.Nutrition. 71, 70 (i960).
17 STEFANELLI, A., "Istologia ed Embriologia", Ed.Ateneo,
Roma 1959, p. 403.
18 SEPPILLI, A., SCASSELLATI SFORZOLJNI, G., SAVING, A.,
PASCASIO, P., "Ricerche *in vivo1 sulla distribuzione
nell*organ!smo dello ione fluoro introdotto per ria di-
gerente", dt press in L'Igiene Moderna (1974).
19 SC1SSELLATI SFORZOLINI, G., SAVING, A., PASCASIO, F.,
"Hicerche sul passaggio dello ione fluoro attraverso la
placenta. Contribute allo studio della fluoroprofilassi
della earie dentaria nel periodo pre-natale", at press
in L »Igiene Moderna (1974).
-------
WECHSELBEZIEHUNGEN
INTERACTIONS
INTERAZIONE
INTERACTIES
'- Chairman - President - Presidents -
J. CARSTENSEN (Denmark)
-------
951
CADMIUM-ZINC INTERACTIONS
M, PISCATOR
Department of Environmental Hygiene, Karolinska Institute,
Stockholm, Sweden
ABSTRACT
Cadmium accumulates with age in the human kidney. "Normal"
exposure causes cadmium concentrations of less than 7 S .ug/g wet
weight in renal cortex at age SO. This "normal" accumulation
is accompanied by an equimolar increase in zinc. It has been
estimated that at a cadmium concentration of about 200 ,ug/g wet
weight in renal cortex tubular function will be disturbed. The
mechanism behind this tubular dysfunction is thought the replace-
ment of zinc by cadmium in certain renal enzymes. There is,
however, a lack of data on zinc in relation to cadmium concen-
trations from 75 to 200 .ug/g wet weight. Animal experiments
have shown that exposure to cadmium will cause an increase in
venal zinc, but exposure has generally been quite high and dur-
ing short periods of time. Normal horses accumulate cadmium
during a life-time, and thus constitute a suitable material for
studies of long-term exposure to small amounts of cadmium.
Cadmium and zinc were analyzed by atomic absorption speotrophoto-
metry in renal cortex from 37 horses. Cadmium concentrations
varied from 5 to 2SO ,ug/g wet weight. At lower concentrations,
i.e. <.-60 ,ug/g wet weight* the increase in zinc was equimolar to
the increase of cadmium. However, with increasing concentrations
of cadmium, zinc did not increase to the same extent, thus indi-
cating a disturbance in the cadmium-zinc relationship. It can
be suggested that cadmium may exert an influence on zinc enzymes
in the kidney at relatively low concentrations, not far from the
present "normal" values.
-------
952
1. Introduction
Cadmium and zinc have many common physico-chemical properties and
occur together in nature. There are, however, large biological differen-
ces. Zinc is an essential metal with short biological half-time, and
occurs in high concentrations in all body compartments. Cadmium is non-
essential, has an extremely long biological half-time, and accumulates
in liver and kidney, the latter organ containing about one third of the
total body burden. The placenta differentiates effectively between the
two metals, so that whereas the newborn has an excess of zinc, cadmium is
virtually absent. Cadmium will accumulate with age i" the human kidney,
and it has bee/i estimated that at a cadmium concentration of about 2UO
jjg/g wet weight in renal cortex, renal tubular dysfunction may occur. In
Europe and the U.S. mean concentrations at age bO are at present about
30 ug/g wet weight, whereas higher levels have been found in Japan. For
further information on metabolism and effects of zinc and cadmium see the
books by Prasad et al. ft] • and Friberg et al. C21 respectively.
The renal tubular dysfunction caused by cadmium is characterized by
the urinary excretion of low-molecular weight proteins - "tubular protein-
uria". The mechanism behind this dysfunction is thought to be a replace-
ment of zinc by cadmium in enzymes taking part in reabsorption, and
in catabolism of proteins. Vigliani pQ reported that simultaneous ad-
ministration of zinc to cadmium-exposed rabbits alleviated the renal
damage caused by cadmium alone.
Several animal experiments have shown that exposure to cadmium will
cause increases in the zinc concentrations in liver and kidney (see for
example Bunn and Matrone [4j , Banis et al. Qj] , Anke et al. (V) ,
Roberts et al. [?J , Cousins et al. [ifif] , bchroeder and Nason CO J. In
these experiments large amounts of cadmium were added to the diet and the
cadmium intake was of the same magnitude as the zinc intake - the con-
centration in the diet or in water being 5u mg/kg or more. This increase
is due to a redistribution of body stores of zinc, since it was found in
a study on calves that the increase in liver and kidney was accompanied
by a decrease in zinc concentrations in muscle and bone. Furthermore,
there was a decrease in intestinal absorption of zinc (Roberts et al.Qj),
Petering et al. 0°J found that when the concentration of zinc was <2 mg
Zn/kg in the diet, and 2 mg/kg of drinking water, cadmium at relatively
low concentration (3.4 mg/kg of drinking water) caused a decrease in the
-------
953
Zn yumol/g dry weight
8-
3
2-
1 -
01 234
6 780 10 11 Cd /umol/g dry weight
Figure 1. Concentrations of cadmium and zinc in renal cortex from 37
Swedish horses.
-------
954
Zn yumol/g dry weight
M8
3 4| 5 6
100 419/9 w*i w*ighi
ft 10 11 Cd/imol/g dry weight
ml wclghi
Figure 2. Regression lines for cadmium and zinc in renal cortex.
1 Normal human beings (age 6-50 years, n = 3fa)
from Piscator and Lind |j2J.
II Normal human beings (age 10-yO years, n = 87)
from Hammer et al. [J3J.
Ill Normal horses with A: cadmium concentration below 2.5 umol/g
(n = 20), and B: above 2.5 |imol/g (n = 17)
IV Experimentally exposed swine (n = 12, whole kidney)
from Cousins et al.
-------
955
zinc concentrations in rat testes, whereas such a decrease was not noted
when the zinc concentration in drinking water was increased to b mg/kg.
These results from animal experiments show that cadmium may cause
alterations in zinc metabolism and that certain tissues may be depleted
of zinc, when cadmium causes an increase of zinc in liver and kidney.
The relationship between cadmium and zinc in organs from human beings
without known industrial exposure has been studied using autopsy cases.
Schroeder et al. fllj showed that there was a parallel increase in cad-
mium and zinc levels in kidney with age. Piscator and Lind £l2) found the
same relationship in renal cortex and could also show that the increase
in zinc was equimolar to the increase in cadmium, the regression equation
being Zn = 1.13 Cd + 0.5 (umol/g wet weight). Furthermore, an estimate of
the base value for zinc in renal cortex was obtained, being about 34 ^ig/g
wet weight {160 jjg/g dry weight). Hammer et al. JJ3J found that the reg-
ression equation was Zn = O.y5 Cd + 29.4 (|imol/g ash), or, transferred to
wet weight, Zn = 0.95 Cd + 0.41. However, the cadmium concentrations in
these studies have been generally below 75 jjg/g wet weight. There was a
need for a study of cadmium-zinc relationships at higher cadmium con-
centrations, i.e. up to 200 jjg/g. Normally exposed horses provided the
necessary material.
i
2. Studies on horses
Liver and kidney were obtained from a total of 37 horses, 1-25 years
of age, killed at two slaughter-houses, one in the South of Sweden, and
one in the North. Cadmium and zinc were determined with atomic absorption
spectrophotometry in the same way as for organs from human beings (Pisca-
tor and Lind [j2] ). In figure 1 zinc concentrations are shown in relation
to cadmium concentrations in renal cortex. It is seen that at concentra-
tions of cadmium above 3 ^imol/g dry weight (about 70 jjg/g wet weight),
there is not a corresponding increase in zinc as seen at Tower concentra-
tions.
In figure 2 the regression lines for the two human studies, the
horse study, and one experimental study are shown together. The horse
material was divided into two groups with 2.5 ^mol/g as border value. It
is seen that at low cadmium concentrations the horses do not differ from
the human groups, but at higher cadmium concentrations there is no longer
an equimolar increase in zinc.
-------
956
3, Discussion
The results obtained and the results from other investigations sug-
gest that at moderate increases in cadmium concentrations to about 75
jjg/g renal cortex (wet weight), there will be an equimolar increase in
zinc and the amount of zinc necessary for normal physiological functions
is probably adequate. At higher concentrations of cadmium the rate of
zinc accumulation will decrease and will no longer be equimolar. The
reason for this may be that the cadmium-binding protein, metallothionein,
which is the main transport and storage protein for cadmium, contains more
cadmium and less zinc than normally, i.e. when there are equimolar amounts
of cadmium and zinc. This would mean that some zinc would still be avail-
able for enzymatic functions. Another possibility is that an equimolar
relationship in metallothionein still exists, in which case less zinc
would be available for enzymatic functions in the renal cortex, and hence
decreases in enzymatic activities can be expected. That very subtle chan-
ges in cadmium-zinc ratios can influence enzymatic activity is suggested
by the experimental fundings of Cousins et al. [&J , who in swine found
that the activity of the zinc enzyme, leucine aminopeptidase, was already
decreased at a cadmium concentration of 78 ug/g in whole kidney (corre-
sponding to about 10U ug/g in renal cortex). It is, however, also possible
that other metals may have been influenced in this experiment, since high
doses were given. Indeed in some of the animals described in the intro-
duction the copper metabolism was also disturbed, which was not the case
in the human studies by Piscator and Lind Qf].
-------
957
References
I PRASAU, A.S. (Ed.), Zinc metabolism. Charles C. Thomas, Springfield.
Illinois (1966)
2 FKIBERG, L., PISCATUR, M., NORDBEKG, G., Cadmium In the Environment.
CRC Press, The Chemical Rubber Co., Cleveland, Ohio (1971)
3 V1GL1ANI-, E.C., "The biopathology of cadmium", Amer. Ind. Hyg.
Ass. J.. 30, 329 (1969)
4 BUNN, CLARA R., MATRONE, G. "In vivo interactions of cadmium,
copper, zinc and iron in the mouse and rat", J. Nutr., 90, 395
(1966)
5 BANIS, R.J., POND, W.G., WALKER, E.F., O'CONNOR, J.R., "Dietary
cadmium, iron and zinc interactions in the growing rat", Proc. Soc.
Exp. Biol. Hed.. 130, 802 (1969)
6 ANKE, M., HENNIG, A., GKOPPEL, B., LUUKE, H., "Der Einfluss des
Kadmiums auf das Wachstum, die Fortpflanzungsleistung und den
Eisen-, Zink- und Kupferstoffwechsel", Arch. Exp. Veterina'rmed.,
25, 799 (1971)
7 ROBERTS, K.K., MILLER, W.J., STAKE, P.E., GENTRY, R.P.. NEATHERY,
M.W., "High dietary cadmium on zinc absorption and metabolism in
calves fed for comparable nitrogen balances", Proc. Soc. Exp. Biol.
Hed.. 144, 9U6 (1973)
8 COUSINS, R.J., BARBER, A.K., TROUT, J.R., "Cadmium toxlcity in
growing swine", J. Nutr.t 103,964(1973)
9 SCHRUEDLR, H.A., NASON. A.P., "Interactions of trace metals 1n rat
tissues. Cadmium and nickel with zinc, chromium, copper, manganese",
J. Nutr. I04, 16/ (1974)
10 PETEKING, H.G., JOHNSON, M.A., STEWER, K.L., "Studies of zinc meta-
bolism in the rat. I. Dose-response effects of cadmium", Arch.
Environ. Health. 23, 93 (1971)
11 SCHROEDLR, H.A., NASON. A.P., TIPTON, I.H., bALASSft, J.J., "Essen-
tial trace metals in man: Zinc. Relation to environmental cadmium",
J. Chron. Pis.. 20, 179 (1967)
-------
958
12 PISCATOR, M., LIND, B., "Cadmium, zinc, copper and lead in human
renal cortex", Arch. Environ. Health, 24, 426 (1972)
13 HAMMER, D.I., COLUCCI, A.V., HASSELBLAD, V., WILLIAMS, M.E.,
PINKERTON, C., "Cadmium and lead in autopsy tissues", 122nd Annual
Convention of the American Medical Association, June 23-27, 1973,
New York City, N.Y.
DISCUSSION
DIEHL (Federal Republic of Germany)
The great concern about cadmium in the environment goes
back primarily to the unfortunate Itat-Itai incident in Japan.
A number of authors have already voiced doubts that the symptoms
seen there could have been due to cadmium toxicity per se. What
we have heard in Dr. Lorke's paper ("Subchronische orale Toxizi-
tat von Cadmium bei Ratte und Hund") strengthened the impres-
sion that cadmium toxicity is not as high as previously believed.
Do you think, on the basis of your studies and of other infor-
mation you have on Zn/Cd interaction, that the Itai-Itai inci-
dent could have been due to a combination of elevated dietary
cadmium intake and insufficient zinc intake?
PISCATOR (Sweden)
Since the pollution was caused by a zinc-mine, the levels
of zinc in rice were above normal and thus the intake of zinc
was sufficient. The calcium intake however, is low in the ac-
tual area and the Itai-Itai disease is caused by cadmium acting
on a sensitive population with calcium deficiency. Cadmium is
highly toxic, the long biological half-time causes an accumula-
tion in the kidney which after many years may reach high levels.
Dr. Lorke's paper stressed what was known before that
short-term exposure to relatively low amounts of cadmium does
not cause any major dysfunction, but that does not mean that
cadmium has a low chronic toxicity.
SCOPPA (Italy)
Have you any idea about the biological half-life of thionein,
or metallothioneins?
-------
959
PISCATOR (Sweden)
No, but the half-times may well be different in liver and
kidney, the latter being a catabolic organ.
van der KREEK (Netherlands)
We learned from one of the earlier introductions that cad-
mium is stored in the thyroid and pituitary glands. You studied
the Cd/Zn balance in the kidney. Did you study also the Cd/Zn
balance in the thyroid and pituitary glands and the possible
influence of excess Cd on the function of thyroid and pituitary
glands?
PISCATOR (Sweden)
We have not studied Cd/Zn balance in other organs. In
connection with experimental studies on the effects of cadmium
on calcium deficient animals also the parathyroids are studied,
but not the thyroid or pituitary glands.
HISLOP (U.K.)
Would you care to comment on the distribution of Cd in
kidney and why did you chose to analyse the cortex?
PISCATOR (Sweden)
The cadmium concentrations are highest in the cortex and
it is easy to standardize the sampling technique.
-------
961
HEPATIC DAMAGE AND ORGANOCHLORINE RESIDUE
CONCENTRATIONS IN BODY TISSUES
P, C, OLOFFS* AND D, F, HARDWICK++
+ Department of Biological Sciences, Simon Fraser University,
Burnaby, B.C., Canada
++ Department of Pathology, Medicine, University of British
Columbia, Vancouver, B.C., Canada
ABSTRACT
Liver specimens, taken from patients with hepatic cirrhosis,
contained significantly higher concentrations of DDT, but signi-
ficantly lower concentrations of heptachlor epoxide, than speci-
mens from controls. Concentrations of dieldrin were not signi-
ficantly different. No significant differences were found between
oirrhotios and controls in the concentrations of any residues in
brain and adipose tissues. None of the specimens contained
detectable amounts of aldrin, heptachlort lindane, or the alpha
or gamma isomers of ohlordane.
There was a highly significant association between lipid
contents and residue concentrations in livers. Cirrhotia livers
with severe fatty infiltration, determined by histopathological
examination, and confirmed analytically, contained significantly
higher amounts of DDT and heptachlor epoxide than did those with
minimal or no fatty changes.
-------
962
1. Introduction
Reportedly, certain diseases, especially those affecting the
liver, have been associated with tissue residue concentrations signi-
ficantly higher than in healthy persons (1). But other workers have
found no association between tissue residue concentrations in humans and
a variety of pathological conditions (2). The purpose of our studies
was to determine whether or not concentrations of organochlorine pesticide
residues in tissues of patients with hepatic cirrhosis were higher than
in those from controls, and to investigate causes responsible for any
differences.
2. Materials and Method
Specimens of liver, brain, and adipose tissue were collected at
autopsy, within 48 hours of death, from patients judged clinically to
have died of hepatic failure with cirrhosis of the liver. For a control
group, an additional series of tissues was collected from patients judged
clinically not to have cirrhosis. Residues were extracted from each
tissue specimen, identified, and quantified as described In detail by
Oloffs e_t^ aJL (3). Lipid concentrations in the liver tissue specimens
were measured gravimetrically (3).
For histopathological examination, sections of each liver, fixed in
Formol Calcium and stained with haematoxylin and eosin, were examined
microscopically for the following: fatty change, fibrosis, central lobular
congestion, and bile stasis. Each was ranked on a scale from 0 (- normal)
to 5 (•= most severe). All slides were examined at the same time in order
to assure consistent judgement. The specimens were not identifiable in
any way and were presented at random for review. Half of the slides were
examined twice in this fashion with consistent results.
3. Results and Discussion
DDT residue concentrations in liver were significantly (*) higher
in the specimens from cirrhotics than in those from controls, but hepta-
chlor epoxide concentrations were significantly (**) lower. No statis-
tically significant differences could be demonstrated for residues in
adipose and brain tissues. Lipid content of liver specimens was higher
(*) in the cirrhotic patients than amoung controls (Table 1). Lindane,
aldrin, heptachlor, alpha chlordane, and gamma chlordane were not detected
in any of the specimens.
Chlorinated hydrocarbon pesticides and some of their metabolites
-------
963
Table I Mean Residue Concentrations (ppb) of Dieldrin,
Heptachlor Epoxide, and DDT in Human Tissues
CIRRHOTICS
CONTROLS
Dieldrin
Heptachlor Epoxide
Total DDT—
Lipid (% b. w.)
Dieldrin
Heptachlor Epoxide
Total DDT-/
Dieldrin
Heptachlor Epoxide
Total DDT-/
X
8.07
19.28**
473.09*
7.97
89.33
91.45
3681.48
2.00
2.29
34.92
,X
LIVER SPECIMENS
(22;40)
(50;12)
(62;0 )
(62;0 )
ADIPOSE TISSUE
(50;12)
(59;3 )
(62;0 )
BRAIN TISSUE
(0;59 )
(4;55 )
(59 ;0 )
X
5.98
34.21
273.64
5.08
113.66
109.31
4248.38
2.32
2.30
46.52
(n ;n )—
p* o'
(21; 28)
(48 ;1 )
(48;1 )
(49;0 )
(38;3 )
(39;2 )
(41;0 )
(1;20 )
(1;20 )
(21;0 )
Pi/
40
0.1
2
5
20
20
50
40
a/
— Z probability that two means belong to the same population;
difference considered significant (*) where P ^ 5, highly significant
(**) where P <_ 1. - DDT + DDE + DDD - n - number of specimens with
concentration above, n » number of specimens with concentrations below
lower limit of quantification: thus n + n
P
obtained and analysed.
total number of specimens
-------
964
are highly lipophilic, and they tend to partition into fatty tissue and
become concentrated therein. The much higher concentrations in adipose
than in liver tissue (Table I) demonstrate this clearly. Furthermore,
since lipid content was significantly higher in cirrhotic than in control
livers, the question arose to what degree the residue levels in liver
tissue may depend on, and vary with, the lipid content, i. e., differences
between residues in cirrhotic and control livers may be due to the respec-
tive lipid contents, rather than to other liver pathology. Linear corre-
lation coefficients indeed indicated a positive correlation between
hepatic lipid and residue concentrations, rather than between liver patho-
logy and residue concentrations. In no case was the correlation negative.
Analysis of the liver data from the cirrhotic patients, grouped
according to tissue Injury determined by hlstopathological examination,
further supports this view. Heptachlor epoxide, total DDT, and lipid
concentrations were significantly (**) higher in livers with moderately
severe and severe fatty infiltration, thus confirming the demonstrated
association between lipid content, as determined gravimetrically in the
laboratory, and residue concentrations. Grouped according to degree of
flbrosis, however, the differences were reversed. Both DDT and lipid
contents were significantly (*) lower in the group with severe fibrosis,
because connective tissue had displaced lipid and thus diminished the
reservoir for the lipophilic DDT residues.
4. Conclusion
We have demonstrated that residue concentrations in livers vary
strongly and closely with lipid content, regardless of the pathology.
Where hepatic cirrhosis was associated with severe fatty infiltration,
the residues were higher than those in the controls or in cirrhotics
having low liver lipid concentrations. In association with severe fibro-
sis or bile stasis, the residue concentrations were below those in the
controls. If altered hepatic function, rather than liver lipid content,
were a major factor contributing to acquisition of pesticide residues In
body tissues, differences should have been found also between residue
concentrations in adipose and brain tissue from the cirrhosis group and
concentrations in the same tissues from the control group. We conclude
that lipid content in the liver was the prime determinant of pesticide
residue concentration.
-------
965
References
(1) Radomski, J. L., Deichmann, W. B., and Clizer, E. E. Pesticide
concentrations in the liver, brain and adipose tissue of terminal
hospital patients. Fd. Cosmet. Toxicol. (1968) 6: 209-220
(2) Morgan, D. P., and Roan, C. C. Chlorinated hydrocarbon pesticide
residues in human tissues. Arch. Environ. Health. (1970)
20: 452-457
(3) Oloffs, P. C., Hardwick D. F., Szeto S. Y., and Hoerman D. G.
DDT, dieldrin, and heptachlor epoxide in humans with liver cirrho-
sis. Clin. Biochem. (1974) Vol. 7, No. 3. In press.
-------
967
INFLUENCE DE CERTAINES DROGUES SUR L'EFFET
TOXIQUE DECLENCHE PAR L'AFLATOXINE Bx DANS LE FOIE
YVONNE MOUlJ
Institut de Recherches Scientifiques sur le Cancer, Centre
National de la Recherche Scientifique, Villejuif, France
ABSTRACT
The effects of a pretreatment of animals with phenobarbital
on the inhibition induaed by aflatoxin B- on RNA synthesis in
liver have been studied in rats and mioe.
Eats are relatively susceptible to aflatoxin action. Pre-
treatment with phenobarbital decreases the inhibition of trans-
cription due to aflatoxin administration.
Mice are much more resistant than rats to toxic and carci-
nogenic effects of aflatoxin B-. However, pvetreatment of
animals with phenobarbital3 enhances the inhibition of RNA syn-
thesis induced by aflatoxin in liver. Thus, phenobarbital may
potentiate the toxic effect of drugs in certain animal species.
Implications of these results are discussed.
-------
968
L'aflatoxine B , mycotoxine secretee par AsperjglUis fla_vus,
est le plus puissant des hepatocancerogenes connus a ce jour. Elle
suscite un interSt particulier en raison de sa presence possible
comme contaminant des aliments de 1'homme et de certaines eapeces
animales ; c'est un danger potentiel se"rieux qui doit fitre pris en
consideration Fvoir la revue de WOGAN (1)1.
En dehors de son action cancerogene qui se manifeste toujours
apres un temps de latence relativement long, 1'aflatoxine B.
declenche a court terme des effets toxiques dans le foie. Ces effets
se traduisent sur le plan morphdogique par des alterations nucle'aires
et cytoplasmiques. Sur le plan biochimique, lea deviations metabo-
liques induites par 1'aflatoxine touchent surtout la synthese des acides
nucleiques (RNA et DNA) et des prote"ines. L'inhibition de la synthese
de RNA correspond k un blocage du me'canisme de transcription
comme le prouvent les diminutions d'activite" RNA polyme"rase des
noyaux de foie isoles a partir d'animaux traite"a par la mycotoxine.
L'aflatoxine est me'tabolise'e par des systfemes enzymatiques
responsables de la transformation des drogues, systemes qui aont
localises dans les membranes du re'seau endoplasmique. II a 6t6
montre" que la molecule biologiquement active n'est pas 1'aflatoxine
elle-mSme mais un de ses metabolites forme's au cours de sa
metabolisation FMOULE et 'FRAYSSINET (2)1.
Le traitement des animaux par certains compos£s,tela lea
barbituriques^eat connu pour induire la synthese d'enzymes
intervenant dans la metabolisation des drogues par 1'interme'diaire du
cytochrome P4_n. Le present travail concerne 1'action d'un traitement
prealable par le phenobarbital sur les propri^t^s toxiques de
1'aflatoxine B. chez deux especes qui pr^sentent de facon constitutive
une grande difference de senaibilit^ k ses effets : le rat et la souris.
C'est ainai que pour le rat, la DL oscille entre 1 et 7 mgAg selon
lea souches de rat alors que pour la souris, elle se situe aux environs
-------
969
de 60 mg/kg. Ces differences de sensibilite se refletent sur le plan
me'tabolique : une dose de 1 mg/kg d'aflatoxine B. donne"e au rat
Wistar (souche Commentry) inhibe la synthese des RNA nucle"aires
de 80 % (temps d'action : 3 heures) alors que dans les m&mes
conditions, une administration de 60 mg/kg a la souris Swiss ne
ddclenche qu'une inhibition de 40 %. Des re'sultats du mSme ordre
sont enregistres au niveau de 1'activite RNA -polyme rase des noyaux
isole*s a partir des foies de cea deux especes (Tableau I).
Le traitement pre"alable des rats au phe"noba rbital a tou jours
comme re'sultat de diminuer 1 'inhibition produite par 1'aflatoxine B.
sur la transcription e'value'e, soit par 1 'incorporation iri vivo de
pre*curseurs dans le RNA nucle*aire, soit par la mesure in vitro de
1 'activit^ RNA polyme"rase des noyaux Isolds (Tableau l). La
stimulation des systemes de me'tabolisation avant 1 'administration
d'aflatoxine B. aboutit done a une diminution des effets toxiques
produits par la toxine. Ces re*sultats confirment les observations de
GUMBMANN et WILLIAMS (3), Mac LEAN et MARSHALL (4) et
NEAL (5) concernant les variations des pouvoirs toxique et
cance"rogene de 1'aflatoxine B. apres action des barbituriques,
Les re*sultats obtenus avec la souris sont totalement differenta.
Le traitement prealable des animaux par le phdnoba rbital produit
dans la plupart des cas une augmentation de 1 'inhibition de la
transcription induite par 1'aflatoxine B. (Tableau I). Ainsi par un tel
traitement, on peut sensibiliser une espece animale normalement
r^sistante aux effets toxiques de certains composes. Des observations
analogues avaient 6t6 faites pour le t^trachlorure de carbon e
[GARNER et Mac LEAN (6)], le i, i, i-trichior^thane [CARLSON (?)j
et la monocrotaline JALLEN et al. (8) chez le rat et pour la re"trorsine
chez le cobaye f WHITE et al. (9)1; cependant, c'est la premiere fois,
a notre connaissance, que la potential! sati on des effets toxiques de
1'aflatoxine B par le phe"noba rbital est
-------
970
TABLEAU I
INFLUENCE DU TRAITEMENT AU PHENOBARBITAL SUR
L'INHIBITION DE LA TRANSCRIPTION DANS LE FOIE
PAR L'AFLATOXINE B CHEZ LE RAT ET LA SOURIS
Inhibition
Synthese in vivo
de RNA * *
Activite
RNA polym^rase
des noyaux isoles
Rats
- Aflatoxine (1 mg/kg)
- Phenobarbital puis
aflatoxine
Souris
- Aflatoxine (60 mg/feg)
- PWSnobarbital puis
aflatoxine
83
35
41
63
74
55
33
48
Temps d'action de 1'aflatoxine : 3 heures
* Le % d'inhibition est calcule" par rapport aux te'moins
respectifs trait^s ou non par le phe*nobarbital.
14
* * Injection d'acide orotique- C, 30 minutes avant le
sacrifice.
L'implication de ces resultats est importante sur le plan de la
metabolisation des drogues dont les me'canismes se re"velent
extrSmement complexes et dont la stimulation peut aboutir a des
situations ambivalentes. D'autre part, elle souligne le danger
potential que peut presenter la superposition d'action de certaines
drogues et, dans ce domaine, on doit eVoquer les effets susceptibles
d'atteindre 1'homme dont le cadre de vie se trouve envahi par la
presence de composes capables d'interf^rer avec son propre
m^tabolisme.
-------
971
REFERENCES
1. WOGAN, C.N., Methods in Cancer Research, 7, 309 (1973).
2. MOULE, Y. et FRAYSSINET, C. , FEES Letters, 25., 52(1972).
3. GUMBMANN, M. R. et WILLIAMS, S. N. ,
Biochem. Pharmacol. , 19. 2861 (1970).
4. Mac LEAN, A.E.M. et MARSHALL, A.,
Br. J. Exp. Pathol., 52., 322 (1971).
5. NEAL, G.E., Biochem. Pharmacol., 2l_, 3023(1972).
6. GARNER, R. C. et Mac LEAN, A.E.M.,
Biochem. Pharmacol., 18, 645(1969).
7. CARLSON, G. P., Life Sciences, 1_3, 67(1973).
8. ALLEN, J.R. , CHESNEY, C.F. et FRAZER, W.J.,
Toxicol. Appl. Pharmacol., 23, 470(1972).
9. WHITE, I.N.H., MATTOCKS, A.R. et BUTLER, W. H. ,
Chem. Biol. Interactions, 6_, 207 (1973).
-------
973
BRONCHITE CHRONIQUE ET PERTURBATIONS FONCTIONNELLES
RESPIRATOIRES EN RELATION AVEC L1EXPOSITION A
DIVERSES NUISANCES : ETUDES DANS DIFFERENTS
GROUPES SOCIO-PROFESSIONNELS
Q. T, PHAM, B, MIRE, J, MARTIN/ P, SADOUL, J. H, KNELSON
Unit€ de Recherche 14, INSERM, Physio-Pathologie Respiratoire,
Vandoeuvre-les-Nancy, France
RESUME
Une etude epidemiologique a port£ BUT 581 ouvriers de sexe
masoulin, ages de 40 4 60 ans, de differents groupes sooio-pro-
feseionnels habitant la meme region de I'Est de la Franoe (ou-
vrierB du batiment,' ouvriers d'une usine 8i.d6rurgi.que, travail-*
leure sans exposition profeeeionnelle particuli&re). Lee eujete
ont ete interregna d. I'aide d'un questionnaire de la bronahite
(questionnaire C.E*C.A.)t examinee du point de vue olinique et
fonationnel. Lee examena fonationnels suivanta ont ete pratiques:
determination de la oapaoite vitale (CV) du volume expiratoire
maximum eeaonde (VEMS)f du volume residuel (VR)t oaloul deo rap-
porte VEMS/CV, VR/CTj Epreuvee au CO, en respiration unique et
en regime stable au repos et au oours d'un effort de 40 watte.
L'analyse dee symptdmes de bronohite ainsi que des perturbations
fonotionnelles observees a ete faite partiouliSrement en fonotion
des nuisances professionnelles reneontrees par les ouvriers du-
rant leur travail. L'exposition profeeeionnelle paratt, oertest
•jouer un rOle non nSgligeable sur les symptdmes de bronohite et
sur lee perturbations fonotionnelles, mais le role des habitudes
tabagiques est apparu beauooup plus important; les nuisances
entratnent q"uelques troubles respiratoires ohez les non fumeurs,
maie oes troubles 8ont beauooup plus frequents et plus importants
-------
974
chez les sujets exposes et gros fumeurs. II apparent un effet
potentialisateur nuisance et fumee de tabac et ceci eat particu-
lierement net lorsque I'on compare les groupes apres standardi-
sation de I'dge.
ABSTRACT
An epidemiological survey was carried out on 581 male wor-
kers aged between 40 and 60 from different social and professional
groups and living in the same region in Eastern France (building
operatives, iron and steel workers, workers with no specific
occupational health risks), The test subjects were given a
bronchitis questionnaire (ECSC questionnaire) and underwent a
clinical and functional examination. The following functional
tests were carried out: measurement of the vital capacity (VC)
of the maximum volume exhaled per second (MVES) and of the resi-
dual volume (UV), calculation of the MVES/VC and PV/TC ratios;
tests for CO by single breath and continuous breathing, both at
rest and under a 40 watt load. The bronchitis symptoms and the
functional disorders that were noted have been analysed with
particular regard to the occupational health risks encountered
by the workers in their jobs. Indeed, occupational health risks
do seem to have a not inconsiderable effect on bronchitis symp-
toms and functional disorders, but the effects of tobacco smoking
habits appeared to be far greater; some respiratory troubles in
the non-smokers are the result of the health risks to which they
are exposed, but these troubles are much more frequent and move
serious in the subjects who are exposed to health risks and are
heavy smokers. Health risks seem to be potentiated by tobacco
smoke, and this is shown especially clearly when the groups are
compared after standardization for age.
-------
975
L1etude du role des nuisances atmospheriques dans le develop-
pement des maladies pulmonaires chroniques doit tenir cotnpte de nombreux
facteurs, tels que la pollution atmospherique generale, les variations
climatiques, les conditions socio-economiques, mais aussi des expositions
professionnelles et des habitudes tabagiques comme le souligne B. FERRIS
(4).
Pour etudier plus particulierement ces deux derniers facteurs,
trois groupes socio-professionnels diffirents habitant la roeme region, done
sounds aux memes ambiances generales mais differents dans leurs expositions
professionnelles et dans leurs habitudes tabagiques personnelles ont ete
examines. Les resultats observes dans ces trois groupes sont ici presentes
et discutes, d'abord en considerant separement les groupes, puis plus
specifiquement en fonction des expositions.
MATERIEL ET METHODE
Les examens ont ete menes dans une ville de 1'Est de la France,
aupres de trois groupes 3 peu pres egaux de travailleurs. Le premier
groupe est constitue d'ouvriers du batiroent sounds professionnellement
aux poussieres et aux intemperies, le deuxieme groupe est constitue
d'ouvriers d'une usine siderurgique sounds durant leur travail & des
polluants multiples (poussieres, fumees, gaz irritants), enfin le
troisieme groupe est form6 de travailleurs peu exposes professionnellement,
constitue essentiellement par les employes et petits commer^ants de
1'agglomeration.
Seuls les bournes de 40 3 60 ans ont ete choisis, dans les deux
premiers groupes par tirage au sort sur la liste nominale professionnelle
apres elimination des sujets ayant une radiographie anormale (sequelles
de tuberculose, silicose, abces du poumon etc ...} et dans le troisieme
groupe, sur la liste electorale apres elimination des sujets pouvant
appartenir aux deux groupes precedents. Le tirage au sort est fait en
sorte que la repartition soit d peu pres identique dans chaque tranche
d'age de 5 ans entre 40 et 60 ans.
Ainsi, 196 ouvriers du batiment (groupe I), 200 ouvriers de
I1usine siderurgique (groupe II) et 185 habitants de 1'agglomeration
-------
FREQUENCE DE LA SYMPTOMATOLOGY DANS LES 3 GRODPES SOCIO-PROFESSIONNELS
Toux 0 Exp. 0
Toux +• Exp. 0
Toux + Exp. +
Toux + Exp. +
Dyspnee d* effort
GROUPE I
Ouvriers bat.
(196 sujets)
(100) 51 Z
(17) 8,5 Z
(38) 19,5 Z
(41) 21 Z xx
(37) 18,9 Z
GROUPE II
Ouvriers sidSrurg.
(200 sujets)
(89) 44,5 Z
(9) 4,5 Z
(38) 19 Z
(64) 32 Z
(43) 21,5 Z
GROUPE III
Habitants
(185 sujets)
** (107) 58 Z
(4) 2,1 Z
** (55) 29,6 Z
xxx (19) 10,3 Z
(39) 31 Z
X
XX
XX
VO
Tableau I
Les astfirisques placfis aprSs les chiffres de la 36 colotme indiquent les conparaisons des chiffres de la ISre
colonne et de la 36, et ceux entre les colonnes, les comparaisons inter-colonnes.
* p < 0,05 xx p < 0,01
xxx p <0,001
-------
977
(groupe III), soit au total 581 sujets, ont 6te examines par la meme
equipe de medecins et techniciens et par les meme equipe de medecins et
techniciens et par les memes appareillages. Un interrogatoire standard de
la bronchite chronique est utilise1 (interrogatoire adopte par fa
Comnunaute Europeenne Charbon et Acier (2), derive de celui du British
Medical Research Council (10}). II pennet de connaitre la symptomatologie,
les habitudes tabagiques, les expositions professionnelles des sujets.
Un examen clinique est ensuite pratique, suivi des epreuves fonctionnelles
comportant : la determination de la capacite vitale (CV), du volume
expiratoire maximum seconde (VEMS), du volume residual (VR) et les calculs
des rapports VEMS/CV et VR/CT ; des epreuves au monoxyde de carbone :
transfert du CO en apnee, transfert et ductances du CO en regime stable
au repos et au cours d'un effort de 40 watts.
RESULTATS
Dans les groupes Studies, les rSponses au questionnaire
permettent de dire qu'il y a plus de sujets se plaignant d'une toux et
d'une expectoration chronique s depuis au mo ins 2 ans (r£pondant ainsi &
la definition de la bronchite chronique) chez les ouvriers de 1'usine
sidSrurgique (groupe II) par rapport aux ouvriers du batiment (groupe I)
et aux habitants de 1*agglomeration (groupe III). Cette difference est
hautement eignificative (p < 0,01 et p < 0,001). C'est aussi dans le
groupe III qu'il y a le plus de sujets sans symptomatologie. II n'y a pas
de difference quant & la frequence de la dyspn£e d'effort qui est signaled
dans les trois groupes A peu pres chez I sujet sur 5.
Les examens fonctionnels montrent que les valeurs spirographiques
sont dans 1'ensemble meilleures dans le groupe des ouvriers de sidSrurgie
(groupe II) par rapport aux deux autres, malgrg les donn£es cliniques
anterieures. Dans les trois groupes, on note une dScroissance de la
capacit€ vitale et du VEMS en Z des valeurs theoriques (ces valeurs
tiennent compte d6j4 des facteurs taille et 3ge des sujets) en fonctlon de
1'Sge. Ceci pennet de dire que les anomalies spirographiques sont plus
frSquentes chez les sujets 3ges, en particulier dans le groupe III
(Fig. I et 2). II n'existe pas de difference significative dans les
fipreuves au monoxyde de carbone pour les trois groupes, d'un groupe d
-------
978
C Wheor.
110, V.
105.
100.
95.
90-
05.
VEMS%fh«or.
110HV.
105.
100^
95-
90.
esJ
Bcfimrnl
40-44
45-49
50-54
55-60 001
~~T
40-44
1
45-49
50-54
55-60
on»
Fig. 1. Modifications de la CV#the- Fig. 2. Modifications du VEMS#-
orique pour un groupe d'ouvriers de theorique pour un group d'ouvriers
eiderurgie.
de siderurgie,
CV*rheor.
DuCO
105.
100.
95-
90-
85-
V.
105.
100-
95
90.
40-44
<5-<9
50-54
55-60 ons
A
Hotnlonti
Fig. 3. Modifications du CO repos-
$theorique pour un groupe d'ouvriere
de siderurgie.
0 1-9 10-19
Fig. *f. Effete du tabac sur la
CV^theorique.
-------
979
1'autre et d'une tranche d'age 3 1'autre lorsqu'on exprime les resultats
en pourcentage des valeurs theoriques (Fig. 3).
En fonction du tabac, on note peu de modifications de la
capacite vitale Z de theorique (Fig. 4) mais par centre le VEMS Z de la
theorique est plus abaisse chez les plus gros fumeurs, ceci dans les trois
groupes socio-professionnels et laisse penser que I'obstruction bronchique
est plus marquee chez ces sujets (Fig. 5). L'efficacite ventilatoire jugee
par la ductance du CO est plus abaissee chez les fumeurs, par rapport
aux non fumeurs, mais il n'y a pas de difference selon I1importance des
habitudes tabagiques (fig. 6).
VEMSMheor.
no-,'/.
105.
100
95
90
\
\
HotMlontt
0 1-9 10-19 «20ci9ar»««
Fig. 5. Effete du tabac sur
le VEMStftheorique.
OuCO repos V. theor.
105. V.
100.
95.
90.
as.
W 10-19
Fig. 6. Effets du tabac sur le
C0-repos#theorique .
La difference dans 1*exposition tenant compte des groupes socio-
professionnels, comme il vient d'etre present§, est relativeroent grossiire.
Au sein du meme groupe, les sujets sont plus ou moins exposes et il est
plus logique de consid€rer les sujets selon leur exposition individuelle.
L'on constate ainsi que le taux de bronchite est toujours plus
eleve chez les sujets ayant une exposition professionnelle, ceci quelie
-------
980
que soit la tranche d'age envisagee (fig. 7). Cependant 1'influence du
tabac est aussi indiscutable : chez les non fumeurs, les sujets exposes
ont certes un taux de bronchite plus eleve (21 %) que les non exposes
(6,5 %), mais ce taux est nettement plus bas que chez les sujets sans
exposition professionnelle mais gros fumeurs (39,5 %). Chez les gros
fumeurs, exposes professionnellement, le taux de bronchite atteint 63,5 Z
(pres des 2/3 des sujets) 'Fig. 8).
so v.
60.
40.
20.
OJ
l~~l non rxfxuri
40-44
45-49
50-54
55-40ons
Fig. ?. Taux de bronchite selon age
et expositions professionnelles.
60,
20.
OJ
I J non fipoitl
A/on A/meurs Gro» /umeura
Fig. 8. Taux de bronchite
suivftnt tabac et expositions
La frequence de la dyspnee est aussi plus grande chez les gros
fumeurs qui sont exposes professionnellement 3 d'autres polluants (Fig. 9),
Quelques unes des donnees fonctionnelles, dont les valeurs moyennes sont
rappelees dans le tableau II, montrent indiscutablement des perturbations
plus marquees chez les sujets fumeurs et exposes professionnellement a des
nuisances. Cette difference est surtout tres significative pour TCO/V.
La plus grande frequence d'un syndrome obstructif (rapport VEMS/CV .<65Z)
est rappelee dans la fig. 10.
La nature de 1'exposition professionnelle joue-t-elle un role ?
Ce point a ete particulierement etudie dans le groupe des ouvriers
siderurgiques ou les expositions sont les mieux caracterisees. Dans ce
-------
CV Z, VEMS % et TCO/m2 EN FONCTION DE LfEXPOSITION, DES HABITUDES TABAGIQUES ET DE I/AGE
CV 5!
Non ExposS
Expos6 profesaionnellement
40-44 ana 45-49 ana
Funeur
Non Funeur
Fumeur
Non Fumeur
Fumeur
Non Fumeur
106,17
(13,4)
105,10
(20,3)
105,00
(14,3)
*
106,01
(28,3)
14,29
€6,1)
*
18,25
(5,5)
95,16
(14,0)
102,73
(17,6)
96,14
(19,9)
108,00
(19,5)
13,33
(3,1)
19,36
(5,8)
50-55 an s 55-60 ans 40-44 ans
96,23
(25,3)
94,83
(13,8)
95,32
(17,5)
99,82
(15,2)
13,17
(2,52)
14,20
(3,6)
91,95
(14,4)
101,00
(29,6)
VEMS %
89,2
(21,5)
99,33
(27,1)
TCO/m2/min . /nonHg
11,92
(2,7)
**x
17,60
(7,4)
100,40
(15,9)
104., 23
(18,8)
100,65
(21,3)
*
115,23
(24,3)
(apnge)
12,67*
. (3,1)
14,29
(6,1)
45-49 ana
98,68
(17,33)
95,40
(17,1)
98,23
(23,9)
104,90
(27,7)
11,62**
(4,0)
13,33**
(3,1)
50-55 ans
96,09
(16,7)
98
(18,0)
95,87
(19,8)
104,84
(18,1)
10,64***
(3,0)
**
13,17
(2,5)
55-60 ans
93,98
(14,54)
92,9
(15,66)
88,57
(20,9)
*
99,15
(20,3)
9,94***
(2,6)
**
11,92**
(2,7)
Tableau II
Lea ast£risquea place's aprSs lea chiffrea des colonnes de droite comparent cellea-ci avec lea colonnes
correapondantes, a gauche. * < 0,05 ** < 0,01 *** < 0,001.
10
oo
-------
982
VEMS/CV < 65V.
Dyspnee
40.
30.
20-
10
1-9
10-19 »20cigar«ll«
Fig. 9. Effets du tabac eur la
dyspnee.
25.
20.
15.
10
5J
0 1-9 10-19 %,20cigar*rt*s
Fig. 10. Effete du tabac sur le
VEMS/CV.
groupe, nous avons distingue les sujets sans exposition, ceux exposes
unique ment aux poussieres, et ceux ayant une exposition multiple, poussieres
fumees et vapeurs chimiques.
La frequence de la toux et de 1'expectoration chroniques est
plus importantes chez les sujets exposes surtout aux poussieres. II n'y a
pas de difference nette dans la frequence de la dyspnee ou des poussees
pulramaires aigues durant les trois dernieres annees. Les habitudes
tabagiques sont comparables dans les trois sous-groupes selon la nature
de 1'exposition (Fig. 11). La comparaison des donnees fonctionnelles de
ces trois sous-groupes est rappelee dans les tableaux III et IV. On ne
constate pas de difference notable dans les valeurs spirographiques. Par
centre, il existe une baisse significative de TCO/V et de la ductance
du CO a 1'effort dans les groupes sounds a une exposition multiple. Ces
resultats font se demander si 1'exposition aux polluants ne deteriorerait
pas plus specifiquement 1'efficacite des echanges, et soulignent 1'interet
pour les etudes epidemiologiques non seulement des techniques spirogra-
phiques classiquement utilisees, mais aussi des epreuves permettant de
juger de 1'efficacite des echanges, en particulier des epreuves au CO.
-------
983
REPARTITION SELON (.'EXPOSITION
60.
40
20
OJ
I
60.
40
20
0
Tbux chr. Exprcl chr. Dyifmff £pwx/« pulm
CD •
Habitude lobagiquf
1a9
10 a 19 2.20 cigar»H«
Fig. 11. Les differents effets pul-
monaires et habitudes tabagiques en
fonction de 1'exposition.
DISCUSSION
Dans cette etude, nous nous sonnnes adresses 3 deux groupemenCs
socio-professionnels bien definis alors que le troisieme groupe est
constitue par les habitants de I1agglomeration. Comme le souligne B. FERRIS
(3) et FLETCHER (5), les groupes de travailleurs constituent des popu-
lations speciales et eelectionnees. Les sujets doivent etre en bonne sante
pour pouvoir obtenir leur travail, d'autre part les personnes qui peuvent
avoir un certain degre d'atteinte pulmonaire ne choisiraient pas le metier
d'ouvrier metallurgiste mais plutot un metier repute peu expose comme
celui de commer^ant par exemple. Ceci pourrait expliquer les differences
que nous avons constatees du point de vue spirographique entre le groupe
des ouvriers de 1'usine siderurgique avec les autres groupes. D'autant que
pour le groupe des habitants de 1*agglomeration, nous n*avons pu faire
-------
RESULTATS EN FONCTION DE L1EXPOSITION DUBANT LE TRAVAIL
I II
aucune exposition exposition aux
n - 31 poussieres
n - 64
CV Z de la thfiorique
M 98,60
o 19,30
VEMS Z de la thSorique
M 103,00
a 27,00
VEMS/CV Z
M 76,50
o 9,20
VR/CT %
M 27,10
a 7,80
99,20
16,70
99,40
21,60
74,40
13,00
26,25
6,70
III Test t
poussieres, I-II I-III II-III
flanges et vapeurs
n - 98
99,90
16,80
101,40
22,5
74,70
10,70
26,39
8,18
vo
00
Tableau III
(Ne figure pas dans ce tableau le sous groupe des 7 sujets exposes aux fumges et/ou aux vapeurs chimiques). II
n'y a pas de difference significative au point de vue spirographique selon les expositions professionnelles.
-------
RESULIATS EN FONCTION DE L'EXPOSITION DURANT LE TRAVAIL
REGIME STABLE
TCO/m2, ml/min/mnHg
Repos
M
a
Exercice
H
a
TCO/V
Repos
M
0
Exercice
M
a
APNEE
TCO/m2
M
a
DuCOZ de la thgorique
Repos
M
a
Exercice
M
a
I
aucune 'exposition
n - 31
13,81
2,66
14,77
3,92
2,11
0,65
2,62
0,72
13,55
2,50
99,10
13,40
105,70
15,90
II
exposition aux
poussiSres
n - 64
12,91
4,17
13,69
3,60
1.94
0,69
2,63
0,83
12,58
2,72
99,70
-10,60
104,70
14,80
III Test t
poussi&res,
fumees et vapeurs I~III II-III
n - 98
12,66
3,59
•f y ^ ^
13,35
3,96
1,80
0,63 *
2,34 * *
0,72
r
12,60
2,73
™ f • •*
98,30
12,00
100,70
12,5 * *
CO
Ul
Tableau IV
Ne figure pas dans ce tableau, le sous-groupe composg de 7 sujets exposes aux fumees et/ou aux vapeurs cbimiques
C'est surtout les mesurea au CO a 1'effort (TCO/V et ductance) qui distinguent le sous-groupe des sujets exposes
aux polluaats multiples. or j Fw.ca
-------
986
lea eliminations d'apres les radiographies comme pour les deux autres
groupes pour lesquels les radiographies systematiques de medecine du
travail ont pu etre faites tous les ans. Afin de rendre le plus comparable
possible les groupes socio—professionnels, nous n'avons choisi que des
sujets homines, de 40 a 60 ans, en echantillons stratifies par 5 ans d'age
et nous avons suppose qu'habitant la me me petite agglomeration les facteurs
atmospheriques seraient les mimes. En fait, selon les vents dominants, la
repartition des fumees et poussicres de 1'usine sur 1'agglomeration peut
etre differente d'un jour a 1'autre. Ceci etant, 1'etude comparative qui a
ete faite ici permet de bien souligner, comme 1'ont deja fait LOWE et coll.
(9), FRAPPIER-DAVIGNON et coll. (6), I1influence importance du facteur
tabagique qu'il faut prendre en consideration chaque fois que 1'on veut
etudier le role des pollutions dans lee maladies pulmonaires chroniques.
Loin cependant de conclure comme LOWE (9) qu'il s'agit du seul facteur
important, les resultats ici presentes montrent que la pollution profesion-
nelle joue un role indiscutable. Les resultats montrent aussi qu'il y a un
effet potentialisateur indiscutable du tabac et les facteurs de nuisance
atmospherique professionnelle. L'interet des epreuves au CO dans les
enquetes epidemiologiques est ici bien souligne par les differences
observees seulement avec ces tests dans les groupes distingues selon leur
type d'exposition professionnelle.
Travail ayant beneficie de 1'aide financiere de la Commission des
Communautes Europeennes.
BIBLIOGRAPHIE
BECKENKAMP, H.W., Chronische Bronchitis und ..Arbeit splat z.
in Thorax Krankheiten. Dtsch Med. J., 5, 23-26 (1971).
BR1LLE, D., VAN DER LENDE, R., SANNA RANDACCIO, F., SMIDT, U.,
MINETTE, A., Commentaires relatifs au questionnaire de la CECA pom-
1'etude de la bronchite chronique et de I'emphyseme pulmonaire.
2eme Edition, Luxembourg (1972).
FERRIS, E.G.Jr., BURGESS, W.A., WORCESTER, T., Prevalence of
chronic respiratory disease in a pulp mill and a paper mill, in
United States, Brit. J. Industr. Med., 24, 26-37 (1967).
-------
987
4 FERRIS, B.C.Jr., Epideroiological studies on Air Pollution and Health.
Arch. Environm. Health, 16, 541-555 (1968).
5 FLETCHER. C.M., ELMES, P.C., FAIRBAIRN, A.S., WOOD, C.H., C.H., The
significance of respiratory symptoms and the diagnosis of chronic
bronchitis in a working population. Brit. Med. J., 2, 257-266 (1959)
6 FRAPPIER-DAVIGNON, L., JEGIER, S., DROUIN, C., MARIER, J., ROY, L.P.,
TOURANGEAU, F.J., Etude de 1'effet combine de la pollution atmosphe'-
rique, de 1'exposition professionnelle et des habitudes de tabac
dans les affections pulroonaires obstructives. I - Methodologie.
Hyg. et Med. Soc., 102, 1537-1541 (1973).
7 HIGGINS, I.T.T., OLDHAM, D.D., COCHRANE, A.L., GILSON, J.C.,
Respiratory symptoms and pulmonary desability in a industrial town
Brit. Med. J. 2, 904 (1956).
8 HIGGINS, I.T.T., COCHRANE, A.L., GILSON, J.C., WOOD, C.H., Popu-
lation studies of chronic respiratory disease. A comparison of
miners, foundry workers and others, in Staveley. Derbishire, Brit.
of Industr. Med., 16, 255-268 (1959).
9 LOWE, G.R., CAMPBELL, H., KHOSLA, T., Bronchitis in two integrated
steel-works. 3 - Respiratory symptoms and ventilatory capacity
related to atmospheric pollution, Brit. J. of Industr. Med., 27,
121-129 (1970),
10 Medical Research Council's committee on the aetrology of chronic
bronchitis (1960). Standardised questionnaires on respiratory
symptoms. FLETCHER, C.M. (Chairman), CLIFYON, M., FAIRBAIRN, A.S.,
FRY, J., GILSON, J.C., HIGGINS, I.T.T., MAIR, A., PEMBERTON, J.,
ROGAN, J.M., SMITH, D.H., and WOOD, C.H. (secretary), Brit. Med.
J., 2, 1665 (1960).
11 MINETTE, A., Le probleroe de la bronchite chez les ouvriers metal-
lurgistes, Cahiers Med. Trav., 4, 71-127 (1966).
12 PHAM, Q.T., GIMENEZ, M., MYRE, M., CHASPOUL, H., MARTIN, J.,
Contribution a 1'epidemiologie de la bronchite chronique chez les
Travailleurs du batiment. Bull. Physio-path. Resp., 8, 769-795 (1972)
13 PHAM, Q.T., MYRE, M., MARTIN, J., KNELSON, J., GRAIMPREY, J.,
Prevalence de la bronchite chronique dans differents groupes socio-
prp_fe8_si_onne_ls_v II Ouyriers d'une usine siderurEique, Respiration
(a paraitre).
14 RAE, S., WALKER, D.D., ATTFIELS, M.D., Chronic bronchitis and dust
exposure in British coal miners, Inhaled Part. Vap., 2, 883-896
(1970).
15 WORTH, G., MYSERS, K., SMIDT, U., GASTHAUS, L., The epidemiology
of bronchopulmonary symptoms in coal miners, foundry workers.
chemical, and bakers, Bull. Physio-path. Resp., 6, 617-636 (1970).
-------
988
DISCUSSION
LEFEVRE (Belgique)
Voulez-vous donner des precisions sur le calcul du facteur
de transfert du CO envers les sujets fumeurs ou non fumeurs.
PHAM (France)
Ce facteur de transfert a 6t6 e"tudi6 par la technique en
respiration unique (single breath) et par la technique en &tat
stable. Pour chaque sujet, 1'analyseur est reglS au point z6ro
sur 1'air expire du sujet (fumeur ou non fumeur) afin d'eViter
les inconvenients de la "back pressure"
-------
989
LARMBELASTUNG, KOHLENMONOXIDEBELASTUNG UND
KOMBINATIONSWIRKUNGEN
M, HAIDER/ E, GROLL-KNAPP/ M, NEUBERGER, H.-G, STIDL
Institut fur Urawelthygiene der Universitat Wien, Osterreich
KURZFASSUNG
FUr die Featlegung von LUrmgrenzwerten aind u. a. Vnterau-
chungen der zeitueiligen HOraohwellenveraohiebung (TTS) und dee
Htfrerholungsvorganga von Bedeutung. In unaeren Experimenten
konnte gezeigt werdenf daaa die Rtlckbildung der H&rerrnUdung bei
gleiohzeitigev Ldrmbelaetung sahon ab 65 dB verzOgert werden kann.
Zwischen 75 und 85 dB steigt diese VerzOgerungswirkung stark an
und sahlagt bei etwa 85 dB in zunehmende HOrermUdung urn.
•f
Uber die Kohlenmonoxiduirkung auf den Menachen konnten wir
eine Reihe payohophysiologiacher Experiments durchftihren, u. a.
konnten dabei Auswirkungen auf die Informationeverarbeitung im
Gehirn mit Hilfe von computeranalyeierten Potentialen nachgewiesen
werden. Urn Aufeahlilaae Uber mOgliche Kombinationsuirkungen zu
erhalteni uurden 20 Vereuohapereonen in einem Doppelblindverauoh
4 Stunden mit 200 ppm Kohlermonoxid belaatet und wtlhrend der letz-
ten 1/4 Stunde einem Ldrm von 105 dB auageaetzt. Die Hdraoh-
uellenveraohiebung und der Verlauf der HSrerholung (1 Stunde)
zeigten keine statistiaoh aignifikanten Unterschiede.
Vahrend fur hohe COHb-Werte synergiatiaohe Wirkungen mit
Larm auf daa Horainneeaystem beaohrieben aind, zeigen dieae
Ergebniaae, daaa bei geringen Doaen die beiden Umweltnoxen in*
different* alao voneinander ueitgehend unabhangigt wirken dtlrften.
Der Ldrm bevirkt die Horechwellenverachiebung duroh Einuirkung
-------
990
auf die Haarzellen im Innenohr, wahrend das Kohlenmonoxid eher
die zentrale akustische Informationsverarbeitung beeinflusst.
(Mit finanzieller UnterstHtzung des Osterreichischen "Ponds
zur FSrderung der wissenschaftlichen Forschung" und der "All-
gemeinen Unfallversicherungsanstalt". )
ABSTRACT
In determining limits for noise levels two of the important
.factors are investigations into temporary threshold shifts (TTS)
and the hearing recovery process. In our experiments we were
able to demonstrate that the regression of aural fatigue from
coincident noise levels could be delayed from 65 dB upwards.
This delaying effect increases sharply between 75 and 85 dB and
at approximately 85 dB suddenly changes to an increase in aural
fatigue.
We were able to conduct a series of psyahophysiological ex-
periments on the effect of carbon monoxide on man. One of our
findings was evidence of the effects on data processing in the
brain, which was obtained with the help of computer-analysed
potentials. In order to obtain data on possible combined ef-
fects 20 subjects were subjected to 200 ppm carbon monoxide for
4 hours in a double blind test, and during the final quarter of
an hour a noise at a level of 105 dB was added. No statisti-
cally significant differences were found between the temporary
threshold shifts and hearing recovery time (1 hour).
While noise-related synergistic effects on the auditosensov-y
system are described for high COEb values, these results show
that the effects of both environmental contaminants, in small
doses, are probably indifferent, and therefore mainly independent
of each other. The noise, acting on the hair cells of the inney
ear, produces temporary threshold shifts, while the effect of the
carbon monoxide is rather on central acoustic data processing.
-------
991
1) Einleitung
LSrm und Kohlenmonoxid sind zwei so welt verbreitete Umwelt-
noxen, daft es sehr wichtig erscheirit, auch ihre mogliohen
Kombinationswirkungen sorgfSltig abzugrenzen. Fur hohe COHb-
Werte konnte eine synergistisehe Wirkunp - wahrscheinlich
aufgrund der hervorgerufenen Hypoxidose im Innenohr - mit
LSrm nachgewiesen werden. Fur geringe Dosen mussen aber die
moglichen Kombinationswirkungen noch eingehend untersucht
werden. Eirien Beitrag dazu sollen unsere Untersuohungen
uber Horerholung unter LSrmbelastunp, sowie uber Kohlenmono-
xidwirkung auf das Zentralnervensystem und schlieBlich uber
das Zusammenwirken von La'rm- und Kohlenmonoxidbelastung bei
der Beeinflussung von Horermiidung und Horerholunp; liefern.
2) Horerholung unter LMrmbelastung
Der iSrmbedingte Horverlust beruht auf der Summierung wie-
derholter Gehorermudungen. Die HorsehSdip.unp, wird durch eine
Stoffwechselinsuffizienz der Innenohrhaarzellen bewirkt und
schreitet umso intensiver fort, je haufiger ein nooh ermii-
detes Ohr neuerlichen LSrmbelastungen ausgesetzt ward. Un-
tersuchungen uber zeitweilige Horschwellenversohiebungen
(TTS) sind sehr wichtig, urn die GesetzmSftigkeiten der Hor-
ermiidung und der Horerholung aufzuklSren und entsprechende
prophylaktische MaBnahmen vorzuschlagen. In einigen expe-
rimentellen Untersuchungen (Schwetz et al. 1, Doooler et
al. 2) konnten wir die Horerholung bei gleiohzeitiger Ein-
wirkung eines weiften Rauschens bzw. eines OktavbandgerSu-
sches mit 2ooo Hz Mittenfrequenz untersuchen. Wir konnten
dabei nachweisen, daft die gleichzeitige Gerauscheinwirkung
die Gehorerholung stark zu reduzieren vermag. Bereits bei
eine^m "Ruheiarmpegel" von 65 dB ist die Verzogerung der
Horerholung nachweisbar, wenn auch erst nach einer Stunde
und nur in geringem Ausmaft. Bei einem "RuhelSrmoegel" von
75 dB Oder zwischen 75 und 85 dB steigt die Verzogerungs-
wirkung stark an und schlagt bei 85 dB und dardber in zu-
nehmende Horermiidung um. Die beobachteten HorerholungskuF-ven
-------
992
dB
30
25-
1Q
65beo.
75,65,30 erw.
3Obeo.
15' 30' 60' 120'
Abb.l Zeitlicher Verlauf der [lorerhol imp; bei gleichzei'
•r LarmeinWirkung von 3o, 65, 75, 85 und 95 dB (au:v
gezoRene Kurvori ) . Die stri chliorten Kurven stellen die
nach Pormeln crrechneten Worte dar. Nach einer Stunde
weicbt nogar die Horerholunp, unter Gerliuscheinwirkunp,
von 65 dB von don berechneteri Erwartungswerten ab.
-------
993
CLICK
EP1
EP2
100msec
O
CO
Abb.2 Akustisch evozierte Potentiale mit und ohne Kohlen
monoxideinwirkung. Die schematische Darstellung der
spSten Potentialanteile ISBt erkennen, daB nur geringe
Amplituden- und Latenzunterschiede bestehen.
-------
994
sind in Abb. 1 den rifjch Bercchnunpyn (Ward 3) erwarteton
K u r ven ge gf-'n fibe rge s te ) 11 .
5) Auswirkungen geringer Kohlomnonox i ddoscn auf Himpoton-
tiale urid Horfunktion
In einigen Versuehsreihen (Grol 1-Knaon ot al . JO konnton
wir nachweison, daft auch goririge Dosen vori Koh 1 en mono x id
Veranderungen bei computeranalyaierton HirnpotonliaIon ho-
wirken konnen. Die dui-ch Cllc:kr, ovo7.ierton Pol,f-nl.i n 1«- 7,eip,-
t,en daboi allerdirip;s riur uohr fori np;o UntorGcbiodp
CO-Bedingunp; und Kontrol Ibocli np-.unp;. Die:-, ir.t in Abb.?
stellt. Die Amplitudon sind untep CO-K i nwi rkunp, Ptwar, re-
duziertv. Mehr Unto rschiedc1 zeip;en die lanp.r.omnn Hirnnoten-
tiale. Solche bilden aich bei spiel swei tie 7.wischon zwo i Roi -
zen, einem Warnreiz und einem imnerat.i ven F^ei?,, in F,rwar-
tungssituationen au:?. Boiapiele mit dout lichen Kndorungpn
unter CO-Einwi rkung frep;en\\b^r dor Kontrollbedi np.unp, mil.
Normalluft sirid in Abb. 3 p;e/,eip;t. Offonsi chtlinh kann die
Informationsverarbeitunp; im Zentralnervonpystem ,durch F,in-
wirkung von Kohlenmonoxid nuch in ger-ingen Donen ger.tort
werden. Die Storung 1st deutlicher bfi e lokt,rint:hen Kirn-
potentialen, die mit komplexen Si tuat i onen, wie F>war>tungci-
vorgangen, verknupft sirid.
Ototoxische Wirkungen des Kohlenmonoxi dt; r^ind r,eit langem
beschrieben, durften aber erst bei hoheren Konzentrationen
auftreten. Horverluste wurderi vor allem nach Kinwirkung von
Generatorgas bei einem bet rScht 1 i chen Pro7.ent.sat7 de r unter-
puchten Pernonen gefunden. (Lumio 5)
4) Kombi nationswirkungen von La'r-m und Kohlenmonoxid
Es gibt eine Heihe von klinischen Beobachtungen uber Arbeits-
situationen, bei denen sowohl La'rmbelaytunp. al? auch Koh-
lenmonoxidbe'lastung vorlagen. Es ist dabei schwierig fest-
zustellen, ob die eine odor die andere Noxe aich in finer
Horstorung auswirkte, oder ob beide Noxen oine kombinierte
Wirkung batten (Wageniann 6). In Tiervorsuchen wurde einer-
seits eine durc-.h I-.a'rm verzogerte Koh lenmonoxidauRscheidung
-------
995
NOmULQEHALT
*0 pprtiCO
VpGRO
VpV»L
Abb.3 Beispiele von Erwartungswellen mit und ohne Koh-
lenmonoxideinwirkung. Die untereinander gezeichneten 3
Kurven jeder Vp. entsprechen den 3 Abschnitten eines
Vigilanztests.
-------
996
TTS 3000
CO
2OdB
16
14
12
10
16
32
64
• in.
Abb.4 Zeitlicher Verlauf der HSrerholungskurven nach 15
Minuten L&rmbelastung (Io5 dB OktavbandlSrm, 2ooo Hz
Mittenfrequenz). Die ausgezogene Kurve mit vorheriger
CO-Belastung und die strichlierte Kurve ohne CO-Be-
lastung zeigen nur geringe^statistisch nicht signifi-
kante Unterschiede.
-------
997
behauptet (Zorn 7) und andererseits treten durch Koblenmo-
noxid bewirkte histologische VerSnderungen an den Haarzel-
len bei gleichzeitiger L&rmeinwirkung friiher ein (Kittel
und Theissing 8). In diesen Fallen 1st also eine syner-
gistische Kombinationswirkung gegeben. Die COHb-Werte lagen
allerdings in den letztgenannten Untersuchungen sehr hoch.
Sie betrugen 5o/8 und mehr.
Urn AufschlUsse fiber mSgliche Kombinationswirkungen auch bei
Einwirkung geririgerer Kohlenmonoxiddosen zu erhalten, ha-
ben wir die Horermiidung (TTS) und die H5rerholung mit und
ohne vorherige CO-Einwirkung bei 2o Vpn untersucht. Sie
wurden in einem Doppelblindversuch entweder einer Atmospha-
re von 2oo ppm CO oder normaler Atemluft fUr je ^ .Stunden
ausgesetzt. WShrend der letzten 15 Minuten wurden sie je-
desmal mit einem OktavbandlSrm von Io5 dB und einer Mitten-
frequenz von 2ooo Hz beschallt. Die zeitweilige HSrschwel-
lenverschiebung wurde mit TesttQnen von 3ooo und 4ooo Hz
nach 4, 8, 16, 32 und 64 Minuten geprUft. Die H8rerholungs-
kurven fQr Testtbne von 3ooo Hz sind in Abb.^ dargestellt.
Die TTS-Werte nach kombinierter CO- und LSrmeinwirkung sind
bei der ersten Messung etwas hoher als in der Kontrollsi-
tuation, bei den weiteren Messungen aber praktisch gleich
und bei der letzten Messung nach einer Stunde sogar etwas
niedriger. Keiner der Unterschiede war statistisch signi-
fikant. Die COHb-Werte liegen zum Zeitpunkt der Beschallung
bei ca. 13?. Bis zu dieser H5he verhalten sich demnach Koh-
lenmonoxidbelastung und LSrmbelastung hinsichtlich der zeit-
weiligen HSrschwellenverschiebung i,ndif ferent. Die Ursaehe
liegt wahrscheinlich darin, dafi> Kohlenmonoxidwirkungen in,
geringen Dosen mehr zentral angreifen, also die akustische
Inf<5rmationsverarbeitung im Rahmen komplexerer Aufmerksam-
keits- und ErwartungsvorgSnge beeintrSchtigen, wShrend LSrm
•hinsichtlich der TTS vorwiegend die Punktion der Haarzellen
im Innenohr herabsetzt. Synergistische Wirkung zwischen
LSrm und Kohlenmonoxid wird offensichtlich erst dann fest-
-------
998
stellbar, wenn auch die Hypoxidose in den Haarzellen des
Innenohres durch Kohlenmonoxid allein ein merkbares Aus-
maft erreicht.
Es wird Aufgabe weiterer Untersuchungen sein festzustellen,
ob, wann und unter welchen Umweltbedingungen mit dem Beginn
synergistischer Wirkungen zwischen Kohlenmonoxid und L&rm
beim Menschen zu rechnen ist.
Literatur :
1) Schwetz P., Donner R., Langer ft. und Haider M. ,
Experimentelle Horermudung und ihre Ruckbildung unter
Ruhe- und Larmbedingungen
Monatsschr. f. Ohrenhei Ikunde , Laryngorhinologie ^_, 162
(197o)
2) Doppler U., Haider M. , Scheiblechner H. und Schwetz P.,
Experimentelle Horermudung und ihre Rilckbildung unter
industrieShnlichen , schmalbandigen La'rmbedingungen
Monatsschr. f. Ohrenheilkunde , Laryngorhinologie 6^, 2^5
(1973)
3) Ward W.D. ,
Temporary Threshold Shift in a Changing Noise Level
J .acoust .Soc.Amer. 32, 235 (196o)
4) Groll-Knapp E., Wagner H., Hauck H. und Haider M. ,
Effect of low Carbon Monoxide Concentrations on Vigilance
Computer-Analysed Brain Potentials
In: Carbon Monoxide - Origin, Measurement and Air Quality
Criteria, 116-119, DUsseldorf (1972)
5 ) Lumio J .3 . ,
Otgneurogical Studies of Chronic Carbon Monoxide Poison-
in Finland
\cta oto.-laryng. (Stockholm), Suppl . 65-112 (19*48)
6 ) Wagemann W. ,
Das otologi_sche Bild der Kohlenoxidvergiftung
Zschr. f. Lar.Rhin.Oto. 38, 691 (196o)
-------
999
7) Zorn H. ,
Die kombinierte Wirkunp; physikalischer und chemischer
Noxen, aufgezeigt am Beispiel der Schadigung durch La>m
und CO
Schriftenr. Arb.Med.Soz.Med.Arb.Hyg.Stuttgart, Bd.27 (1968)
8) Kittel G. und Theissirig G.,
Histologische Ur^tersuchungen dei- Cochleaan HSutchenprg.-
£ajraten und Treppenserienschrii tten nach hochgradiger pro-
trahlerter Hypoxldose
Arch. f. klin. u. exp. Ohren-, Nasen- und Kehlkopfheilkunde
191, 53*1 (1968)
DISKUSSION
JANSEN (Bundesrepublik Deutschland)
Ihre Beobachtungen iiber Horermudungen stimmen nicht mit denen
Von Ward viberein. Dies 1st auch anderen Forschern aufgefallen.
Ward erklarte, dass seine Beobachtungsformeln nur fiir einmalige
Schallereignisse gelten. Frage: Haben Sie nur "einmalige" Oder
"intermittierende" Gerausche in Ihren Untersuchungen angewandt?
HAIDER (Oesterreich)
Wir haben zwar zur Erzeugung der Horermiidung dieselben
Versuchsbedingungen wie Ward angewandt, haben aber die Horer-
holung langer beobachtet als Wart dies urspriinglich tat. Daraus
ergaben sich die Unterschiede. Wart hat in neueren Untersuchungen
unsere Ergebnisse bestatigt.
-------
1001
REVALUATION OF EPIDEMIOLOGICAL HEALTH EFFECTS FORMERLY
ATTRIBUTED TO MEASURED LEVELS OF NITROGEN DIOXIDE IN
VIEW OF SYNERGISTIC EFFECTS DUE TO CO-POLLUTANTS
PETER 0, WARNER AND LAZIER STEVENS
Wayne County Department of Health, Air Pollution Control Division
Detroit, Michigan, USA .
ABSTRACT
Data have been collected to -indicate the presence of several
synergistically interfering air contaminants in ambient air in
the immediate vicinity of Chattanooga* Tennesseet where epidemi-
ological studies have classically related observed human respir-
atory effects to measured elevated levels of NO, alone.
Such data appear to support recent evidence showing the re-
lationship between levels of small particle sulfate aerosol and
indicence of respiratory disease.
The possible synergism between sulfate aerosol and NO^ is
discussed and compared to data from the recent U.S. Government
Community Health Environmental Systems Study (CHESS) partiaulate
in the particle size range below 3.5 microns.
Levels of aerosol sulfate recently measured in an urban in-
dustrial area are presented together with sulfate distribution
as a function of particle size range.
It follows from these data that adverse health effects clas-
sically attributed to nitrogen dioxide may be accounted for either
totally or in part by the presence of epidemiologically interfering
compounds such as aerosol eulfate or acid mist whose specific meas-
urements have not previously been a part of epidemiological surveys.
-------
1002
Introduction
Between November 1968 and Apr±l 1969, a study of "acute
respiratory illness" was conducted among families residing in
the areas of the Greater Chattanooga School District which in-
cludes Hamilton County, Term., and Walker County, Ga. These
areas are in close proximity to a large TNT plant which may be
considered responsible for elevated levels of nitrogen dioxide
for which reason the area was chosen as a single-pollutant re-
lated population study. [1]
Conclusions flowing from this study have been employed as
classical evidence of human health effects attributable to ex-
posure to ambient levels of nitrogen dioxide. Evidence con-
tained in this discussion would, however, suggest that in ad-
dition to NO 2 emissions there were, in fact, significant quan-
tities of other TNT process-related contaminants (acid mists
such as nitric and sulfuric) on a scale which would preclude
the assumption that the study area represents an environment
in which observed respiratory symptoms may be related exclu-
sively to nitrogen dioxide exposure.
Consideration 1 : Discussion of the Physiological Effects of
Human Exposure to Nitrogen Dioxide as These
Relate to the Chattanooga Study
Since ventilatory performance has been related to NOo ex-
posure by reason of a number of studies! 2 J and because toxic
properties of NOg are usually attributable to irritation of
mucous membranes , it was apparently determined that ventilatorv
performance would be used in the Chattanooga Study as an index
of NOo exposure in school children together with information
gained through questionnaires.
While a number of socioeconomic factors in addition to
home exposure to cigarette smoke was considered and resolved
by Shy e_t al. , [I] exposure to other TNT production related co-
pollutants which included the synergistically related particu-
late-SO ?(SO ) couple was apparently considered negligible or
equivalent in comparing test areas.
Consideration 2; Discussion of the Physiological Effects of
~~ " Human Exposure to Sulfuric Acid Mist, Nitric
Acid Mist, and Sulfur Dioxide as Physiolog-
ically Interfering to the Effects of Nitro-
gen Dioxide
-------
1003
As described in Air Quality Criteria for Sulfur Oxides,
physiological response to sulfur dioxide and sulfur trloxide
are as follows: [3)- Incidence of chronic pulmonary disease
- Prevalence of respiratory symptoms
- Changes in ventilatory function
Here it appears from reference to Consideration 1, that
ventilatory functions are affected in the same manner by sul-
fur oxides as by nitrogen dioxide. In this regard, reference
to the difficulty in obtaining even interference-free sulfur*
oxide atmospheres for the purpose of studying specifically
SOX community epidemiology is presented in Air Quality Criter-
ia for Sulfur Oxides.[3]
Such combined exposures which are also typified by "in-
crease in pulmonary flow resistance,"[3] would be expected to
produce a sensitized individual on the basis of laboratory ex-
periments which show, "A three to four fold potentiation of
the irritant response ..." [3] when conditions of synergistic
exposure are observed.
Such sensitizatlon has appeared as a result of synergis-
tic exposure tests involving sulfur dioxide where successive
exposure to contaminated air has produced a post exposure per-
iod of 1-5 hours during which pulmonary resistance continued
at test level or even increased slightly.[3]
As a further example of post exposure sensitization such
contaminants as carbon monoxide which are retained by the body
in equilibrium for several hours after exposure are known to
produce human systemic imbalance as measured by elevated blood
carboxyhemoglobin, where several researchers have related this
imbalance to sensitizatlon or potentiation to develop high
blood chlolesterol levels[4] of the degree associated with
arteriosclerosis. Indeed, it is the post exposure sensitivity
of siblings and their families to contract respiratory illness
which is the basis upon which Part II of the classical N02
population exposure study in Chattanooga was founded.[5]
Consideration 3i Major Non-Nitrogen Oxide Emissions in the
Control Area
In evaluating the presence and degree of symptoms attri-
buted purely to WO^ in this classical study, it is important
-------
1004
to note that according to a Summary Report of Air Pollution
Evaluations prepared by the U. S. Army Environmental Hygiene
Agency, Edgewood Arsenal, the boiler plant proximate to the
TNT plant at Tyner, Term., which is adjacent to the test area,
produces daily the following non-nitrogen oxide emissions:
25,500 Ibs of particulates - daily [6]
16,100 Ibs of sulfur oxides - daily [6]
while the Sellite stack emits:
23,900 Ibs of sulfur dioxide - daily [6]
2,120 Ibs of 1OO$ sulfuric acid mist - daily [6]
Equally important, as noted among the conclusions of this
emissions inventory,[6] is the statement, "Oxides of nitrogen
sulfuric and [ sic] nitric acid mist, sulfur dioxide, and par--
ticulates are all being presently emitted in quantities suf-
ficiently large as to have air pollution potential."
In brief, while a maximum of only 96,400 Ibs/day of N02
emission is accounted for, 1.5 times this quantity or 151,1OO
Ibs/day of other respiration relatable contaminants are co-
emitted.
Evidence of these concentrations of equally or more harm-
ful air contaminants exists in the community as measurable
levels of sulfur dioxide in the range of 0.0,32 to O.047 ppm ac-
companied by sulfuric acid mist at levels of 1.1 to as much as
13.0 mg/m3.[6] These levels may be the result of recorded
Oleum-Sellite plant source emission volumes of 9,3OO ppm sulfur
dioxide together with 326,000 pg/m3 of total acid mist.[6]
Supplementary to these measurements are percentage sul-
fates found in suspended particulates, reported during the
1969 Study by Shy, et al.[1] These findings show high levels
of suspended sulfate in the NOg area which vary from an overall
mean of 11.2 Pg/m3 particulate sulfate to a 90th percentile
average high of 20.7 wg/m3. Since these relatively high sus-
pended sulfates are measured in a low sulfur dioxide air shed,
it is certainly a valid inference that otherwise unaccounted
for sulfuric acid mist is responsible for the balance of sul-
fate and at least a portion of respiratory symptoms classical-
ly attributed purely to nitrogen dioxide. The inference at-
-------
1005
tached prlma facie, to the presence of sulfate aerosol is dis-
quieting in the light of recent evidence observed in the re-
ports of the U. S. EPA Community Health Environmental Systems
Study (CHESS). Published data resulting from this study of
five major U. S. areas indicate significant correlation between
incidence of respiratory health effects and measured elevated
levels of sulfate aerosol.[7] To date, no certain lower limit
of sulfate aerosol has been attributed to observed health ef-
fects. Detroit, Michigan measurements show sulfate concentra-
tions averaging 11 percent of suspended dust in the respirable
range below 10 v diameter over the five year period ending in
1970.
Assessment of epidemiological symptoms attributed to ni-
trogen dioxide may be accounted for by one or more of the fol-
lowing alternatives.
Possible Explanations of Observed Respiratory Symptoms Attri-
buted to NO.,
a. Symptoms of air-related respiratory disorders are the
product of a synergistic effect resulting from ex-
posure to a combination of pollutants, i.e., nitrogen
dioxide and combined sulfuric acid and nitric acid
mist.
b. Symptoms which result from exposure to levels of
nitrogen dioxide greater than O.O83 ppm are observed
in a population which is rendered sensitive by virtue
of background exposure to steady and/or intermittent
high levels of combined acid mist (HNOo and/or H2SO^).
c. Symptoms are a result of exposure to acid mist only,
rather than to actual nitrogen dioxide levels measured.
Conclusion
We conclude that the basis for epidemiological health ef-
fects representing long term exposure of populations to WOp
at ambient levels of 0.05 ppm or less is faulty and founded
upon inappropriate evidence.
As a result, we conclude that the degree of hazard at-
tached to long term, low level exposure to NO, is unnecessari-
ly strict and represents rather the sensitivity of individuals
who are coincidentally exposed to substantial levels of other
non-nitrogen dioxide pollutants (H-pSO^ and HNO~ mists) which
are equally or more hazardous than NOg?
We encourage further research to better elucidate popula-
tion health effects attributable to nitrogen dioxide; mean-
while, we would identify as premature any extraordinary mea-
sures to drastically reduce or eliminate exclusively atmos-
pheric emissions of nitrogen dioxide, in view of evidence
which suggests the equal or greater need to limit respiratory
burden of coambient aerosol particulate sulfate and acid mist.
-------
1006
References
1. C.M. Shy, et al., "The Chattanooga School Children Study:
Effects of community exposure to nitrogen dioxide,
J. Air Poll. Control. Assoc. 20 (8): 539 (1970)
2. Air Quality Criteria for Nitrogen Oxides, Environmental
Protection Agency, APCO Publ. No. AP.84, 1971. Ch. 9.
3. Air Quality Criteria for Sulfur Oxides, Environmental
Protection Agency, APCO Publ. No. AP-5O, 197O Ch. 9.
4. "Concern grows over CO Pollution, "Chem. Eng. News 48 (3):
7 (Jan. 19, 197O)
5. C.M. Shy, et al., "The Chattanooga School Children Study:
Effects of community exposure to nitrogen dioxide. II
Incidence of acute respiratory illness," J. Air Poll.
Control Assoc. 2O (9): 582 (197O)
6. Summary Report of Air Pollution Evaluations, Volunteer
Army Ammunition Plant, Tyner, Tenn., U.S. Army Environ-
mental Hygiene Agency, Edgewood Arsenal, Md., 1965-67.
7. j.G. French, et al., "The Effect of Sulfur Dioxide and
Suspended Sulfates on Acute Respiratory Disease," Arch.
Env. Hlth. 27, 129-133, 1973.
DISCUSSION
STEENSBERG (Denmark)
The paper is of particular interest because the Chattanooga
studies have been an important basis for considering criteria
for nitrogen oxides. Even on this basis - which now seems to
be too weakly founded - the working party behind the WHO tech-
nical report no. 5O6 did not find sufficient basis for estab-
lishing NO criteria. I am therefore interested to know whether
other studies are under way on the influence of nitrogen oxides
on human health.
WARNER (U.S.A.)
It is my understanding that the Chattanooga area is being
restudied. In addition, another study, EPA's Community Health
Environmental Systems Study (CHESS) is presently in progress,
and it is hoped that this study will better elucidate the re-
lationship between measured levels of air contaminants and
observed health effects.
Here, the investigation of the health effects attributable
to sulfate aerosol forms an especially interesting aspect of
the CHESS study.
-------
1007
VELI-PEKKA LEHTO (Finland)
Is it reasonable to conclude that the NC>2-liinit is too
strict, if it would lead to raising the permited level of N02-
Is it not better to say that the N02-limit seems to be safe if
we want to develop safe limits.
WARNER (U.S.A.)
This is a question of degree. What degree of control of
N0» must be exercised in order to achieve the degree of safety
required for reason of human health? Perhaps future studies
will show that our efforts could be better spent controlling
air pollutants exclusive of NO9 that are more obviously health
related. ^
BATES (Canada)
Was the source of S02 in Chattanooga sufficiently close to
the source of NO , that the contours of the NO levels in
Chattanooga woulS also be the contours of maximal distribution
of sulphate?
WARNER (U.S.A.)
The sources of S02, sulphate (as acid) and NOX were from
the same plant. Damage to vegetation characterized as "acid
damage" was reported in the U.S. Army industrial Hygiene Study.
This particular acid-sulphate damage was reported within a 1
mile radius of the plant. High and moderately high suspended
sulphates and nitrates were also observed; these over most*of
the study and control areas of Greater Chattanooga.
In other words measured levels of sulphate and nitrate as
well as acid damage to vegetation are reported in the study area.
FREEMAN (U.S.A.)
The data presented refer to stable sources of NO2 emissions
only in view of the N02 dependent levels of ozone on photo-
chemically generated pollutants, can the interdependent NOj
level alone be considered by itself circumspectly?* Ozone, de-
pendent upon N02 , is 10-20 times as toxic as NO, itself. What
is the thinking in this regard?
-------
1008
WARNER (U.S.A.)
This is an interesting question. Stationary or stable NO_
sources, rather than traffic sources of NC>2, would hopefully
lead to a better, more ozone free examination of N02 effects.
It is not altogether clear from the study data whether or not
traffic related hydrocarbons were eliminated as as possible
ozone producing interference.
-------
1009
ON THE INTERACTION OF ATMOSPHERIC POLLUTANTS
K, A, BUSTUEVA+ AND I, V, SANOTSKY+H"
+ Central Institute for Advanced Medical Training, Moscow, USSR
++ Institute of Labor Hygiene and Occupational Diseases, Moscow,
AMN, USSR
ABSTRACT
In evaluating the health effects of ahemiaal and physical
factors, -it is of great importance to know the possible inter-
actions between different pollutants. The biological effects
of interactions, when present* may be synergistic, antagonistic
or additive. Each type of interaction calls for a different
evaluation and different practical measures. As yet the under-
standing of such effects is not clear, probably because of dif-
fering definitions of terminology. For example, the combined
effect of eulfur dioxide and particulates is interpreted as a
synergistic effect; in the author's opinion, this is an aggra-
vating effect. The type of interaction depends on the levels
of concentration observed - for example, the synergism shown at
high levels of concentration is not always demonstrated for low
levels of concentration. In fact there is little evidence of
synergistic effects from ambient air pollutants; the more com-
mon type of interaction is additive in effect.
Examples of each type of interaction will be presented.
-------
1010
Recent years have witnessed a noticeable growth in the
interest towards the interaction of different atmospheric
pollutants and industrial toxins. More and more facts are
being amassed proving that the combined presence of two and
more substance in the air that is inhaled can significantly
change the degree of the toxic effect that is characteristic
of each component of the mixture. Moreover, the ultimate
effect of the combined action may be characterized as sum-
mation (total or partial), potentiation or antagonism.
It is quite understandable that the nature of the com-
bined action is of considerable practical significance,
first and foremost, when establishing air quality standards,
both for atmospheric air and air in industrial premises.
The character of the combined action (summation,
potentiation, antagonism) depends not only on the direction
of the effect of each component in the mixture, but also on
the level of active concentrations, for instance, lethal
concentration levels and chronic action threshold, or even
maximum permissible concentrations (MFC).
An experimental investigation aimed at verifying this
supposition has revealed that narcotic substances of one
group, in case of combined action, usually sum up, bothat
lethal concentration levels and at chronic action threshold
level. As for substances from different groups, for in-
stance, narcotics and irritants, then the transition from
lethal level to threshold level proceeds according to various
laws, depending on concrete toxins: either a summation is
observed, or an "independent" action according to Elkins
classification. Independent action, according to our data,
may sometimes be classified as sub-clinical antagonism.
-------
1011
Experience points to an exclusively rare appearance of
potentiation in case of chronic combined action at chronic
action threshold level. As an example, we may refer to the
clinical observations when there was a combined action of
ethanol and arsenic, ethanol and thiuramdisulfide, ozone and
sulphuric acid aerosol by A.I. Nevskaya and T.A. Kochetko-
va [13.
The significance of the active levels may be demon-
strated using the example of the interaction of SQg and HOp.
For the range of concentration in the open atmosphere, this
is summation of effects (0*1. Shalamberidze [2] ), and for
a higher level it is antagonism (L.S. Mitina [3] ).
The experience gained by Soviet hygienists in their
study of the combined action of different chemical sub-
stances within the range of concentrations typical of the
outside atmosphere (25 combinations), has led to the con-
clusion that the most frequent character of this action is
total summation of effects (K.A. BuStueva [4] ). Only for
certain mixtures was there independent action (S02 + CO).
The nature of the combined action of several atmos-
pheric pollutants has been taken into consideration in
sanitary legislation for practical purposes.
The most complicated question is that of evaluating
the character of the combined action of gases and aerosols.
One of the prime difficulties in making such an evaluation
is that besides the changes in the organism's reaction, the
presence of aerosol can alter the depth to which the gas
penetrates, and only on account of this, the character of
the effect may change. And the latter can no longer be con-
sidered the result of the genuine interaction of the com-
ponents.
In particular, an example of the latter can be seen
in the combination of the inertly suspended sulfur dioxide
cinders in the period of toxic fogs in London.
-------
1012
It is known that sulfur dioxide is a compound that dis-
solves well in water, as a result of which it is absorbed in
the main in the upper respiratory tracts. Even in case of
high concentrations, the absorption of S02 in the upper
respiratory tracts varies from 48$ to 81.3$, giving an
average of 61.6S& (I.D. Gadaskina [5] )• In the presence of
aerosols and high humidity, S02 dissolves in the moisture
that condenses around the aerosol particles. On the aerosol
particles the S02 is "conducted" to the alveolae where it
is released in view of the great affinity in the lung
tissue, creating a high local concentration* This, as could
have been expected, lies at the basis of the unfavorable con-
sequences of toxic fogs*
The first experimental proof that an aerosol could alter
an organism's response to an Irritant gas was presented by
Dautrebande in 1939* He termed such an aerosol a "vector",
and concluded that the adsorption of a gas on aerosol
particles boosts the number of gas molecules that reach the
lung alveolae.
This process was studied more intensively in the 1950s.
The significance of size of particles was noted (Dautre-
bande L.,et al. [6] ), as well as the physico-chemical
peculiarities of gases and aerosols (La Belle C., et al*),
the level of active components (Amdur M., [7] ), the affin-
ity of gases to aerosol particles in lung tissue (Goetz A.,
[83 ).
Therefore, in the period of toxic fogs there was an
intensification of the effect because of the change in the
site where S02 came into contact, and not due to interac-
tion in the scientific understanding of this process. Never-
theless, it is necessary to emphasize that for practical
purposes, the very fact that the effect is intensified is
what is important, not its scientific interpretation.
Another explanation of the types of combined action of
gases and aerosols should be sought in the reactions of the
organism. For example, in case of intratracheal introduction
of copper oxide the fibrosia process in the lungs weakened
-------
1013
if this was followed by prolonged inhalation of low concen-
trations of nitric oxides by the animals (I.V. Sanotsky,
I.P. Ulanova, N.M. Karamzina, T.A. Kochetkova [91 ).
The presence of copper oxide in the lungs weakened the
irritating action of nitric oxides. In other cases, for in-
stance, under the action of coal dust and carbon monoxide
(V.K. Navrotsky [103), or silicon oxide and radon (V.S. Ku-
shneva [11 ]) pulmonary fibrosis intensified.
The simple summation effect was illustrated upon the
interaction of sulfur dioxide and sulfuric acid aerosol in
low concentrations (K.A. Bustueva [121),
In industrial hygiene, when dealing with relatively
constant mixtures of substances whose composition is not
fully known, the standards are figured out according to the
leading component which determines tho clinical character of
the mixture's action as a whole. Simultaneously it is
necessary to take into consideration the need for hygienic
standardization according to the most typical component
which characterizes the source from which the toxins are
emitted.
A number of formulae and charts have been proposed for
evaluating the nature of the combine action of different
chemical substances. However, the main shortcoming of the
majority of these formulae for interaction of substances, in
our opinion, is that they are based on the false supposition
that at different quantitative levels the direction of the
combined action of the toxins is the same. However, their
practical usage is very limited without taking a differen-
tiated approach, with due consideration to the latter factor.
-------
1014
Rei'erences
1. MSSKAYA A.I. and KOTCHETKOVA T.A. , "Occupational hvgiena and
diseases" (USSR) 2, 20-9 (1961)
2. SHALAMBEHYDZE O.P. "Hygiene and Public Health" (USSR) No. II, 1969
3. T'TYTIM L.S. "Hygiene and Public Health" (USSR) No. 10, 3-8 (1962)
4. BlJiVTUEVA K.A. et al. "Proceedings of the XIV All-union Congress of
Hygienists and Public Health Doctors", Moscow 26-29 September 1972,
122-5
5. GADASKYNA I.O., "Pharmacology and Toxicology" (USSR) £, 51-53 (1964)
6. DAUTREBANDE, L., SHAVER, I., CAPPS, R., Arch. Intern. Pharmacodynamie.
1951, 85, 17-48
7. AMDUR, M., Int. J. Air Poll.. 1959, v. 1. 170-183
8. G03TZ, A., Int. J. Air, Water Poll., 1961, v.4, No. 3/4
9. SAWOTSKY I.V. ULANOVA, I.P. KARAMZYNA, N.M. and KOTZETKOVA I.A. "The
toxicology of the new industrial chemical products" 1969i 2nd adition*
47
10. NAVROTZKI V. K., "Qcjaipat icinal. Ji^giejie_and Ojccupat ional diseases" 1961 f 9
11. KUSCHNEVA V.S., "Occupational hygiene and occupational diseases" I960, 1
12. BUSTUEVA K.A. "Maximum allowable concentration of atmospheric pollutants"
9th Edition 1967
-------
EXPOSITIONS!NDIKATOREN
INDICATORS OF EXPOSURE
INDICATEURS DEPOSITION
INDICATORI DI ESPOSIZIONE
INDICATOREN VAN EXPOSIE
VoTs-itzender - Chairman - President - Presidente - Voorzitter
J.L. MUNKMAN (Canada)
-------
1016
DISCUSSION
JOOSTING (Nederland)
Please would you be so kind as to tell us something more
about the ways inhaled aerosols can influence the effect of
other inhaled substances, e.g. gases. It seems to me that the
uptake of gases by the lung and their effect would not be
influenced by e.g. the harrowing effect of an inhaled aerosol
on the lumen of the airways, because breathing will continue.
Please would you correct me if I didn't understand you well?
BUSTUEVA (U.S.S.R.)
The interaction of inhaled aerosols and gases has an
appreciable influence on their biological effectiveness.
The overall effect depends not only on the depth of pene-
tration into the lungs, but also on the speed of adsorption and,
most important, of desorption of the toxin from the surface of
the particles (Sanockij, I.V. (1969) Toksikologija novyh
promyslennyh vesestv (Toxicology of new industrial substances)
v. II, p. 6) .
The combined effect usually, diminishes when there is
significant interaction between gas and aerosol (hydrocarbons 4
A2°3>'
Litau, V.G. & Solov'ev, V.I. (journal Gigiena turda i
Profzabolevanija, 1973, No. 9, P. 58) detected a sharp increase
in the action of steam products of thermal destruction of oils
(ethers, aldehydes, ketones, organic acids and carbon monoxide)
in the presense of oil vapour, which cannot easily be explained
as being due to deep penetration of this mixture into the lungs,
or to the processes of adsorption and desorption of the toxin
from the surface of the particles. The indirect effect of oil
vapour on the phospholipid marginal layer of the respiratory section
of the lungs (sulfactant) is of significance.
-------
1017
MERCURY AND OTHER ELEMENTS IN BLOOD OF THE
DUTCH POPULATION
E, M, DEN TONKELAAR+, G, J, VAN ESCH+, B, HOFMAN+,
P, L, SCHULLER* AND J, H, L, ZWIERS++
+ National Institute of Public Health, Bilthoven, Netherlands
++ Central Laboratory TNO, Delft, Netherlands
ABSTRACT
For a number of years problems have existed about the tox-
icity of heavy metals and other elements occurring in the envi-
ronment in higher levels than in the past. Concern about this
started with the Minamata disease in Japan, caused by methyl-
mercury in fish. Also reports from Sweden about fish caught
in lakes polluted with mercury caused some alarm. To see
whether the Dutch population, especially people eating much
fish, were in danger of (me thy I)mercury intoxication, the mercu-
ry content of blood and hair was determined in a number of people.
In this population the relation between fish-eating habits and
mercury content was studied. People Here asked whether they
ate fish at least once a week, at least once a month, at least
once a year or never and were divided into 4 groups accordingly.
Questions about fish eating were asked for sea-fish and for
freshwater fish, because the mercury content of the latter is
generally higher. From the results a clear correlation was
found between fish eating habits and mercury content of the
blood. This correlation was found especially when freshwater
fish was considered. However, the mercury content of the blood
was, even for the heaviest fish eaters, relatively low compared
with the results for Swedish and Finnish fish eaters and much
lower than the level at which clinical symptoms can be expected.
-------
1018
The mercury content of hair did not correlate very well with
blood, although in oases of relatively high mercury content of
blood, the level in hair was also higher. But in general, for-
screening purposes of a population, mercury content of hair is
not a valuable parameter. From this study the conclusion can
be drawn that as yet no necessity exists for limiting the fish
consumption of the Dutch population.
-------
1019
Introduction
In order to evaluate the possible risk of (methyl)mercury
intoxication resulting from fish consumption, blood and/or hair was
collected from people with known fish eating habits for determination
of the total mercury content. Because people with a high and a low
fish consumption were taken, it could be studied whether fish eating
caused an increase in body burden of mercury. In the meantime we
wanted to get an impression about the mercury content in blood of the
Dutch population, compared with people from other countries. Hair was
Included in this study to examine whether mercury in hair could be
used as a reliable index for determination of the total body burden
of mercury.
2, Materials and methods
Blood was obtained from 127 and hair from &f persons. People
were questioned about their fish eating habits, graded in eating sea-
fish at least once a week, at least once a month, at least once a year
or never. The same was asked for the consumption of freshwater fish,
since this is known to have in general a higher mercury content than
sea-fish. A special effort was made to get persons who told to ate
much or on the contrary never fish. The people involved were partly
from our own institute or found with the help of some hospitals. In
that way we also got samples from a little fishing-town called Volen-
dam, where people are known to catch and eat much eel (freshwater fish).
Blood was sampled in heparinized tubes and the mercury
content was determined in whole blood by means of neutron activation
analysis. Mercury in some samples of hair was also determined by
neutron activation analysis, but in most samples by atomic absorption
apectrometry. These samples were washed thoroughly before analysis.
J. Results
Blood
Prom the frequency distribution (fig.1) it can be seen that
mercury in blood does not show a normal distribution. Therefore the
-------
1020
number of samples BLOOD
35
Frequency distribution of the mercury content in whole blood.
30
25
20
15
10
Total number of samples: 127, PPb = ng/ml.
PI
•
n
12 16 20 24 28 32
mercury content
Pig I.
number of samples
HAIR
25
20
15
10
Frequency distribution of the mercury content in hair.
Total number of samples 87»
n n n
n
0.6 1.8 3.0
Pig 2.
12 16
mercury content
-------
1021
Table 1. Mercury content in whole blood compared with consumption
of sea-fish or freshwater fish
sea-fish
number of samples
median value (ppb)
range ( ppb)
% > 4.0 ppb
freshwater fish
number of samples
median value (ppb)
range (ppb)
relative fish consumption x )
3
49
4.4
1.3-40.5
53
20
7.3
1.8-40.5
2
46
2.4
1.0-10.7
24
32
3-2
1.3-32.5
1
18
2.0
0.8-9.9
11
40
2.4
1.0-10.7
0
13
1.5
0.3-4.0
0
34
1.9
0.3-7.3
x) 3 = at least once a week, 2 = at least once a month, 1 = at least
once a year and 0 = never
Tabel 2. Statistical evaluation of the results from table 1 after
logarithmization of the figures
sea-fish
number of samples
log lOx + SD
P gr 3 / gr 2, 1 and 0
P gr 2 / gr 1 and 0
freshwater fish
number of samples
log lOx + SD
P gr 3 / gr 2, 1 and 0
P gr 2 / gr 1 and 0
relative fish consumption
3
49
1.69+0.33
-
-
20
1.86+0.32
-
•
i
2
46
1.43+0.23
< 0.001
-
32
1.54+0.28
< 0.001
-
1
18
1.32+0.28
< 0.001
N.S.
40
1.41+0.24
< 0.001
< 0.01
0
13
1.16+0.26
< 0.001
< 0.001
34
1.31+0.29
< 0.001
< 0.01
-------
1022
median value instead of the mean was estimated. This was 2,9 ppb
(ng/ml), range 0.3-40,5 ppb.
From the frequency distribution it was found that
4# is 0-1 ppb
32# is 0-2 ppb
68# is 0-4 ppb
8356 is 0-6 ppb
is 0-8 ppb
is 0-10 ppb
Only 8 values were higher than 10 ppb, varying from 10.7-40,5 ppb.
To correlate the values in blood with fish consumption the samples
were divided into 4 groups, of which group 3 consisted of people
who according to their own statement ate fish at least once a week.
Samples of people eating fish at least once a month, once a year
or never were divided into group 2, 1 and 0 respectively. This was
done for sea-fish as well as for freshwater fish. The results of the
mercury content in blood for each group are summarized in table 1.
Because the values do not meet the requirements of a normal distribu-
tion statistical analysis was carried out with the logarithms of the
figures. After multiplying the figures by'a factor 10, Student's -t-
test was carried out on the logarithms. Hie results are given in
table 2.
From the tables it is obvious that a dose response relationship is
found between mercury in blood and fish consumption. There' is a marked
difference in median and log value, especially between group 3 and the
other groups. According to statistical analysis the mercury content
in blood of people in group 3 is significantly higher than that of
the other groups, for sea-fish as well as for freshwater fish. In
addition the mercury content in blood of people in group 2 is significant-
ly higher than that of group 0 for sea-fish and of group 1 and 0 for
freshwater fish. In all cases the differences are more marked when
freshwater fish is taken into consideration. This was expected since
it is generally known that the mercury content of this type of fish,
like fte\,pike and pike-perch is higher than that of sea-fish.
-------
1023
Table J. Mercury content in whole blood of people eating much sea-fish,
but less freshwater fish, compared with people eating little
or no fish at all
number of samples
median value (ppb)
range (ppb)
log lOx + SD
|P gr 3 / gr 1 and 0
relative sea-fish consumption
3.X)
30
3.6
1 - 33-5
1.57+0.28
m
1+0
31
1.7
0.3 - 9.9
1.25+0.27
< 0.001
x) Only people eating sea-fish at least once a week, but less fresh-
water fish
Table 4. Mercury content in hair compared with fish consumption
(sea- and freshwater fish combined)
number of samples
nedlan value (ppm)
range (ppm)
% > 0.9 ppm
log lOx + SD
P gr 3 / gr 2, 1 and 0
relative fish consumption
3
17
1.6
0.6-13.2
94#
1.26+0.29
-
2
35
0.5
0.2-3.5
17#
0.71+0.28
< 0.001
1
21
0.5
0.2-5.2
19*
0.76+0.34
< 0.001
0
9
0.3
0,1-2.6
22$
0.64+0.44
< 0.001
-------
1024
Because a number of people with a high consumption of sea-
fish were also eating much freshwater fish, it is difficult to estimate
the share that sea-fish has in the ultimate mercury content of the
blood. Therefore a group was made of people belonging to grpup 3 for
sea-fish, but to a different group for freshwater fish. This group
was statistically compared with a combination of group 1 and 0 for
sea-fish (table j). It is obvious that also the consumption of much
sea-fish causes a statistical significantly higher mercury content
in blood than when fish is eaten very little or never.
Hair
The frequency distribution of mercury in hair is given in
fig. 2. Also in this case no normal distribution is found. The median
value is 0,6 ppm (ng/g), while it can be calculated that:
6Q% is 0 - 1 ppm
83$ is 0 - 2 ppm
92$ is 0-3 ppm
95$ is 0 - 4 ppm
Only 4 values were higher than 4 ppm. Of these probably 3 values
(resp, 12, 16 and + 200 ppm) have nothing to do with fish consumption,
but can be caused by external contamination (use of shampoo containing
mercury). These values have been omitted from the calculations. One
value of 13.2 ppm belongs to the same person having a very high con-
sumption of both sea- and freshwater fish and a blood content of
40.5 ppb.
In table 4 the results of the mercury content in hair are
given in relation to fish consumption (sea- and freshwater fish taken
together). Also the statistical evaluation of the logarithmized values
is shown. Prom this table it is obvious that hair of people with a
high fish consumption contains more mercury than that of people eating
less fish. Of the former group 94$ has a mercury content of 1 ppm or
higher contrary to 18$ for the latter groups. Between the groups
.that eat fish at least once a month, at least once a year or never
no differences exist in mercury content of hair.
-------
1025
mercury in hair (ppm)
3.5
3.0
Fig- 3
group 3
group 2
group 1 or 0
40.5"
t
13,2
2.5
2.0
1.5
1.0
0.5
0
V
A
A *
A *
0
A
A
A
• • • A
• 0 •
s ••/
a» •
• **
1
6
8
10 11
12
mercury in blood
Comparison of the mercury content in blood with that in hair
in 47 samples. Group 3 consists of people eating fish at least
once a week, group 2 at least once a month, group 1 at least
once a year and group 0 never.
number of samples
100
80
20
Fig.
D Data from Finland and Sweden
EU Data from the Netherlands
Frequency distribution of the mercury content in whole blood
of people in this study compared with data from Finland and
Sweden including people with high fish consumption (data
derived from Berglund et al.,
-ft-
25 50 75 100 12S 150 175 200 225 650
mercury content in blood
-------
1026
Correlation between_blood and hair
According to Japanese and Swedish studies (Berglund et al.,
1971) a correlation exists between mercury content in blood and hair.
Prom 47 persons in our study samples of both blood and hair were ob-
tained. The results for the mercury content are represented in fig.3
to study whether a correlation could be found. From this figure it
is obvious that no clear correlation is present between values of mer-
cury in blood and hair. Although a slight correlation was found by
using Kendall's rank correlation test, this was caused mainly by the
values of the people with a high fish consumption. Among people eating
less fish sometimes relatively high values in blood are combined with
low values in hair, but also the opposite is found.
For the whole group with a mean value of 1.3 PP"i in hair
and 4.7 ppb in blood a factor of about 280, but showing a large range,
could be calculated. This Is of the same order of magnitude as the
factor of about 250 found by Berglund et al. (1971).
Discussion
Although the material selected for this study does not
constitute a random test sample of the Dutch population, a number
of valuable conclusions can be drawn. There is a clear correlation
between fish consumption and mercury content in whole blood. This
applies especially for people that according to their own statement
consumed much sea- and freshwater fish. Some of them were even eating
fish twice a day.
The mercury content in blood was less than 10 ppb in 94$ of the people.
In general the increase caused by fish consumption amounted to a few
ppb's only. The mercury content in hair also showed a relation with
(high) fish consumption. However because there was only a slight
correlation between mercury in blood and hair (except for people with
high fish consumption) determination of mercury in hair can not be
used for the estimation of the mercury body burden of the Dutch popu-
lation. Regarding to the mercury content of hair possibly other factors
(like external contamination) play a role.
Although an obvious correlation was found for mercury in
-------
1027
blood and fish consumption, the values obtained In this study are very
low compared to values obtained In other countries. In the Swedish re-
port of an expert group (1971) a frequency distribution is given of
the mercury content in blood cells of people without clinical symptoms
but with a high fish consumption . These data have been modified to
whole blood (by dividing them by a factor 2) and in fig.4 they are
compared with our results. It can be concluded that 98% of our data
fall within the lowest group of the Swedish frequency distribution.
Recently a study was published (Skerfving, 1974) in which
mercury levels in blood and hair of Swedish people consuming contami-
nated fish were compared with the health status. Clinical observation
of people with mercury contents in blood of up till 200 ppb and in one
case even 650 ppb did not show any abnormalities that could be ascribed
to (methyl)mercury intoxication. Comparing our data with those mentioned
In Berglund et al. (1971) the conclusion can be drawn that the mercury
content in blood and hair in our study was relatively low or of the
same order of magnitude as found in other studies for a normal popula-
tion.
The preliminary results of a study in which 1200 blood
samples of l8 year old Dutch men were analysed, show even a lower median
value. In this case no data about fish consumption are known. It can
be concluded that as yet it is not necessary to limit fish consumption
in the Netherlands.
References
P.Berglund, M.Berlin, G.Birke, R.CederlSf, U.von Euler, L.Friberg,
B.Holmstedt, E.Jonsson, K.G.LUning, C.Ramel, S.Skerfving, A.Swensson
and S.TeJning (1971).
Methyl mercury in fish, a toxicologlc-epldemlologic evaluation of
risks.
Report from an expert group. Nordisk Hygienisk Tidskrift Suppl. 4.
S.Skerfving (197^). Methylmercury exposure, mercury levels in blood
and hair and health status in Swedes consuming contaminated fish.
Toxicology 2 J - 2J.
-------
1028
DISCUSSION
CLEMENTE (Italy)
1. Could you please say what were the average Hg concentrations
in the fish eaten by the Dutch population?
2. Were the hair samples washed carefully before analysis?
If so it is hard to attribute the high Hg content in the hair
to contamination by shampoos containing Hg. Hg in shampoos may
in fact cause external contamination in the hair but this would
surely be eliminated by proper rinsing.
DEN TONKELAAR (Netherlands)
1. The average Hg concentration in sea fish is approximately
0.1 to O.3 ppm in freshwater fish approximately 0.5 ppm, occasion-
ally higher, up to 1 to 2 ppm.
2. The hair was thoroughly washed before the analysis. Be-
cause hair contains many S-containing proteins it probably forms
chemical bonds with the mercury contained in some shampoos which
is therefore not eliminated by rinsing.
Van der KREEK (Netherlands)
Shampoos contains Hg as a preservative, not only in the
Netherlands, but in most other countries too. The concentration
in the shampoo never exceeds 3O ppb. I wonder if it is possible
that this 30 ppb can lead to a Hg content of the hair of 200 ppm,
the case you mentioned in your introduction.
DEN TONKELAAR (Netherlands)
The Hg concentration in shampoo is low, but our analysis
mainly concentrated on the ends of the hair.
The high levels were found among girls with very long hair.
In these cases the ends were perhaps as much as four years old
and thus have been washed very frequently. In view of the fact
that Hg can form chemical bonds with hair such a high level could
be possible after a number of years.
-------
1029
DENNIS (Canada)
In a Saskatchewan survey it was found that recency of fish
eating (fresh water fish within two weeks of sampling) was more
closely correlated to higher blood mercury levels than frequency
of fish eating.
In the same Saskatchewan survey involv-ing 679 blood samples,
month of sampling was a significant variable, as the fishing
lakes are only free of ice for some 4-5 months of the year;
during these months blood mercury levels are significantly higher
and so is the consumption of fresh-water fish.
DEN TONKELAAR (Netherlands)
We did not consider this point in our study. Most of the
blood and hair samples were collected during the summer and
autumn. Sea fish is eaten throughout the year in the Netherlands,
and freshwater fish mainly in summer (roughly up to October).
Since the Hg found in fish is usually in the form of methylmer-
cury, which has a fairly long half-life in man (approximately
70 days), I feel that the season in which most fish is eaten
cannot have a particularly great influence on the results of
measurements of mercury levels in the blood and hair.
BERNSTEIN (Canada)
1. Were whole strands of hair collected for analysis, and if
so, were any of the hair samples with high mercury levels seg-
mented to compare levels in proximal and distal segments?
2. In reference to one of the former queries from the audience,
could not exogenous mercuriferous substances applied to hair bind
quite firmly to hair protein and therefore be difficult to remove
by washing the hair sample?
DEN TONKELAAR (Netherlands)
1. In most cases the ends of the hair were collected and in
no case were whole strands analysed.
However, in one case a girl was found to have 20O ppm Hg
in the ends of her hair. Individual hairs from her neck were
later analysed and found to contain 2.3 ppm.
2. I fully agree with this observation.
-------
1030
SANTARONI (Italy)
I should like to know what the author thinks about the
possible value of measuring levels of Hg contamination from the
food intake of representative population groups and the amounts
of Hg inhaled, given that blood levels are too variable in time,
according to what our Canadian friend said, whilst measurements
of hair can, as you said yourself, be affected by contamination
from extraneous agents which are not necessarily metabolized.
DEN TONKELAAR (Netherlands)
Mercury levels in various food stuffs should certainly be
determined so that an estimate of daily Hg intake may be made.
On the other hand, according to English data, average daily
intake can also be estimated from the mercury content of the
blood.
-------
1031
THE CELL CULTURE AS TEST SYSTEM IN APPLIED
ENVIRONMENTAL HYGIENE
E, G, BECK, N, MANOJLOVIC AND ANNA B, FISCHER
Hygiene-Institut, Oekologisches Zentrum, Justus Liebig-UniversltSt,
Giessen, and Med.Institut f.Lufthygiene und Silikoseforschung,
Diisseldorf, BRD
ABSTRACT
The results of epidemiological studies in the field of en-
vironmental hygiene are the basis for controlled experimental
research to identify the relevant effective factors. Human
beings can only be used as test persons under certain circum-
stances and with special safety precautions^ to test the biolo-
gical effect of these substances singly and in combination. The
laboratory animal and the cell culture are the appropriate test
systems. The cell culture test system has been developed to
such an extent that it is suitable for examining both the acute
and the chronic biological effects of foreign matter from the
environment. The harmfulness of a substance in vitro indicates
its harmfulness also in vivo. The laboratory animal and the
cell-culture are closely connected with regard to method and
validity of results.
The object of the research is the use of the two test systems
as screening processes for rapid assessment of environmental pol-
lutants (toxicity, carcinogenicity). It is intended that these
processes contribute to the elucidation of epidemiological find-
ings and serve to facilitate decisions on preventative measures
in the field of environmental protection.
Results will be presented which have been obtained with the
cell culture test system. The substances tested were atmospheric
dust (extracts^ components) from immissions in industrial conglo-
merations and urban and rural areas.
-------
1032
In pharmacological and toxicological research the test system cell
culture has attained a firm place. Quite apart from the consider-
able saving of time and cost, this system offers the following ad-
vantages: 1. the possibility of studying events on the cellular level;
2. the use of homogeneous cell populations; 5« experimental conditiond
which are easy to survey, i.e. defined, quantifiable and reproducible;
*4. due to the merits stated under points 1 and 2 a good possibility
for statistical affirmation. The limits of this method should,
however, also be pointed out: 1. lack of those integration mechanisms
(humoral, nerval) to which the cell is subjected in the whole orga-
nism; 2. lack of metabolization of offered substances in the sense of
their activation or detoxification; J>. difficult and limited extrapo-
lation of results on animals or man, though correlations do exist.
As an example, studies in the field of pneumoconiosis research should
be cited which showed a good correspondence of the relations between
the frequency of silicosis of mine dusts, their fibrinogenic action
in vivo, and their cytoxicity in vitro (1,2).
In slides the test objects, i.e. diploid and permanent proliferating
and nonproliferating cells in vitro and the test parameters like cell
morphology, physiology, functions, and specific faculties are pre-
sented (figs. 1 to 5).
Alveolar macrophages are a test system providing a link between in
vivo- and in vitro-conditions. The exposition can take place in
vivo as well as in vitro, while for the evaluation of the effects
the advantages of the in vitro system can be used. Alveolar macro-
phages play a decisive role in the defense of the lung against
particulate air pollutants. As it is possible to cultivate them
as a homogeneous cell population, the cell culture offers the oppor-
tunity for investigations which are non-existent in vivo. AB an
instance, experiments were conducted by BRUCH et al. regarding the
question whether the faculty of macrophages to metabolize benzo(a)-
pyrene is influenced by inhaled lead (J>). After exposition to a lead
aerosol, the alveolar macrophages of the experimental animals were
obtained by pulmonary lavage, cultivated in vitro, and incubated with
benzo(a)pyrene. It was noted that the quantity of benzo(a)pyrene
metabolized by the alveolar macrophages of lead exposed animals is
-------
1033
CELL TOXICOLOGY
I. Test objects:
cells in vitro
II. Parameters:
a) morphology
b) physiology
c) functions
d) special functions or abilities
Fig. 1
CELL TOXICOLOGY
I. Test objects In vitro:
a) diploid cells (mammalian,
b) permanent cells
c) macrophages
d) lymphocytes
proliferating
Including human)
(mammalian, non-proliferating
including human) <«"hout stimulation)
Fig. 2
CELL TOXICOLOGY
II. Parameters In vitro;
a) Call morphology:
1. Form and structures: nuclear and nucleotar structure
endoplasmic retlculum
lysosomes
lipid droplets
vacuoles
degenerating formi
2. Morphoklnetica:
motility
membrane activity
plasma flow
necrobiosls
Fig. 3
-------
1034
CELL TOXICOLOGY
II. Parameters in vitro:
b) Cell physiology!
1. Energy metabolism:
2. Structure metabolism:
Fig.
lactate production
glucose consumption
0,,-consumption
ATP/ADP quotient
RNA synthesis
protein synthesis
CELL TOXICOLOGY
II. Parameters In vitro:
c) Cellular functions:
1. Reproduction or cell kinetics: cell number
mltotic rate
plating efficiency
generation analysis
2. Permeability:
3. Phagocytosis/Pinocytosis
Fig. 5
(DNA metabolism)
enzyme release
dye uptake
only a fraction of the control values proving impairment of this
specific cellular function. These experiments confirmed earlier
findings concerning the disturbance of benzo(a)pyrene metabolism of
alveolar macrophages following in vitro exposition to lead or ambient
dusts (4). In the course of the investigations on the cytotoxicity
of particulate air pollutants it was found that in this case dose-
effect relationships must be regarded from the point of view of the
hit theory. Microkinematographic observations have shown that one
lead particle is capable of causing lysis of a cell (1 particle + 1
cell = 1 hit = cell death). As regards the lung, one should also
proceed from the assumption that punctual damages occur during the
-------
1035
inhalation of particulate matter, which, upon accumulation, can be of
pathogenetic significance. BINGHAM and colleagues demonstrated a
distinct reduction in the number of alveolar macrophages which could
be obtained by lung washings from rats having inhaled lead for a
longer period of time (5). Moreover, BRUCH et al. detected a reduced
lung clearance in lead exposed experimental animals (6).
Alveolar macrophages are differentiated postmitotic cells which also
do not multiply under normal tissue culture conditions in vitro.
However, with the use of diploid fibroblasts and permanent cells the
essential property of the cell to proliferate can be tested. Dose-
response relationships concerning one substance can be established
and also different substances can be compared with each other. To
give an example, the influence on cell growth and metabolism of the
environmentally relevant heavy metals lead, cadmium, mercury, zinc,
and tin was studied in a mouse fibroblast line and clear concentration
dependencies were found. Different doses of these metals cause a
509» reduction of replication* with Sn showing the lowest and Cd a
1000-fold higher cytotoxicity, while Pb, Zn, and Hg are in between.
Thus in this model a Toxicological rank order could be established (7).
Fibrous materials gain increasing importance in the context of en-
vironmental toxicology. In animal tests it was proved that besides
asbestos also glass fibres induce the formation of foreign body
granulomas and malignant tumours (8,9). In cell cultures fibriform
material causes a specific incorporation mechanism which is dependent
on form and length of the fibre (*O. The delayed and incomplete
phagocytosis leads to a localized leakiness of the cell with persis-
tent but compensated loss of enzymes and increased energy metabolism.
The engagement between the cell and the fibre results in a chronic
irritation which ia being discussed as one of the causes of tumour
formation. In this context it is of interest that fibrous material
induces the formation of giant cells in vivo as well as in vitro.
In vitro it was proved that the polycarocytes caused by asbestos or
glass fibres are the product of cellular fusion (10).
Cellklnetic and biochemical studies contribute to the elucidation of
the modes of action. Such fundamental investigations are often only
-------
1036
possible with the aid of cell cultures. Following assessment of the
relations between the results obtained in cell culture as a screening
system for the evaluation of the toxicity of novel environmental
noxious substances.
It has been known for many years that cells in vitro can be trans-
formed into tumour cells by a number of DNA- and RNA-viruses. The
first report concerning the oncogenic transformation in vitro by
chemicals was given by BERWALD and SACHS in 19&? (11). In the
meantime, tissue culture cells are being used more and more in tumour
research for the evaluation of the oncogenic capacity of chemical
substances and their modes of action (12,13)* This test system
offers several advantages as compared to the whole animal. The
starting point of cancer is the malignant change of the cell. The
cell culture makes possible the direst study of this event with regard
to morphological and functional aspects.
The test objects and parameters for oncogenic transformation in vitro
are presented in figs. 6 and 7«
CELL TOXICOLOGY
II. Parameters in vitro:
d) Specific functions or abilities
1. Drug metabolism
2. Stimulation e.g. by PHA (lymphocytes)
3. Bactericidal action, virus susceptibility (interferon
production)
4. Migration of cells in the electric field
Fig. 6
In the context of air pollution research, the question is of para-
mount importance whether particulate air pollutants or their extracts
are capable of transforming cells in vitro. In the literature only
a few papers are known under this aspect. Special mention should be
made of the studies executed by FHEEDHAN and collaborators on the
transforming action of extracts of "city smog" OA). The authors
-------
1037
ONCOGEN1C TRANSFORMATION IN VITRO
I. Test systems;
CHEMICAL CARCINOGEN
CELL
(rat, mouse, hamster)
/ foetus in vivo
culture In vitro culture in vitro
INOCULATION INTO
ADEQUATE ANIMAL
Fig. 7
could demonstrate that the city smog extracts contained one or more
factors with a 600-fold oncogenic capacity than pure benzo(a)pyrene.
As the decisive proof for transformation in vitro to have occurred
the capability of the cells to form tumours in an adequate animal
was considered.
The difficulty that some carcinogenic substances have to be activated
or metabolized in vivo limits the use of in vitro/in vivo experiments
ONCOGENIC TRANSFORMATION IN VITRO
II. Parameters:
multllayered growth
(loss of contact inhibition)
loss of specific growth patterns
Increased multiplication
(decreased generation time)
increased plating efficiency
altered metabolic behaviour
(Increased glycolysls)
altered karyotype
unlimited proliferatlve capacity in vitro
tumour formation after Inoculation
in adequate animal
Fig. 8
-------
1038
like the one described above. DI PAOLO et al. developed an elegant
test system combining in vivo- and in vitro-methods in such a manner
that exposition occurs in vivo while the transformation takes place in
vitro (15). To date this test system has yielded the expected results
in 12 carcinogenic substances. The new test promises to become a
suitable screening method for the assessment of the carcinogenicity
of environmental pollutants or their metabolites.
The investigations regarding the toxicity and oncogenicity of en-
vironmental chemicals in vitro demonstrate the increasing significance
of the cell culture as a test system in applied research. It is the
aim of these studies to employ the cell culture, also in connection
with the experimental animal, as a screening system for the rapid
detection of noxious substances in the environment.
LITE RATUR
l) LAUFHUETTE, D.W., K. ROBOCK und W. KLOSTEBKOETTER "Untersuchungen
iiber die cytotoxische Wirkung von Grubenstauben aus dem Saarkar-
bon" t ErgebniBse von^ Untersuchungen auf dem Gebiet der Staub- und
Silikosebekampfung im Steinkohlenbergbau, Vol. 8, S. 131-138,
Verlag Gliickauf , Essen (1971)
2) BECK, E.G., R. HOLUSA, D. JIRAKOVA, B. KYSELA, K. ROBOCK und
V. SKODA "Ueber die unterschiedliche Wirkung von zwei Quarzen im
Tier - und. Zellversuch und ihre physikaliechen Halbleitereigen-
schaften", Staub- Reinh. Luft 33, 3-7 (1973)
3) BRUCH, J., A. BROCKHAUS und W. LEHNEN "Elektronenmikroekopische
Beobachtungen an Rattenlungen nach Exposition mit partikelf8rmi-
gem Blei", Internationales Symposium; Die geeundheitlichen Aepekte
der UmweltverBchmutzung durch Blei, Amsterdam, ".-6. Oktober 1972,
published by the Commission of the European Communities, Luxembourg,
pp. 221-230 (1973)
4) BECK, E.G., N. NANOJLOVIC, A.B. FISCHER "Vergleichende Untersuchungen
fiber die Zelltoiizitat atmOBpharischer Schwebestaube" , Proc. 3rd
Intern. Clean Air Congress. Dtisseldorf, VDI-Verlag, pp. A3 - A
5) BINGHAM, E., E.A. PFITZF.R, W. BARKLEY, E.P. RADFORD "Alveolar macro-
phages* reduced number in rats after prolonged inhalation of lead
sesquioxide" , Science, 162, 1297 - 1299 (1968)
-------
1039
6) BRUCH, J., A. BROCKHAUS, W. DEHNEN "Ueber lokale Effekte inhalierter
Bleiverbindungen auf die Lunge", Internationales Symposium Umwelt
und Gesundheit, Paris 24-28. Juni (1974)
7) FISCHER, A.B. unveroffentliche Ergebnisse
8) POTT, P., K.-H. PRIEDRICHS "Tumoren der Ratte nach i.p. - Injektion
faserfbrmiger Staube", Naturwissenschaften, 59, 318 (1972)
9) STANTON, M.F., C. WRENCH "Mechanism of meeothelioma induction with
asbestos and fibrous glass", J. Nat. Cancer Inst., 48, 797 - 821
(1972)
10) BECK, E.G., S. SETHI, W. HILSCHER "Vergleichende in vivo- und in
vitro-Untersuchungen zur Riesenzellbildung durch Aebest- und Glas-
fasern", Verh. Dtsch. Gee. Path., 56, 662 (1972)
11) BERWALD, Y., L. SACHS "In vitro transformation with chemical carcino-
gens", Nature, 200, 1182-1184 (1963)
12) HEIDELBERGER, C., "Chemical carci no genesis, chemotherapy: cancer* s
continuing core challenges - G.H.A. Clowes Memorial Lecture",
Cancer Res., 30, 1549-1569 (1970)
13) THUST, R., T. SCHRAMM "Neoplastische Alteration durch chemische
Karzinogene in vitro", Arch. Geschwulstforsch, 39» 249-263 (1972)
14) FREEMAN, A.E., P.J. PRICE, R.J. BRYAN, R.J. GORDON, R.V. GILDEN,
G.J. KELLOFP, R.J. HUEBNER "Transformation of rat and hamster em-
bryo cells by extracts of city smog. Proc. Nat. Acad. Sci. USA,
68, 445-449 (1971)
15) DI PAOLO, J.A., R.L. NELSON, P.J. DONOVAN, C.H. EVANS, Host-mediated
in vivo-in vitro assay for chemical carcinogenesis, Arch. Pathoi.,
95, 380-385 (1973)
DISKUSSION
BIANCO (Italien)
Ich habe drei Fragen:
- Welche Technik wurde bei den rnhalationsbelastungsversuchen
benutzt? '
- Haben Sie die Konzentration der verschiedenen Partikel
in der fur die inhalative Belastung verwandten Luft bestimmt?
c^ ^~ J*?811,818 die Korn9rossenverteilung und die geometrische
Standardabweichung der Partikel ermittelt?
-------
1040
BECK (Bundesrepublik Deutschland)
Die zitierten Tierversuche wurden von Dr. Bruch und Mit-
arbeitern (Diisseldorf, BRD) durchgef uhrt.
BRUCH (Bundesrepublik Deutschland)
Die Technik der Ver suchsdurchf iihrung wurde in dem Vortrag
"Ueber lokale Effekte inhalierter Bleiverbindungen auf die
Lunge" beschrieben. Das Bleiaerosol wurde durch Kondensation von
verdampftern Bleioxid erzeugt. Die Bestimmung der Konzentration
erfolgte an Membranfiltern mit Hilfe der Atomabsorptionsspektro-
metrie. Die Grosse der Teilchen war kleiner als 0,5,urn.
HINE (U.S.A.)
Haben Sie Schleimhautzellen oder Lymphocyten von Personen
untersucht, die Expositionen gegenuber Cadmium, alkylierenden
Agentien oder schwer verunreinigter Stadtluft aufwiessen?
BECK (Bundesrepublik Deutschland)
Solche Untersuchungen sind geplant. Als vorbereitende
Versuche haben wir Zellen von Tieren untersucht, die liber
langere Zeit atmospharischen Feinstaub einzeln oder in Kombina-
tion mit Fremdgasen (SC>2/ N02) inhaliert hatten. Wir stellten
fest, dass die Zahl der aus der Lunge gespulten Makrophagen
dieser Tiere reduziert und dass spezifische Funktionen der Al-
veolarmakrophagen (z.B. drug metabolism) beeintrachtigt waren.
Von Dr. Manojlovic (Dusseldorf, BRD) wurden zur Prufung der
individuellen Disposition Lymphozyten von Versuchspersonen in
vitro mit Quarz und PHA inkubiert und anschliessend Mitose-
sowie Transformationsrate der Lymphozyten untersucht.
ZIELHUIS (Niederlande)
Wie erklart Herr Dr. Beck die von Cooper (USA) verbffent-
lichtan Angaben, aus denen sich kein Hinweis einer erhohten
Sterblicnkeit infolge von Erkrankungen der Atemwege ergibt, und
was sagt er zu der allgemeinen Erfahrung, dass es keine erhohte
Sterblichkeit durch Erkrankungen der Atemwege gibt, im Blick-
winkel seiner Versuche und insbesondere der "Treffer-Theorie"?
Mit anderen Worten, wie vereinbart er seine Versuche mit
dem weiten Erfahrungsschatz der Humanmedizin?
-------
1041
BECK (Bundesrepublik Deutschland)
Ziel des Dbersichtsreferates liber die Verwendung der Zell-
kultur in der Umweltmedizin war, den Stellenwert dieses Test-
systems aufzuzeigen und bestehende Korrelationen zwischen Er-
gebnissen von in vivo- und in vitro-Untersuchungen darzustellen.
Die Untersuchungen liber die Wirkung von partikelformigem Blei
in der Zellkultur und im Tierversuch sind also hier in diesem
Zusammenhang zu sehen.
Ira Falle des Bleis wird besonders von Toxikologen die
Meinung vertreten, dass erst der Nachweis einer chronischen^
Toxizitat gesundheitspolitische Massnahmen rechtfertigt, Praven-
tivmedizinern geniigt fur solche Massnahraen dagegen bereits der
begriindete Verdacht fur eine Gesundheitsgefahrdung vor allem der
Personen, die durch Krankheit oder 'Alter in ihrer Widerstandskraft
geschwacht sind, besonders, wenn berucksichtigt wird, dass Blei
innerhalb der "toxischen Gesamtsituation" nur einen Faktor unter
vielen darstellt.
In der Zellkultur ist bei Applikation von partikelformigen
Stoffen die Dosis-Wirkungs-Beziehung unter dem Gesichtspunkt
der Treffer-Theorie zu betrachten. Dies gilt letztlich auch
fiir die Zellen in der Lunge nach Inhalation partikelformiger
Stoffe. Jeder aktive oder passive Kontakt eines Staubteilchens
mit der Zelle ist als ein Treffer zu betrachten. Jeder Treffer
wird eine Reaktion der Zelle auslosen. Die Summation der Ein-
zelereignisse kann pathogenetisch bedeutungsvoll sein.
DANIEL (G.B.)
Gewebekulturen bieten eine Moglichkeit, Verbindungen auf
ihr toxikologisches Potential hin zu untersuchen. Sie stellen
ferner eine verfeinerte Methode dar, die sich zur tiefergehenden
Untersuchung des Wirkungsmechanismus von Verbindungen auf der
Zellebene eignet. Ich mochte den Referenten fragen, welche
biocheraischen Parameter seiner Meinung nach als die empfindlichsten
Indikatoren toxischer Wirkung anzusehen sind. Ich spreche nicht
von histochemischen Verfahren; mir geht es darum, mehr quanti-
tative Angaben zu sammeln, so dass die Dosis-Wirkungs-Beziehung
festgestellt und damit die Beurteilung des jeweiligen Risikos
erleichtert werden kann.
BECK (Bundesrepublik Deutschland)
Auch wir sind der Meinung, dass das Testsystem Zellkultur
inzwischen soweit ausgereift ist, dass es sich zur Prlifung
sowohl der akuten als auch chronischen biologischen Wirkung von
Fremdstoffen der Umwelt eignet. Beziiglich Ihrer Frage, welche
biochemischen Parameter unserer Meinung nach als die empfindlichs-
ten Indikatoren einer toxischen Wirkung anzusehen sind, mochte
-------
1042
ich in erster Linie die Zellpermeabilitat nennen. Ein intakter
Zellstoffwechsel ist die Voraussetzung zur Aufrechterhaltung
einer physiologischen Permeabilitat. Wird Z.B. der Energiestof f-
wechsel durch toxische Einwirkung gehemmt, so ist ein Enzymverlust
die Folge. Eine Storung der Zellpermeabilitat wird aber nicht
nur durch einen toxischen Eingriff in den Energiestoffwechsel
verursacht, sondern ebenso durch direkte Wirkung auf die aussere
Zellmembran. Als Kriterium fur den Nachweis einer erhohten
Permeabilitat wird von uns die Freisetzung intrazellularer Enzyme
herangezogen, gemessen an ihrer Aktivitat im iiberstehenden Nahr-
mediurn. Die extrazellulare Enzymaktivitat ist ein reprasentativer
Indikator fur die Permeabilitat der Zelle. Dabei sollte besonders
hervorgehoben werden, dass wegen der geringen benotigten Proben-
menge kontinuierliche Messungen der Enzymaktivitat und parallel
dazu morphologische Untersuchungen der Zellen im Versuchsablauf
mbglich sind. Als weiterer Indikator fur eine Storung der Per-
meabilitat dient die Aufnahme saurer bzw. der Verlust fluoro-
chromer Farbstoffe. Die Fluorochromasie kann quantitativ erfasst
werden, entweder mit Hilfe der Impulszytophotometrie oder aber
durch Messung der Emissionsenergie des in den Oberstand freigesetz-
ten Farbstoffes.
Als weitere empfindliche Parameter fur eine toxische Wirkung
auf zellularer Ebene eignen sich der ATP/ADP-Quotient und der
O^-Verbrauch (kontinuierliche polarographische Messung) als
Ausdruck des Energiestoffwechsels. Von besonderem Wert als
Kriterium fur eine Schadstoffwirkung ist schliesslich die
Aufzeichnung des Habitus und der Motilitat der Zellen im Zeit-
rafferfilm. Auf diese Weise lasst sich das Verhalten von Zellen
(Vermehrung und Motilitat) kontinuierlich registrieren und
quantitativ auswerten.
-------
1043
EPIDEMIOLOGY OF PESTICIDE AND METAL RESIDUES
ANTHONY V, COLUCCI
Environmental Protection Agency, Research Triangle Park, NC, USA
(Editorial Note: Paper presented by J.A. Santolucito)
ABSTRACT
Studies relating to the epidemiology of pesticide and metal
residues involve the formulation of appropriate research models.
These models must be formulated in order to quantitate the impact
of exposures involving multiple routes to man and to test speci-
fic hypothesis relating these pollutants to adverse health effects,
Crucial to all of these studies is the investigation of hu-
man body burdens of these residues using both the general popu-
lation or selected subgroups at greater risk either from occupa-
tional or geographic exposure.
The studies we have conducted have been designed to address
a number of questions in several areas. The first series of
investigations was aimed at determining which tissues are most
suitable for monitoring body burdens of the residues in human
populations. Thus blood, urine, hair, feces and placenta were
analyzed for pesticide, trace metal and synthetic organic resi-
dues. In addition, multiple tissue sets were collected at
autopsy and analyzed.
The second series of studies was designed to test specific
hypothesis relating disease risk to pollutant levels. Typical
of the questions investigated were the relationship between met-
als and organic burdens and cardiovascular disease and toxemia
of pregnancy.
-------
1044
The final phase of our research efforts focused on linking
tissue burdens with refined laboratory indices of health impair-
ment. Examples of these studies include the effects of metals^
organics and pesticides on placental enzyme activity, hematolo-
gical indices and detoxification mechanisms along with changes
in other critical clinical parameters.
This linkage of clinical and laboratory approaches to a
coordinated epidemiological system allows not only a current
assessment of risk in these populations but, in addition, a move
predictive posture as new pollutant problems arise.
To date these studies have yielded interesting preliminary
results. Whereas easily collected specimens such as hair and
blood have proved utility in estimation of environmental expo-
suret these tissues cannot, as yet, be considered a good quan-
titative indicator of many body pollutant burdens. Studies of
occupationally exposed groups or groups of individuals with
clinically evident disease have failed to clearly implicate
pollutant burdens with either disease production or aggravation
although, in general* the blood levels of these individuals do
reflect increased exposure to pollutants. Studies of multiple
tissue sets collected at autopsy are encouraging inasmuch as it
appears these tissues will provide useful information about the
pollutant burden for constructing predictive models and, in
addition, provide flashback capabilities.
-------
1045
1. Introduction
Studies relating to the epidemiology of pesticide and metal residues
involves the formulation of appropriate research models. These models must
be formulated in order to quantitate the impact of exposures Involving mul-
tiple routes to man and to test specific hypothesis relating these pollu-
tants to adverse health effects.
Crucial to all of these studies is the investigation of human body
burdens of these residues using both the general population of selected sub-
groups at greater risk either from occupational or geographic exposure.
To those of us concerned with health effects, clear definition of the
linkage between pollutant burdens and biological response becomes a matter
of great importance. This is particularly true because human pollutant
burdens can elicit a broad spectrum of biological responses that are not
clearly separable. Specifically, a pollutant burden can exist with no
associated physiological changes or it can be associated with physiological
changes that are sentinels of disease and have a strong association with
morbidity or mortality.
2. Approach
In addressing Itself to these problems, our group has currently mount-
ed projects in several areas. The first series of investigations is de-
signed to determine what tissues are most suitable for monitoring pollutant
burdens 1n large populations. Thus, blood, urine, hair, excreta, and pla-
centa are assayed for selected trace metals and synthetic organic com-
pounds. In addition, multiple tissue sets are being collected at autopsy
and analyzed. Whenever possible, study groups are chosen because of their
exposure to an appropriate pollutant gradient. Another aspect of this
first series of studies 1s the establishment of a national tissue bank to
provide flashback capabilities as new problems eirerge, and to allow accu-
rate assessment of tissue distribution profiles of the various pollutants
in the human population.
The second series of studies we are conducting is designed to test
specific hypotheses that relate disease risk and pollutant levels. Typi-
cal of the questions investigated are such problems as the relationship
of specific metals to Indices of cardiovascular disease and the trans-
placental transfer of metals and toxemia or pregnancy. The rationale is to
correlate pollutant burdens with known disease symptoms and assess their
role 1n disease production or aggravation. Focusing on selectively vul-
nerable or occupationally exposed groups permits direct assessment without
-------
1046
the large dilution factors operative in general population studies.
The final phase of our research efforts is focused on linking pollutant
burdens with refined laboratory indices of health impairment. Specific
examples of these studies include the effects of metals and organic com-
pounds on profiles of placental enzyme activity and pollutant-induced
changes in detoxification mechanisms.
Selection of tissues for evaluation of pollutant burdens is currently
under active investigation by our group. A number of studies have indicated
a correlation between trace metal content in hair and exposure; an example
is shown in Table 1. As can be seen, levels of arsenic, cadmium, and lead
in hair closely follow the exposure gradient, whereas zinc and copper
levels do not. The latter two metals, it should be recalled, are essential
to man, and homeostatic mechanisms for their metabolism are known to exist.
Studies of occupationally exposed groups such as men exposed to cad-
mium dust from superphosphate production have established the utility of
blood as an index of exposure. In these workers (Table 2), plasma levels
of cadmium and zinc were elevated in both the intermediate- and high-expo-
sure groups. By contrast, urine levels failed to show any correlation with
exposure.
Attention has been focused on organic pollutants as well. By exam-
ination of the plasma of over 700 healthy individuals, polychlorinated bi-
phenyl (PCB) residues, ranging in concentrations up to 29 parts per billion
(ppb), could be found in 43%. In addition, residues of pp'DDDT and DDE
were found in 84% and 63%, respectively, of the individuals tested. Inter-
actions involving ethnic differences and ethnic residence were noted for
most residues as well. Residues of polychlorinated biphenyl were found more
frequently and at higher levels in white, urban residents and rare (4.1%) in
rural blacks (Table 3). In refuse workers potentially exposed to poly-
chlorinated biphenyl from incineration of numerous materials, residues of
PCB were identified in the plasma of 81% (32 of 37 workers) while only 11%
(6 of 54) of the control group showed abnormal evidence of these residues.
Median levels of PCB for detectable samples were 2.6 ppb (exposed), 3.7 ppb
(controls), and maximum levels in plasma were 14.1 ppb (exposed) and 20.2
ppb in the contrcls. Scalp hair in this study was found to be of no util-
ity in estimating exposure to PCB. (Table 4)
-------
1047
TABLE I. DISTRIBUTION OF TRACE METAL LEVELS IN HAIR
Metal
Arsenic
*•
Cadmium
Copper
Lead
Zinc
Exposure*
Ranking (City)
5
4
3
2
1
5
4
3
2
1
5
4
3
2
1
5
4
3
2
1
5
4
3
2
1
Arithmetic
Mean, ppmt
10.6
5.2
1.7
0.8
0.4
3.5
2.0
1.3
1.3
0.9
25.7
15.3
11.8
12.6
22.5
107.1
44.3
14.3
12.1
7.6
154.0
145.2
156.6
155.4
154.2
Standard
Deviation, ppm
7.00
6.00
1.48
0.33
0.26
4.94
K54
0.99
1.30
0.58
28.1
7.5
3.0
6.0
34.7
131.8
49.3
14.1
11.4
5.0
33.7
30.8
26.0
36.9
32.5
Sample
Size
31
16
32
13
28
45
85
37
21
37
45
37
25
21
37
45
25
37
21
38
45
25
37
21
38
*Exposure Ranking 5>4>3>2>1.
tParts per million.
TABLE II. AVERAGE CADMIUM AND ZINC LEVELS IN URINE
AND PLASMA OF WORKERS EXPOSED TO CADMIUM DUST
Exposure
Category Cadmium Zinc
Plasma Concentration, yg/100 ml
Low 0.42+0.7 54+32
Intermediate 2.7 +2.7 136+68
High 1.9 +2.0 104+64
Urine Concentration, pg/Liters
Low 1.04 630+550
Intermediate 1.14 440+350
High 1.10 630±40Q
-------
1048
TABLE III. PLASMA RESIDUES OF SELECTED CHLORINATED HYDROCARBONS*
Race-Residence
Grouping
Rural black
Urban black
Rural white
Urban white
Total DDT,
ppbt
20.90
12.50
5.57
4.71
Mean
Percent
Measurable
100
98.3
97.0
99.0
Plasma Residue
Total PCB,
PPb
0.35
1.97
3.18
2.35
Percent
Measurable
4.1
49.3
50.9
55.7
*Sample taken among residents of Charleston County, South Carolina.
tAbbreviations: DDT, dichlorodiphenyl trichloroethane; ppb, parts per
billion; PCB, polychlorinated biphenyl.
TABLE IV. PLASMA LEVELS OF POLYCHLORINATED BIPHENYLS
IN REFUSE WORKERS AND CONTROLS
Group
Control
Exposed
refuse
workers
No. of
Observations
54
37
No. With
Measurable
Level s
6
32
%
Measurable
11
81
Mean Levels,
ppb*
3.7
2.6
Maximum
Levels
PPb
20.2
14.1
*ppb - parts per billion
As can be seen from studies of this type, easily collected tissues,
such as hair end blood, can be of utility in estimation of exposure. How-
ever, a great need exists to relate these levels of pollutants in the hair
and blood to actual tissue burdens.
To begin this task, our group has conducted feasibility studies fo-
cused on trace metal analysis of multiple tissue sets collected at autopsy.
Preliminary results have revealed some trends between the amounts of lead,
copper, iron, cadmium, and zinc in scalp hair and the amounts in bone,
kidney, liver, lung, and aorta tissue, but more data will be needed before
rigorous, predictable relationships can be established.
When we have investigated the relationship between pollutant burdens
and selected populations at risk or subjects with disease, the problems
have become even more manifest.
-------
1049
Studies in maternal-fetal tissues have provided evidence for accumu-
lation and transplacental transfer of metals. Analysis of these sets
(Table 5) demonstrated that placental cadmium levels were one to two times
the levels found in maternal or cord blood. It was observed also that
erythrocyte cadmium levels were roughly three to five times plasma cadmium
levels, and that maternal erythrocyte cadmium levels were somewhat higher
(27%) than those noted for the fetus. In addition, cadmium levels in the
meconium were very similar to those found in maternal blood. The data on
lead is only partially complete, but its transfer and accumulation is al-
ready known. The problems relating mercury to fetal Minamata disease are
familiar.
TABLE V. RELATIONSHIPS BETWEEN CADMIUM AND LEAD LEVELS OBSERVED
IN TISSUES FROM MOTHERS AND NEWBORN CHILDREN
Arithmetic Mean
Tissue
Placenta
Cord Plasma
Maternal plasma
Placenta
Maternal scalp hair
Maternal perineal hair
Maternal whole blood
Maternal erythrocytes
Cord blood
Cord erythrocytes
Merconium
No.
of Pairs
100
100
100
25
25
25
25
25
25
25
25
Cadmium Lead
(uq/100 qm) (yfl/100 gin)
7.3
1.4
1.5
6.8
374.0
309.0
3.7
7.1
4.6
5.6
8.6
96
« • •
• * *
• • *
2,933
1,236
33
58
44
46
26
Ratio,
Lead to Cadmium
13.2
• • *
* * •
• * »
7.8
4.0
8.9
8.2
9.6
8.2
3.0
These data are thus clearly indicative of transplacental transfer of
cadmium, yet metal levels could not be causally linked to increased blood
pressure or decreased weight of the newborn infant, nor could they be
shown to increase in toxemia of pregnancy as has been alluded to by other
workers.
Studies in persons who died of cardiovascular disease demonstrated
no consistent elevation in renal cadmium concentrations, and the apparent
elevations in renal cadmium concentrations previously observed may have
been occasioned by the confounding effects of the widely varying renal
cadmium levels noted in cancer deaths.
Similarly, although elevated levels of plasma cadmium were observed
in superphosphate workers, these elevated levels could not be linked to
changes in hemoglobin levels, hematocrit readings, blood pressure, and
cholesterol or serum lipid levels in the exposed groups.
-------
1050
Thus, it is clear that the mere presence of a pollutant in tissue
is not always sufficient evidence of disease potential, and one must be
careful in ascribing the cause of disease to pollutants fortuitously
associated with clinical symptoms. Pharmacological experience has taught
us that the toxicity of an agent is not always expressed at the site of
maximum concentration, and it may be manifested only by subtle changes.
Thus, our final series of studies currently underway is designed to link
the effects of pollutant burdens with refined laboratory indices of health
impairment. These studies are designed to answer several questions. First*
what subtle biochemical changes occur in individuals under certain pollu-
tant burden stresses? Second, what changes in readily assayable tissue
can correlate or quantify these effects? And finally, what is the dose-
response relationship between pollutant burdens and changes in critical phy-
siological and biochemical systems? These studies are recent in conception,
but the preliminary results appear promising. One promising index current-
ly under investigation utilizes the profiles of placental enzyme activity.
This two-part project examines enzymes that may be induced or altered by
specific pollutants such as hydroxylases and metalloenzymes and, in
addition, other enzymes that are rate-limiting for selected metabolic
cycles.
Assessment of changes in nonenzymatic areas is also necessary. Thus,
we examine protein profiles and critical substrate levels in blood and
other tissues as well. Bioenergetic and immunologic systems may also
reflect effects of pollutant burden. In our laboratories, preliminary
studies are underway, and attention is being focused on development of
mitochondrial and microsomal assays that reflect such pollutant burden
sensitivities.
Clearly, the problems encountered in linkage of refined laboratory
indices to pollutant burdens are even more complicated than those encount-
ered in other studies. Inhibition of any enzyme by a pollutant is an effect
worthy of note, but relation of this effect to disease production in man
requires careful documentation. Similarly, direct or indirect changes In-
duced by exposures to pollutants in areas known to be sentinels of disease
must be carefully evaluated. Adverse effects must be separated from
normal or even beneficial changes and dose response is a prime concern.
Transient changes in any dimension can have a cause far removed from
pollutant effects themselves, and clear patterns in controlled studies
-------
1051
must be obtained. It Is our belief* however, that these biochemical and
physiological Indices hold great hope for the future. Factors such as
these are applicable to living human populations In a practical sense and
have the advantage of being relatively Inexpensive to assay and are be-
coming very well standardized.
In the design and Implementation of all these studies, a critical need
exists for multiple tissue analysis. In response to this need, we have
Initiated on a pilot scale a national tissue bank designed to permit pre-
sent and future evaluation of pollutant levels. By accumulation of tissues
from humans at autopsy, we can assess the relationship between pollutant
burdens 1n various tissues. Similarly, this tissue bank will permit
collection of sufficient tissues from cases of verified disease and
allow more comprehensive Investigation of correlations between patterns
of pollutant burdens and these disorders. In addition, this tissue bank
will provide flashback capabilities should, as almost certainly will be
the case, new pollutant problems arise in the future.
This discussion has given only a brief overview of our efforts In
the area of assessing and characterizing effects of pollutant burdens, but
we feel it represents an Integrated approach to the solution of these com-
plex problems.
DISCUSSION
SILBERGELD (U.S.A.)
With reference to the first slide, showing levels of various
metals, is there any relationship, direct or inverse, between the
lead and zinc levels observed in your sampled population?
SANTOLUCITO (U.S.A.)
The data presented were preliminary and because of the small
sample size inferences on a relationship between lead and zinc
levels were not made.
-------
1053
STORAGE MAP OF ORGANO-CHLORINE COMPOUNDS (OCC) IN HUMANS
EPIDEMIOLOGICAL DEDUCTIONS FOR FURTHER MONITORING
M, WASSERMAN+, L, TOMATIS++, DORA WASSERMAN+
+ Department of Occupational Health, Hebrew University-Hadassah
Medical School, Jerusalen, Israel
++ World Health Organization - International Agency for Research
on Cancer, Lyon, France
ABSTRACT
It is characteristic of living organisms to carry the foreign
compounds which enter them through continuous cycles of activity
like their own components. Some of the compounds on the environ-
ment accumulate to some extent in the animal body. This storage
constitutes a dynamic process.
In the framework of a joint programme WHO-International
Agency for Research on Cancer and the Hebrew University-Hadassah
Medical School, Department of Occupational Health, Jerusalem* the
OCI storage in humans has been investigated in various populations
in Africa^ Asia and South America.
Data obtained in these studies led to the conclusion that the
age group 25-44 years stored the highest amount of OCI in both
sexes for most populations. Since after the age of 45 years a
change in the amount of OCI stored was observed, it is suggested
that the age group of 25-44 years may characterize the OCI storage
level of a community and can be compared with the mean storage
level of stillborns in order to assess the increase in man's OCI
impregnation from his fetal life.
The paper presents a map of the storage level of OCI in hu-
mans throughout the world according to data reported in literature.
Suggestions for the way of further monitoring are presented.
-------
1054
The importance of the assessment of the OCC body load in the
general population and in occupationally exposed workers evolves
from the circumstances of continuous pollution of the environment
with remanent, biologically active compounds.
The OCC map we intend to draw has this justification. More-
over/ it may constitute a reference point for further epidemic-
logical studies.
A quarter of a century has passed since the first publication
of DDT storage in man (Howell, 1949).
The study of OCC storage in the general population and
occupationally exposed people, which began in the U.S., spread
to Europe, Asia, and the other continents and has now reached
great development in Japan.
From the data available on OCC storage, the following facts
emerge:
1. The storage of OCC in man is a widespread phenomenon,
in fact they are a current constituent of humans living in the
second half of this century.
2. The storage of OCC is proportional to the degree of ex-
posure, since occupationally exposed people store the highest
levels (128).
3. The storage of OCI is higher in men, and there is a
direct relation to age, starting with intrauterine life up to
the age of 45 years. In some countries, (USA, Israel) this
direct relationship exists for all the age groups, while in
others a decrease in the storage level occurs after 45 years of
age.
4. The geographical location of the groups samples seem
to have an influence on OCI storage. The storage is higher in
south and east Europe when compared with north and west Europe.
The same applies when we compare Canada to the United States.
The cause of these geographical differences may be local agri-
culture or sanitary practices.
5. The physiological state of the body (e.g. pregnancy),
obesity, loss of weight and pathological conditions like liver
disease (86), carcinoma (85), seem to influence the storage
level of OCC in opposite directions. (The milk of obese women
contains lower amounts of OCI than that of women with normal
weight (Germany, DDR (70)).
6. Race also seems to influence the storage level. Davies
(30) found higher concentration in Negroes of the United States.
In South Africa, a lower level of OCI storage was reported by us
in non-white people (127). It is possible that socio-economic
conditions may explain such differences. In a study of OCI serum
levels in a multiracial population, significant differences were
found among the various ethnic groups: sera from Chinese con-
tained the highest levels of p,p'-DDT and 0-BHC. Koreans had
the highest levels of Dieldrin, and people from triracial back-
grounds had the highest levels of y-BHC (64).
-------
1055
7. There is a bidirectional relation between OCX. and some
drugs which influence their storage level by activation or in-
versely by inhibition of enzymatic systems involved in the meta-
bolization of these compounds. (Volunteers receiving diphenyl-
hydantoin (31) as well as patients receiving phenobarbital and/
or diphenylhydantoin (137) had a lower OCI storage level when
compared to controls. In an OCI plant, one of the workers who
took phenobarbital and diphenylhydantoin over 25 years for post
traumatic epilepsy had no or trace amounts of DDT and DDE residues
in his serum (73).
8. The banning of special OCC which reached too high a
level in special areas (e.g. 3-BHC in Japan) led to decrease in
storage of the banned compound (B-pHC in Japan decreased 20-50%
(89)).
9. The exposure of the general population to OCI occurs
especially from contaminated food but also from household use
(B-BHC residue was higher in the milk of non-farm women in
Japan (9,114)). Mothers in non-agricultural families consume
larger amounts of beef and milk daily, when compared to farm
women (49).
1O. The greatest exposure of the general population, non-
occupationally exposed to OCCr is that of infants fed by mother's
milk. Mother's milk has a higher OCC residue than cow's milk
and in some regions the exposure is high enough to cause bio-
logical effects. (Inhibition of corticoids synthesis, gluconeo-
genic enzyme activity, and interference with calcium, vitamin
D and sex hormone metabolism (7)). In Guatemala, OCI residue
of mother's milk is 25-30 times the average level found in the
USA, England, and Sweden (Lofroth, 76).
In conclusion, the epidemiological and analytical findings
regarding the OCC storage in man provide, at this time, a
reasonable basis for adopting a more uniform approach in assessing
the storage levels of these compounds in the general population.
Accordingly, we propose:
1> The age group 25-45 years should constitute a reference
group characteristic of the OCC storage of a community.
2. A uniform method for the entire analytical procedure of
OCC assessment should be adopted.
3. The findings should be reported both as OCI in extracted
lipids and in whole tissues or biological fluids.
4. The measurement of OCI residues in the human body should
be considered as a component of a national program in public
health, since it proved to be a valuable indicator of the ex-
posure to OCC and consequently a basis for implementing suitable
preventive programs.
-------
1056
Table 1. Storage of OCC in fat tissue of humans (ppm). North America
Authors
Laug et al .
Hayes et al.
Dale & Quinby
Hoffman et al.
Quinby et al.
Hoffman et al.
Hayes et al.
Ket
52
29
55
100
54
51
Fisherova Bergerova 43
et al.
Zavon et al.
Shafer & Campbell
Edmunds on et al.
Morgan & Roan
Da vies & Edmundson
Curley et al.
Warnick
Price & Velch
, * n PS
Jpbs, A.R.
Biros and Walker
Durham et al.
Cassaret et al.
Kadis et al.
Read & McKinley
iitcey et al.
Mastromatteo. E.
143
108
37
82
30
24
136
98
59
14
35
20
62
102
103
80
Country
USA
USA
USA
USA
USA
USA, Chicago
USA, New Orlean
USA
USA
USA
USA, Florida
USA, Arizona
USA, Florida
USA, Atlanta
USA, Utah
USA, Michigan
USA
USA
USA, Alaska
USA, Hawaii
CANADA
CANADA
CANADA
CANADA, Ontario
T.DDT BHC
5.3
11.7
6.7
10.3
12.7
10.4
slO.3
10.6
7.6
2-31
6.5
12.4
8.8
9.0
7.2
5.3
3.0
5.7
4.3
4.9
4.8
9.2
0.20,,
0.57*
0.48*
0.60*
0.55
1.07*
0.01*
Die!.
0.15
0.11
0.14
0.29
0.22
0.31
0.22
0.14
0.24
0.20
0.15
0.15
0.01
0.12
0.22
PCB
2.0
»2 0
ba • \J
HCB
Year
1950
*•* vw
1955
*•* W ti
1961
*«^W J
1964
* W V
196]
*JU J
1963
1965
A. J \J ^
1966
1964
*• Jv "
1964
J-7D*t
IQfiR
*J\JiJ
1967
J.JU /
1966
±J\J\J
1969
1968
1969
1970
1972
1Q7P
ij 1C.
1Q7rt
1-7/U
1960
*.j\j\j
1968
±Tj\j\j
1967
10 CO
x?97
1969
1970
Table 2. Storage of OCC in fat tissue of humans (ppm). South America
Authors
Wassermann et al.
Wassermann et al.
Fernandez et al.
Astolfi et al.
Ref.
129
126
42
10
Country
BRAZIL
ARGENTINA
ARGENTINA
ARGENTINA 2
T.DDT
7.9
13.2
6.6
.7-30.
BHC
0.25*
2.43
0
Diel.
0.13
0.30
PCB
HCB
Year
1970
1969
1970
1970
Only one isomer.
-------
1057
Table 3. Storage of OCC in fat tissue of humans(ppm). Europe,
Authors
Abott et al.
Abott et al.
tobinson et al .
tobinson et al.
•lunter et al.
:gan et al.
Cassidy et al.
Widmark & Jensen
Bjerk, J.E.
Wei he, M.
Vlieger et al.
laier-Bode, H.
^ngst et al.
•ngst & Knoll
WUncher & Acker
Acker & Schulte
Jorneff, j.
Jarchet, R.
layes et al.
:ournier, E.
:ournier et al.
Ref.
1
2
105
104
58
38
21
140
15
138
120
79
40
39
142
4
17
11
50
44
45
Jonezyk & Bojanovska 60
Juszkiewisz & Stecy
)chynski & Bronicz
Jogusz, M.
'rojanowska et al.
61
91
16
116
'askovskaya&Komarova!19
Uracheva
ialacka et al.
tosival et al.
)enes, A.
Denes & Tarjan
Jerend et al.
Soos et al.
'esendor/er et al .
Adamovi c et al .
ftizicovici et al .
tondroiu & Jordache-
(aloyanova, F. scu
-------
1058
Table 4. Storage of OCC in fat tissue of humans (ppm). Asia.
Authors
Dale et al.
dassermann et al.
Jassermann et al.
iJassermann et al.
tfassermann et al.
lughal & Rahman
Jassermann et al.
lishimoto et al.
iizutani et al.
)oguchi et al.
Curley et al.
Ui, I.
hurley et al.
Casai , A.
Suzuki et al.
fatsumi et al.
Kawanishi et al.
Ref.
28
121
123
135
133
83
131
88
81
27
23
117
23
66
110
112
68
Country
INDIA
ISRAEL
ISRAEL
ISRAEL
ISRAEL
PAKISTAN
THAILAND
JAPAN, Kochi
JAPAN, Kyoto
JAPAN, Tokyo
JAPAN
JAPAN
JAPAN
JAPAN
JAPAN, Hiraga
JAPAN, Usaqa
T.DDT
12-31
19.2
15.4
14.4
25.0
13.0
6.9
9.7
3.7
2.5
8.1
4.5
2.1
4.0
4.2
6.4
BHC
0.86
-1.7
0.47
12.2
11.7
3.2
1.5
4.3
2.4
3.0
4.0
2.4
2.7
Diel.
0.03
-0.06
0.12
0.46
0.19
0.33
0.13
0.16
0.21
0.43
0.16
0.13
PCBs
2.75
4.7
5.0
0.8
HCB
0.08
Year
1965
1963
1965
1969
1973
X973
1970
1970
1972
1972
1973
1972
1973
1972
1970
1971
1972
1971
1973
Table 5. Storage of OCC in fat tissue of humans (ppm). Oceania.
Authors
Jick, M.
tassermann et al .
Luqq, R.
Jrotiy & Siyali
Jrewer & Grath
Darcre, J.C.
Copplestone et al.
Dyment et al.
Ref.
13
124
77
19
18
26
22
36
Country
AUSTRALIA
AUSTRALIA
AUSTRALIA
AUSTRALIA
NEW ZEALAND
NEW ZEALAND
NEW ZEALAND
NEW GUINEA
T.DDT
9.5*
9.4
3.1
5.8
5.4
3.9
BHC
0.68
0.50
Diel.
0.05
0.67
0.21
0.27
0.41
0.27
PCBS
0.0
Fable 6. Storage of OCC in fat tissue of humans (ppm). Africa.
Authors
tassermann et' al.
tfassermann et al.
nlassermann et al.
Jassermann et al.
ufassermann et al.
Ref.
127
125
132
130
134
Country
SOUTH AFRICA
NIGERIA
NIGERIA
KENYA
UGANDA
T.DDT
6.38
8,75
6.50
4.60
2.90
BHC
2.41
0.68
0.30
0.29
0.08
Diel.
0.04
0.22
0.18
0.10
0.04
PCB$
HCB
1.25
Year
1965
1968
1969
197?
196fi
1963
1965
1969
1971
HCB
Year
1969
1967
1970
1970
1970
* Arithmetic mean.
-------
1059
Table 7. OCC residues in mother's whole milk (ppb).
Authors
Bjerk, J.E.
Westoo & Noren
Egan et al.
Knoll & layanaman
Acker & Schulte
Engst & Knoll
Kontek et al.
Bogusz, M.
Gracheva, G.V.
Komarova, L.F.
Komarova, L.F.
Mandroiu &Jordaches cu
Adamovi c et al .
Adamovic et al
Laug et al.
Quinby et al.
Curley & Kimb rough
Mi Ison et al .
Savage et al.
.of roth, 6.
Ref
15
139
38
70
3
39
72
16
47
71
71
78
5
7
74
99
25
141
107
76
Olszyna Marzys et al. 92
Lugg, R.
Newton & Greene
Horn ab rook et al.
Gejvall et al.
Takeshi ta & Inuyama
Takeshi ta & Inuyama
Kato et al .
Oura et al.
Anonymous
Anonymous
Narafu, T.
Hidaka et al.
77
87
56
46
111
111
67
94
9
9
85
53
[Tattori Res.Hyg.Inst.115
Kamata, I..
Osaka Pref.
Osaka Pref.
lay ash i, M.
layashi , M.
Yishimoto et al.
Yagai , I .
lagai, I.
lagai, I.
pagai, I.
64
93
93
49
49
90
84
84
84
84
Country
NORWAY
SWEDEN
ENGLAND
GERMANY
GERMANY
GERMANY
POLAND
POLAND
USSR
USSR, Urban
USSR, Rural
T.DDT
50-100
113.0
130.0
320.0
112.0
230.0
280.0
715.6
0-1000
100.0
190.0
ROMANIA, Vrancea
SERBIA
SERBIA
USA, Black
USA
USA
USA, White
USA
207.5
587.1
130.0
145.0
70.0
170.0
7.4950
CENTRAL AMERICA 310QP
GUATEMALA
AUSTRALIA
AUSTRALIA
NEW GUINEA
GHANA
JAPAN, Agric.
JAPAN, Nonagr
JAPAN
JAPAN
JAPAN
JAPAN
JAPAN
JAPAN
OAPAN
JAPAN
JAPAN
JAPAN
JAPAN, Agric.
28633
170.0
142.0
29.0-959
29.0
BHC
13.0
18.0
Diel.
6.0
Q- 560.0
5.0
14.0
7.0
79.0
6.0
o-sqo! o-ii.q
.0-
10QO
2.0
X.O
79 J3 142.9
, 660250.9
19-105.0] 18:
33.0
60.2
56.2
740
49.0
115.4
96.0
15.0
0-12J)
0-43P
0-
12.0
20-400.0
95.0
124.0
90-1800
21.0
43.0
56.3
JAPAN, Nonagr. 63.5
JAPAN
JAPAN, Oshima
JAPAN, Nagato
JAPAN, Yanai
JAPAN, Ass a
20.0
31.0
12.0
33.0
120.0
109.3
5.0
4.7
70-160
161.5
180.0
92.6
143.4
163.0
219.0
94.0
247.0
1.0
2.0
3.7
PCBs
1030
40.0
.100.0
HCB
153JD
75. (W
52. a
<5.q86D
30.0
3.4
3.1
50.0
30.0
Year
1972
1968
1965
1971
1971
1970
1971
1972
1964
1970
1970
1971
1969
1972
1951
1965
1967
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1971
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1972
1972
1972
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1060
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DISCUSSION
VAN DER KREEK (Netherlands)
I quote your fact 10 "the greatest exposure of the general
population, is that of infants fed by mother's milk."
Your showed us a slide with data on concentrations of OCC
in fat of infants and neonates. These concentrations in infants
do not differ from the concentrations in adults. Therefore I
do not understand your fact 10, which is not confirmed by the
residues in the fat of the infant. Do you have an explanation?
WASSERMAN (Israel)
At the birth, the OCI storage level in newborns is about
50 percent of their mothers' OCI storage level. There is a
continuous increase of the OCI level - a positive age correlation
until the age of about 45 years. In the mother's milk, the OCI
concentration is rather high (in Guatemala it was reported at
about 3000 ppb) which means a hazardous non-occupational exposure.
OLOFFS (Canada)
1. Considering the difficulties and problems in PCS quantifi-
cation (number of components and differential metabolism), how
valid is it to compare data from the many sources - especially
some older ones - and to base conclusions on such data.
2. During a visit to DDR 2 years ago, I had the impression
that human tissue burdens of DDT should be rather high in this
area as DDT use seemed to be rather prevalent. I find it inter-
esting that the data you presented, Dr. Wasserman, confirm this.
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1066
WASSERMAN (Israel)
The LGC method for PCBs assessment is rather more laborious
than uncertain. The LGC peaks of PCBs were identified by a
number of experienced laboratories.
The present data on stored levels of PCBs in man reported
in the literature, which show differences from country to
country, may be interpreted in the light of local socio-geogra-
phical conjunctures to which the general population is exposed.
REUTER (U.S.A.)
Has any attempt been made to correlate DDT consumption
(by nation and /ears) with tissue levels observed?
WASSERMAN (Israel) &
Hayes et al[AMA Arch. Ind. Hlth, 18, 519 (1972)] had found
lower levels of stored DOT in vegetarians than in meat eaters,
in U.S.A. The same phenomenon was reported by Dale et alfBull.
WHO, 33:471,465] in India. To some extent, a similar situation
for ElHcimos in Alaska, was also reported by Dale.
HOLL (Federal Republic of Germany)
1. Apart from the fact that the toxicity of DDT is still an
open question, and evaluation will have to take into consideration,
that the Federal Republic of Germany placed a legal ban on the
use of DDT on man (Law on the Trade in DDT of 7 August 1972).
Exceptions are only applicable to entomology and parasitology.
2. The question of increased concentrations of HCH in mother's
milk and cow's milk (vice versa in the infant) was of some im-
portance in the south-western border area of Germany at the
beginning of 1974, the reasog.grobably being bound up with waste
disposal problems (via soil^water)• Special studies are being
carried out and results so far obtained can be made available.
Have you become aware of similar relations? They show the
urgency and importance of international arrangements concerning
environmental contamination across national frontiers.
WASSERMAN (Israel)
No comment.
-------
1067
EVALUATION OF THE HEALTH EFFECTS OF NITRATES IN WATER
NACHMAN GRUENER AND HILLEL I. SHUVAL
Environmental Health Laboratory, Hebrew University-Hadassah Med-
ical School, Jerusalem, Israel
ABSTRACT
The standard for nitrates in drinking water was established
as 45 mg/1 based on limited epidemiological and experimental evi-
dence. We initiated a broad range of toxicological and epidem-
iologioal studies to provide a scientific basis for evaluating
the health effects of nitrates in drinking water. The two epi-
demiologieal studies to be reported upon here are part of this
project.
One of the studies which was done among 408 healthy infants
who consume appreciable amounts of tap water in powdered milk for-
mula showed that those infants who consume water with nitrate con-
centrations over 45 mg/l exhibited significantly raised methemo-
globin levels in their blood. The second epidemiological study
was done under controlled conditions in a hospital to attempt to
determine the threshold value of nitrates in water which can cause
a significant increase above "normal" methemoglobin levels in in-
fants. In this study with 115 infanta we showed that nitrate
levels in drinking water of about 100 mg/l can cause a signifi-
cant increase in infants' methemoglobin levels.
The possible health significance of a slightly raised or
subclinical methemoglobin level as found in the two studies is
discussed in light of results from toxicological studies and
general considerations in evaluating the risk of population ex-
posure to chemicals in the environment.
-------
1068
The standard for nitrates in drinking water was initially established
based on limited epidemiological evidence that indicated that no cases of
infant methemoglobinemia occurred in areas with less than 45 ppm of
nitrates (as NO,) in the water [1]. Since the standard was established
there has been considerable controversy on the subject. Some European re-
searchers have reported that they were able to detect raised methemoglobin
levels in so called normal infants in areas where occasional clinical
cases of the disease were reported [2], In addition, clinical disease was
also reported among infants exposed to water having less than 45 ppm of
nitrates. Suggestions that a stricter standard be enforced have been made
as a result of such studies.
On the other hand, extensive areas in the U.S. and Europe supplying
water showing nitrate levels above the standard have reported little or no
clinical cases of the diseases. Since nitrates are difficult to remove
from water by economically feasible means moves have been made for a more
liberal standard reflecting the lack of clinical disease in such areas.
Since the epidemiological and toxicological base for establishing the
nitrate standard was relatively limited, the project reported upon here
was initiated to clarify some of the basic questions concerned with the
health effects of nitrates in drinking water.
The following general questions served as guidelines in carrying out
the evaluation of the suitability of the current nitrate standard in
drinking water:
1. Can a dose-response relationship be established between intake of
nitrates in drinking water and the development of raised methemo-
globin levels in infants?
2. At what threshold level of nitrates in water is the first effect
detectable?
3. What environmental, nutritional, physiological or genetic factors
influence this relationship?
4. What is the health significance, if any, of chronic subclinical or
slightly raised levels of MetHb?
5. Are there direct toxic effects of exposure to nitrates and/or
nitrites other than raised MetHb?
6. Can new sensitive parameters be used to detect health effects due
to exposure to nitrates other than raised MetHb levels?
-------
1069
In many areas of the coastal plain of Israel concentrations of nitrates
in the water supply range from 50-200 mg/1. On completion of the epidemio-
logical study involving 2473 infants in the environs of Rehovot having a
mean NO, concentration of 70 ppm, it became apparent that there are no sig-
•j
nificant differences between the mean MetHb levels in the infants in this
area as compared to infants of the control area (nitrate level of 5 mg/1
[3]. A possible explanation to this lies in the infant feeding practices
where only 6% of the infants included in the study received appreciable
amounts of tap water together with formula prepared from powdered milk. The
Gaza area was considered to be favorable for such a study since preliminary
investigations indicated that well over 50% of the infants up to two years
of age received powdered milk formula with tap water.
The Gaza area study has provided some confirmation that infants in
areas with water supplies having concentrations over 45 mg/1 of nitrates
who consume appreciable amounts of tap water in powdered milk formula show
raised MetHb levels. As can be seen in Fig. 1, infants in the low nitrate
group (under 45 mg/1) show similar MetHb levels (mean = ,74%) regardless of
milk regime while infants who consume powdered milk only in the medium and
high nitrates group (46-200 mg/1) have a significantly raised mean MetHb .
level of 1.18%while those consuming powdered milk and other milk show MetHb
of .99%. Although no clinical cases of infant methemoglobinmeia were re-
vealed among 285 infants in the medium and high nitrate group, 26 of them
showed significantly raised MetHb levels of over 1.8%; of them 23 (10.7%)
received either only powdered milk formula or powdered milk formula in
addition to other types of milk. Only 3.2% of the infants in the low
nitrate group showed raised MetHb.
Under normal field conditions many difficulties hamper the possibility
of detecting a dose-response relationship between nitrate intake and met-
hemoglobin levels especially in the low range. To overcome some of these
problems a controlled experiment was carried out in a hospital located in
an area normally supplied with water high in nitrate,content. In this study
we attempted to determine the threshold value of nitrates in water which
can cause a significant increase above "normal" MetHb levels in infants.
For five days 104 hospitalized infants ranging from one week to ten
months were exposed exclusively to water whose nitrate content was exactly
controlled. The infants received mainly formula prepared from milk powder.
The exposure schedule was: the first and fifth days, low nitrate content
-------
1070
I Z
1.0
0.8
< .
< .
0.4
02
Powdered milk only
Powdered milk + other milk
Other milk only
n= (408)
a b c
MEDIUM HIGH
NITRATES
46 - 200 mg/l
FIGURE 1.
LH.'AN HETHEMOGLOBIN LEVELS IN INFANTS ON DIFFERENT MILK REGIMES,
CONSUMING OF LOW ANT) MEDIUM TO HIGH CONCENTRATIONS OF NITRATES - GAZA.
(15 mg/l), and the second, third and fourth days, high nitrate content
(108 mg/l). The high nitrate water was from a well that served as the nor-
mal supply to the hospital. There was a significant rise on the second day
compared to the first one i.e., following the first exposure to nitrate.
The mean MetUh level on the third day decreased almost to the original
level in spite of the fact that the high exposure continued. It remained
constant on the fourth day (high nitrate intake) but dropped even lower
than the first day on the fifth day (low nitrate again). This may hint of
an adaptation mechanism.
This work indicates that nitrate levels in drinking water of about
100 mg/l can cause a significant increase in infant Metllb levels. If this
exposure is stopped recovery is rapid. It is worthy to note that the mean
Metllb level of 22 infants in the Gaza area on "milk powder only" and who
received water having high nitrate concentrations (mean 82 mg/l) had a
mean Metllh level of 1.37% as compared with a mean of 1.30% for the 104
hospitalized infants exposed to approximately the same level of nitrates
in their powdered milk formula.
-------
1071
The Gaza study appears to provide support for the present maximum
recommended standard of 45 mg/1 of NO, in drinking water. The fact that
•J
the first signs of raised MetHb levels clearly appear in infants exposed
to water just above the standard (45-55 mg/1 NO ) suggests that little if
any safety factor is provided by this standard.
The full health significance of slightly raised MetHb levels such as
reported upon here is yet to be established. Whether such subclinical met-
hemoglobinemia is deletrious in itself or whether such exposure is only of
importance to the extent that clinical cases of the disease develop re-
quires further study.
The question of why and how subclinical methemoglobinemia develops in
certain individuals into the clinical form of the disease still remains
unanswered. Other possible direct toxic effects of nitrates and nitrites
cannot be overlooked as well. Our lexicological studies with rats and mice
pointed out the following findings:
1. Nitrites can pass the placenta and cause raised MetHb in the fetus
[4].
2. Pregnant rats are particularly sensitive to exposure to nitrites
and the pups showed poor growth [5].
3. Rats chronically exposed to sodium nitrate and nitrite in their
drinking water for 16 months show distinct deviations in heart
blood vessels even at the level of 200 ppm [6],
4. Exposure of mice to nitrites in drinking water causes behavioral
effects such as lowered motor activity and an increase in isolation
induced aggression [7].
CONCLUSIONS
The results of the two epidemiological studies which encompassed
2891 infants up to 24 months of age indicate that there is a relationship
between intake of nitrates in drinking water consumed mainly as powdered
milk formula and raised MetHb levels. The effect was detectable and signi-
ficant even in the group of infants exposed to water containing 45-55 ppra
of NO^. Even though no clinical cases of methemoglobinemia were detected,
it is felt that the appearance of a significant increase above the normal
MetHb levels in infants when exposed to water with nitrate concentrations
slightly above the current standard of 45 ppm is sufficient to provide
direct epidemiological support for the current standard. The health signi-
ficance of such subclinical levels of MetHb is unclear and its is still
-------
1072
unknown why only a small number of infants exposed to such levels of
nitrates in water develop clinical cases of the disease.
There is still much to be learned about the association of nitrates in
drinking water and raised MetHb levels in infants. What role does the
powdered milk formula actually play in the development of MetHb? How ef-
fective are vitamin rich foods such as citrus juice and tomato juice in
preventing raised MetHb? What is their mode of action? Why do only a small
percent of infants exposed to high nitrates in water develop clinical
cases of methemoglobinemia? A fuller understanding of these questions is
still required.
However, despite the many questions that remain unanswered it is
apparent that nitrates are potentially more toxic than generally assumed.
Until those questions are fully elucidated the standard would best be
maintained as is while being kept under constant scrutiny and review.
References
1. Walton, B., "Survey of the literature relating to infant
methemoglobinemia due to nitrate contaminated water",
A.J.P.H., 41, 9861 (1951)
2. Knoteck, Z., Schmidt, P., "Pathogenesis, incidence and
possibilities of preventing alimentary nitrate methemo-
globinemia in infants", Pediatrics,34, 78 (1964).
3. Shuval, H.L., Gruener, N., "Epidemiological and toxicological
aspects of nitrates and nitrites in the environment", A.J.P.H
62, 1045 (1972). '
4. Gruener, N., Shuval, H.I., Behroozi, K., Cohen, S., Shechter,
H., "Methemoglobinemia induced by transplacental passage
of nitrite in rats", Bull. Env. Cont. Toxicol., 9, 44, (1973),
5. Shuval, H.I., Gruener, N., "Health aspects of nitrates in
water", U.S. Environmental Protection Agency Report, to be
published (1974).
6. Gruener, N., Yarom, R., Shuval, H.I., "Histological changes
in rat heart arteries after chronic administration of
nitrates and nitrites", Proer Eur. Svmp. on Impact of
Ecological Factors on Peripheral Vascular Disease,
September, 1973,
7. Gruener, N., The effect of nitrites on isolation-induced
agression in mice", Pharmacol. Biochem. Behav. (in press)
-------
1073
RECHERCHES CONCERNANT L'INFLUENCE DE L'ENVIRONMENT
POLLUE AUX RADIATIONS IONISANTES OU SUBSTANCES
CHIMIQUES SUR LES NOYAUX LEUCOCYTAIRES
GH, ROSCA, STELA ROSCA/ E, T, BARSAN
Facultfi de MSdecine, Institut de MSdecine et de Pharmacie, Cluj,
Roumanie
RESUME
Les auteura ont e^tudie1* BUT un grand nombre de aujeta, lea
modifications de I'appendice nucl6aire au niveau dea leucocytes
du aang pSripherique.
D'une part, lea recherohea ont ports aur 500 aujeta prove-
nant de:
a) maladee expoaSa d. dea vadiationa ioniaantea (vayona X ou
gamma) au aouva dea pvocedurea the'rapeutiquesj
b) cadres mSdicaux et auxiliairea tvavaillant dana dea Bevvioea
de radiothevapie ou la vadio-diagnoatique;
c) diffdrenta aujeta venant pour I'examen ou le control pevio~
dique medical.
Lea analyaea cytologiquea ont m-ia en 6widenoet d. un pourcen-
tage varie"* mate eurtout dana le caa b)j la pv&aence de nombreuaes
excroiaaonceanucl&airea aoua la forme d'appendicea nucl&airea.
D'autre part, on a fait dee investigations ches lea pevaonnea
qui trayaillent dans un environnement ohimiquement pollu6 (benzdne
colorants ohimiquea et alooola), dont on a pratique" dea examena
h&matologiquea> Lea analyses ont Sgalement mis en Evidence lea
aapecta dea mSmea modifications oytologiques.
-------
1074
Dans toutes les deux cat6govi.ee dee sujets on a compare lea
donn&es en Tea rapportant a celles dee sujets de contrdle, tout
en relevant lee aspects qualitatifs et quanti.tati.fB de diverses
formes d'excroissances nuclSaires.
ABSTRACT
The authors have carried out tests on a large number of per-
sons in order to study the changes in the nuclear appendix in
peripheral blood leukocytes.
Research was carried out on 500 test subjects taken from the
following groups:
a) patients exposed to ionising radiation (x-rays or gamma rays)
ae part of therapeutic treatment;
b) medical and auxiliary staff working in therapeutic and diag-
nostic radiology units}
c) various persons coming for examination or periodical medical
checks.
The cytological analyses revealed the presence in varying
degrees, particularly in the case of group b), of numerous nuc-
lear growths in the form of nuclear appendices.
In addition, blood tests were carried out on persons working
in a chemically polluted environment (benzene, chemical dyes and
alcohols). Various aspects of the same cytological changes were
also revealed by these analyses.
For both categories of test subjects, the findings were
compared with those of the control subjects, special attention
being given to the qualitative and quantitative aspects of various
forms of nuclear growths.
-------
1075
1. INTRODUCTION
Les recherches cytologiqu.es dStailleee de Davidson et Smith;
Kosenow et Scupin, de meme que celles des plusieurB autres chercheurs
(Caratzali; Porter; Ruhren; Popovici; De Bernard! et Griva; Asley et
Jones; Capra P. Marzani, etc.) Bur la frequence et la morphologic de
certainee ezcroissances nucleaires des leucocytes Banguins ont releve
des aspects particulierement interessants d'une grande utilite pour la
pratique medicale (2, 3, 4, 7, 17* 18, 19, 21, 23, 25, 28).
Un grand nombre d*autres recfaerches experimentales et de laboratoire
ont demontre la liaison qu'ont ces appendices nucleaires, non seulement
avec le sexe des porteurs (drumsticks), mais egalement avec 1*action
nocive de certaines substances chimiqueB ou de facteurs physiques
(mutantes?), qui, se sont montres susceptibles d'induire, dans certaines
conditions, des modifications de la structure nucleaire. (l, 4, 5, 6, 7i
10, 11, 15, 16, 19, 22, 24, 26, 27, 31).
2. HYPOTHESE DE TRAVAIL
Vu les donnees de la litterature medicale de specialite quant a
1*action nocive de certains facteurs chimiqueB physiques ou biologiques,
Bur 1'organisme huraain en general, et en particulier sur ses structures
cellulaires, ainsi que Bur la maniere du comportament cytologique
(microscopique), nous nous Bommes proposes d'etudier:
- la frequence et 1'aspect morpho-cytologique des excroissances
nucleaires (appendices nucleaires) au niveau des leucocytes;
- les m§mes valeurs, obtenues chez des personnes travaillant dans
des milieux pollu^s par dee rayons ioniBants ou des substances chimiques
nocives, par rapport a un lot temoin.
3. KATERIBL ET KETHODE
Le travail a comport6 I'&tude des 1000 personnes, des deux sexes
agSes de 18 - 60 ans, qui nous furent envoy^es afin d'examiner leur
tableau eanguin.
-------
1076
On a fait des recherches sur 3 grands groupes de personnee.
Groupe A, comportant 200 jeunes sujets, sur lesquels on a effectu4 les
examens imposes par leur engagement dans la production.
Groupe B. comportant 500 sujets ayant travaille effectivement 1-20
ans dans des milieux polluee avec diverses substances chimiques telles
ques le benzene, lee colorants synthetiques, lee alcools, I1acetone etc.
Groupe C. comportant 300 personnes divisees en deux Bous-groupes:
le Boue-qroujje C-l. comprenant des sujete exposes a I1action des rayons
ionisants (rayons gamma, rayons x) a 1'occasion de certains precedes
therapeutiques ou bien dans un but diagnostique, et le jpus-groupe C-2.
dont les sujets £taient represent^ par les medecins et dee techniciens,
qui developpaient leur activite quotidienne dans les services de radio-
diagnostic et de radiotherapie.
Les frottis de sang peripherique furent colores par la methode
classique de Kay-Criinwald-Giemsa et furent ensuite examines et photo-
graphies au microscope (imersion).
Sans tenir compte de leur signification, nous avons enregistre les
types suivants d'excroissances nucleaires (sur 100 neutrophilee examines
chez chaque sujet):
l) Le type "A" (Kosenow et Scupin) dit aussi celui de "1'appendice
pedicule" qui a la forme d'une "baguette de tambour", de "goutte sus-
pendile" ou de "mollet de poule" ("drumstieke", d'apres Davidson et
Smith). II s'agit d(une granule chromatique, d'un diametre de 1,5 microns,
bien delimitee et liee au noyau par un mince filament chromatique
(fig. 1-a).
2) Le type "B" (Kosenow et Scupin) dit aussi celui des "appendices-
sessiles", ou bien "tags" (d'apres Davidson et Smith). Ceux-ci apparais-
saient comme des granulations chromatiques similaires, comme formes et
dimensions a celles de la premiere catSgorie. Elles sont depourvues de
filament d'union, sont eessiles et acollees au noyau (fig. 1-b).
3) Le type "C" (Kosenow et Scupin) ou bien celui dee small-clubs"
(d'apres Davidson et Smith), etant donnfi qu'elles apparaissaient comme
des excroissances nucleaires pediculees, termineeB per une t8te en forme
de massue, dont les dimensions se trouvent toujours au-dessous d'un micron.
Leur aspect est similaire a celui d'une "^pingle". (fig. 1-c).
-------
1077
4) Les excroiseances nucleaires ayant la forme de certains lobes
(Davidson et Smith), sesailes ou plus rarement liees au noyau par 2 ou
3 filaments, moins chromatiquete et moins homo genes. Lea lobes toujours
un diametre de 2 microns (fig* 1-d).
5) Des excroissances nucleaires de forme conique qui sont des
filaments chromatiques ayant une extremitS algue I1aspect d'une aiguille
ou de stalactite, jamais granulees ou lobule'es (fig. 1-e).
6) Nous en avons ajoute" le groupe des excroissances nucle'aires diffi-
cilement ou bien impossible S. classifier 4 cause de leurs formes atypi-
ques, non caracte'ristiques.
4. HESULTATS ET DISCUSSIONS
Nous avons enregistre" les r^sultats de nos observations dans des
tableaux et des graphiques, et nous les de'montrons par des microphoto-
graphiee.
De 1'analyse des donne'es statistiques offerte par nos tableaux et
graphiques, il est a mentionner les suivants aspects plus particuliers:
Le lot temoin (gronpe A)
Des excroissances nucleaires apparaissent seulement sur 38,47 % des
noyaux leucocytaires surtout pour le type "B" (16,76$). Le type "A"
touche des valeurs plus grandes chez le sexe feminin (6,98$) Par rapport
au sexe masculin (0, 16%). Rapportant les indices de fr6quence des
divers types d*appendices nucle'aires sSpar^ment a chaque groupe d'Sge,
1'on constate une croissance, parallele a 1'fige, des noyaux modifies,
(de 34,40?J pour le groupe de 18 - 20 ans jusqu'au 41 ( 91% pour le groupe
de 51 - ^0 ans). (tabl. I, graph, nr. l).
Pour ce qui concerne les personnes travail 1 ant dans un milieu pollue"
par des substances chimiques nocives, le nombre des cellules depourvues
de modifications nucleaires est diminue d'une maniere significative,
jusqu'au pourcentage de 40,93$. Le nombre des noyaux comportant diverses
excroissances est egalement SlevS, pour les deux sexes, ayant une fre-
quence presque double par rapport au lot temoin et aux types 2, 3 et 4.
Par centre, le nombre des appendices pe'dicule's diminue, tandis que les
formations sessiles augmentent serieueement et atteignent des valeurs
importantes, notamment chez les personnes ayant une activite" prolongee
dans le milieu pollud (tabl. II, graph. 2, 5).
-------
1078
Figure la
Figure Ib
Figure IG
Figure Id
Figure le
FIGURE 1. Les microphotographies avec 1'aspect
des divers types d'excroissances nucleaires.
(la "drumsticks"; Ib "tags"; le "small-clubs";
Id "lobes"; le "Aiguille")
-------
Le tableau n° I
represente les indices de frequence des divers types d1appendices
nucleaires ohez les personnee du lot "A" (temoin) sSpare en 5
groupes, par Sge et sexe.
1'aoe Cellules
sans
18-20
21-30
3MO
41-50
51-60
MED.G.RAI
appenoices
•* f Toul
66, 65,20 65,60
64,25 60,70 62,1.8
59, 63,70 61,35
62,66 57,70 60,16
55,66 60,40 58,03
.61,50 61,5*. 61,53
Cellules avec des divert tjpii d'appndices
t
•? f Total
1,00 6, 3,50
0,5 8, 4,25
0, 7,70 3,85
0,66 7, 3,83
1,66 6,20 3,93
0,76 6,98 3,87
•
r7 | total
22, 14,60 18,30
18, 16,80 17,40
17, 14, 15,50
16,50 18,70 17,60
16,35 13,60 14,98
17,97 15,54 16,76
t
•* | Total
1, 5,20 3,10
3,50 4, k 3,75
It, 5,70 7,85
7, 5,60 6,30
7,33 7,22 7,26
6,17 5,14 5,65
m
** • Total
3, 5,50 4,25
6, 6, 6,
9, 8,30 8,65
5,33 5,80 5,56
12, 8,60 10,30
7,07 6,84 6,95
A
•? • Total
7, 2,50 4,75
6,25 3,70 4,97
3, 1,30 2,15
7, 4,50 5,75
7, 2,80 4,90
6,05 2,96 4,50
P
** • Total
+
0, 1, 0,50
1,50 0,80 1,15
0, 1,30 0,65
0,85 0,70 0,78
0, 1,20 0,60
0,47 1,00 0,74
o
>J
vo
-------
Le "tableau n° II
comprend les indices de frequence des divers types d1excroissances
nucleaires pour les 3 groupes des personnes examinees.
Gr.
A
B
C-l
C-II
Cellules
sans
jmnpnrtir.M
r* T . .1
W 1 ot i I
61,50 61,54 51,53
38/0 43,45 40,93
50,52 42,76 46,64
1.2, 56, 39
Cellules avec des divers types d
*
Total
f •
0,76 6,98 3,87
1,60 4,95 3,28
1,70 4,56 3,04
1, 1,90 1,45
•
Total
f •
17,97 15,5* I6,7f
27,90 ZS,1.? 27,8:
ZO, 32,36 26,18
25, 30,18 27.5S
t
Total
•- f
6,17 5,U 5,65
12,40 7,78 10,09
8, 6,74 7,34
5, 15,07 10,03
appendices
A
-* Total
«r 9
7,07 6,84 6,95
14,60 9,97 12,28
14, 10,18 12,09
13, 13,18 13,09
A
*• . '""
6,05 2,96 4, 0
4, 3,30 3,65
6, 2,42 4,21
13, 2,67 7,8".
0
Total
•* t
0,87 1, 0,74
1,80 2,08 1,94
0,02 0,98 9,50
1, 1, 1,
o
oo
o
-------
1081
:
, i.
.-
C-l
'
IOH«UtUT» . C-I
-------
1082
Chez les personnes irradiees pour des fins diagnostiques ou thera-
peutiques, les tests cytologiques indiquent une valeur elevee de 1'in-
dice general de frequence des noyaux ayant des modifications de surface
(53|36$). Les types 2-3 et 4 d1appendices nucleairea presentent toujours
des valeurs presque doubles par rapport au lot temoin. De mSme 1'on a
enregistre une diminution des formations pediculees, mSme pour celles
caracteristiques au sexe feminin (le sexe nucleaire femelle). (tabl. II,
graph. 3, 5).
Chez les personnes qui travaillent dans un milieu pollue par des
rayonnements ionisants: medecins, personnel technique et cadres moyens
auxiliaires (1'Sge variant entre 25 - 50 ans), les aspects morpholo-
giques et de frequence des excroissances nucleaires, mentionne's pour le
sous-groupe anterieur C-l, sont encore plus marques et plus significa-
tifs, A notre avis il s'agitt dans ce groupe, d'une action chronique
cumulative du milieu pollue par des rayons sur 1'organisme humain.
Generalement, voir-me'me chez les femmes, les excroissances pediculees
diminuent d'une maniere manifesto, tandis que les formations sessiles,
atteignent, pour ce sous-groupe C-2, les valeurs les plus eleveas
(tabl. II, graph. 4, 5)-
Les donnees que nous avons obtenues par des analyses morpho-cytolo-
giques et statistiques, effectuees sur un grand nombre de cas, t^moignent
de la possibilite de deceler certaines modifications de la surface nucle-
aire. Ces modifications de la surface nucleaire, permettent, outre 1'eta—
blissement du sexe nucleaire, des estimations sur le degre d'action de
plusieurs noxes chimiques ou physiques du milieu pollue BUT I'organisme
humain en general et notamment sur les cellules leucocytaires. L1augmen-
tation marquee du nombre d'excroissances nucle'ajres chez les personnes
qui travaillent dans un milieu pollue (gr. E-C), se trouve favorise'e ou
bien constitue 1'expression, la manifestation cytologique, de 1'action
des agents nocifs, sur la surface et le contenu nucleaire. Les modifica-
tions peuvent 3tre dues a des me'canismes differents, tel quej I'ipaisse-
ment de la membrane nucleaire, le plissement de la membrane nucleaire,
la pycnose, la karyolyse, la n^crobiose.
-------
1083
En guise de conclusion nous pouvons dire que les tests cytologiques
effectues chez un grand nombre de personnes venues en contact avec des
milieux pollues, indiquent:
1. L1existence, au niveau des noyaux leucocytaires, de plusieurs
types d'excroissances nucle'aires, dont certaines refletent par leur
forme et frequence le degre de nocivite du milieu pollue1 sur 1'organisme
et notamment sur les cellules leucocytaires.
2. Ces excroissances nucleaires ayant la forme dfappendices ont une
frequence elevee, et modifient leur forme (en predominance lea formations
sessiles) en rapport direct avec l'9ge et le temps passe dans le milieu
pollue par des substances nocives chimiques ou par des rayons ionisants.
REPERENC E S
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2. BAMFORD SOPHIA, MITCHELL G.W, CASSIN CLAIRE, GORMAN ANITA, FRENCH
BREDA, "Purhter Observations on Neutrophil Appendage in Sex Chromo-
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3. BARR M.L., "Cytological tests of sex - The Lancet, 19>6/7
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1964, pg. 417
6. BENVENUTI A, BUAITTI A, D'AHATO P, RAGOZZINI R, "Mutazioni somatiche
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7. CARATZALI A. "Nuclear Sex Appendages of Leucocytes - Genital Anoma-
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8. COMINGS D.E. "The Tationale for an Ordered Arrangement of Chromatin
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1968/5, pg. 440
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1084
12. GROUCHY J., Cytogenetic Studies in Irradiated Marrow and Blood Cells
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13. KAPLAN A.R., "The use of cytological data in heredity counseling
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meristemului activ din radacinile de bob (Vicia faba L). - Comunic.
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17. MARBANI CAPRA P. "L'esame morfologico dei polinucleati quale metodo
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18. MICHAELIS P. "Genetical Interactions between nucleus and cytoplasinic
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-------
EXPOSITIONSINDIKATOREN
INDICATORS OF EXPOSURE
INDICATEURS D'EXPOSITION
INDICATORI DI ESPOSIZIONE
INDICATOREN VAN EXPOS IE
(Continued)
Voraitzender - Chairman - President - Presidente - Voorzitter
W.J. LLOYD (U.S.A.)
-------
1087
STANDARDIZATION OF ALA-D ACTIVITY DETERMINATIONS AT
THE EUROPEAN LEVEL; INTERCALATION AND APPLICATIONS
A, BERLIN"*", K.-H, SCHALLER++, j, SMEETS+
+ Health Protection Directorate, Commission of the European
Communities, Luxembourg
++ Institut fur Arbeits- und Sozial-Medizin und Poliklinik fur
Berufskrankheiten, Erlangen, BRD
ABSTRACT
In view of the difficulties with blood lead analysis^ at
levels corresponding to a normal environmental exposure to lead,
the possibility of using ALAD activity changes for the assessment
of such exposures has been explored.
A "European Standardized Method" has been developed, cali-
brated and tested in an intercomparison programme. The results
of these tests compare very favourably both with blood lead and
ALA in urine determinations. The interlaboratory coefficient of
variation in the ALAD intercomparison programme was of 10%.
The method has been applied within the framework of a small
population survey conducted by nineteen laboratories. The pre-
liminary results of this survey seem to indicate that statisti-
cally significant differences might exist in ALAD activity levels
between similar population groups in different cities.
-------
1088
Introduction
Lead is amongst the priority pollutants to be dealt with,
within the Environmental Action Programme of the European Com-
munities approved by the Council of Ministers on 19 July 1973.
Its universal presence in the environment makes a system-
atic monitoring of this element in the various media an extreme-
ly arduous task.
Attempts have been made to assess man's exposure to lead
by measuring the blood lead levels. This approach has led to
fruitful results in cases of professional and accidental expo-
sures. The analytical techniques for blood lead determinations
at these levels are sufficiently accurate, and most often only
likely to be in error by excess, to serve as a diagnostic tool
to detect abnormally high exposures to lead.
In order to assess the accuracy and reliability of blood
lead measurements at concentrations corresponding to a normal
exposure to environmental lead, we have completed two interna-
tional intercomparison programmes on blood lead measurements
designed for this purpose (1, 2). These programmes, carried
out at a two year interval, have not shown any marked improve-
ment in the results, in spite of the efforts made by the labora-
tories in the interval to improve the reliability of the measure-
ments .
It is still possible to conclude that none of the technlques,
as currently used by the laboratories, seem to be accurate and
precise enough to detect, on one hand the small differences in
blood lead levels that might result from different environmental
exposures to lead, and, on the other, to compare results ob-
tained by different laboratories.
The discovery that the activity of delta-aminolevulinic
acid dehydratase (ALAD) in red blood cells is affected by small
changes in blood lead levels, at concentrations normally found
in unexposed populations (3, 4) has led us to consider the
possibility of using ALAD activity as a bio-analytical tool to
assess small changes in environmental exposure to lead.
-------
1089
The recent confirmation (5) that the depression of ALAD
activity by lead is an in-vivo effect and not simply an in-vitro
artifact strengthens our suggestion that the decrease in the
value of the correlation coefficient between blood lead levels
and ALAD activities, at low blood lead levels, is mainly due to
the increased uncertainty in the accuracy of blood lead deter-
minations.
Chronological development and main features of the "European
Standardized Method" for ALAD determination
A small study performed in June 1972 with the collaboration
of nine laboratories has shown that ALAD activity ratios can be
determined with good reliability, especially in comparison with
blood lead or ALA in urine (1), even when each laboratory used
its own method.
These results were sufficiently encouraging to set out with
the development of a "standard method" having a general accepta-
bility. For comparing results obtained by different laboratories
a standard method is needed, since activity determinations are
not absolute determinations and since control groups in different
areas may have different base lines.
In June 1973 a preliminary common procedure was elaborated
by the participating laboratories. From June to September 1973
it was tested by each laboratory with respect to the ease of
manipulation, accuracy and reliability. During these tests the
sensitivity to light of the porphobilinogen formed was discovered.
In September 1973 at a technical seminar in Erlangen all
the participating laboratories had the possibility to test
jointly the method, including minor modifications. Some of the
results obtained are reported further on. At the end of the
seminar the final, currently used, method was approved.
The calibration of the method and some of the results ob-
tained to date in population surveys are described below.
The principle of the method adopted for the determination
of delta-aminolevulinic acid dehydratase activity is well known (6)
-------
1090
It is based on the incubation of the enzyme on an excess sub-
strate of delta-aminolevulinic acid. The porphobilinogen formed
within a fixed time is mixed with modified Ehrlich's reagent and
the colour developed measured photometrically against a blank.
The quantity of porphobilinogen produced is a measurement of the
delta-aminolevulinic acid dehydratase activity.
A schematic diagram of the procedure adopted is given in
Figure 1. The details of the procedure are described elsewhere (6)
SAMPLE PREPARATION BLANK PREPARATION
o,2 ml venous blood o,2 venous blood
+ 1,5 ml dist. water + 1,5 diat. water
Hemolysis by 57°C for 10 rain.
+ 1 ml ALA-sol. + 1 ml TCA-HgCl2-80l.
+ 1 ml ALA-sol.
Incubation by 37°C for 1 hr.
+ 1 ml TCA-HgCl2-sol.
Centrifugation and Filtration
1 ml of supernatant
+ 1 ml Ehrlich's reagent
5 min. reaction time
Extinction measurement of sample and blank at
555 nm in 1 cm cell.
Calculation:
P.P. corr. x 55x 2 x 100 = /imols ALA • min"1 • LRBC"1- U/L
Hct (j) x GO x 0,062'
Figure 1: Diagram of the procedure for the determination of
ALAD activity in blood with the "European Standard-
ized Method".
-------
1091
The present method is aimed at assessing activity levels in
blood corresponding to low levels of exposure to environmental lead.
The steps normally leading to an enhancement of the activity,
(i.e. hemolysis with Triton X 100) necessary when determining the
ALAD activity of persons exposed to lead, have been omitted.
The method has been designed in such a way that two techni-
cians should carry out the sampling in the field; the number of
tasks that have been assigned to be performed during the sampling
will reduce considerably the workload to be performed in the lab-
oratory .
For normal blood the optical density values obtained range
from 0.5 to 1.0, giving ALAD activities in U/L from 30 to 60.
Calibration of the method and intercomparison study
A first test of the reproducibility of the method was carried
out during the technical seminar in Erlangen where the technicians
of each of the 16 laboratories analysed a series of 10 identical
samples. The average coefficient of variation for these analy-
ses was of only 3% with a standard deviation of 2% (Table 1).
Ub.
1
ft
3
4
5
6
7
B
9
10
It
12
13
14
15
16
17
18
19
1C
+ S.D
No. precision
Innerics
C.V.94
2,56
2,56
2,4
2,8
7,2
2,16
<,1
0,517
4,8
1,3
6,7
3,45
0,6
0,6'
1,73
4,16
2,98
2,02
precision froa precision from day to day, precision from day to day,
week to week fro* lab to lab from lob to lab, fron technician
(two blood snapl.) to technician
(two blood samples)
C.V.* C.V.* C.V.X
}
20,30
21,58
11,7
11,26
8,25
6,2
14,01
15,22
7,49
10.2O
7,09
2,10
6,29
2,73
5,00 j
\
1 11,7
or
12,5
/
9,5
or
13,5
9«»7 12,1 11,50
5,83
Table 1: Comparison of the variation coefficients for the
ALAD determination with the "European Standardized
Method".
-------
1092
Following the technical seminar each laboratory was re-
quested to select five persons to act as internal standards.
An ALAD activity analysis was to be carried out at least weekly
for a minimum of three weeks. A statistical analysis of these
week to week results shows a coefficient of variation of 10%
with a standard deviation of 6%.
These results were considered sufficiently satisfactory to
undertake the next step of the study: an international inter-
laboratory comparison programme.
The lack of stability in-vitro of ALAD prevented, as in
the case of the internal standardization, the simple shipment of
blood samples. Two persons, Miss J. Trotter and one of the
authors (K.H.S.) volunteered to act as international references
and to visit all the participating laboratories. A programme
was set up in which in addition to donating blood, they would
carry out the ALAD activity determinations alongside the local
laboratory and also verify the calibration of the spectrophoto-
meters with standard filters.
The activity values found were as follows:
Blood Trotter Blood Schaller
Average as analysed by Trotter 45.9,- 36.0-
3 b
Average as analysed by the local
laboratory technicians 46.1- 36.6,.
The calculated coefficients of variation are presented in
Table 1, column 4 for the analyses carried out by Trotter and
column 5 for the ones performed by the local laboratory techni-
cians. The slightly larger coefficient of variation obtained
by Trotter (12.1 vs 11.5%) can be easily explained by the un-
familiar conditions under which she had to work each time; it
can only be concluded that the method, as elaborated, is not
sensitive to the technician.
At the same time as determining ALAD activity values on
the two international references, a number of laboratories also
measured the blood lead values; a coefficient of variation of
over 20% was found for these determinations. While already
significantly higher than the coefficient of variation calculated
for ALAD activities (12%) it is quite acceptable when compared
with the interlaboratory coefficients of variation of 43, 52 and
75% obtained in the intercomparison programme (2).
-------
1093
,. 2-5-
k
1
TJ
O
Z
_«
1
S
5
£1.5-
**
<
D
0 '
*« "
n ^
cr <
O.5
European Standardized Method
Analyst. Technicians (1973)
xi 1,26 ^_j
^-" "^
I
o
European Standardized Method
Analyst: Trotter (1973)
x = 1,27 f_/-
| '
1
2.5-
1,5-
OS-
_T
x.1,84 ^jj
J
^
Different ALA -D
Methods'(1971)
Ana ly st :lechn icians
CM
•D
§
2
o
HI
£
„.
?
o
Z
|-
t>
<
Q
1
^
J
laboratories
laboratories
laboratories
Figure 2: ALAD ratios obtained by different laboratories and
Miss Trotter with the "European Standardized Method"
(1973) and during an ALAD intercomparison programme
(1971) with different ALAD methods.
The comparison, in Figure 2, of the activity ratios ob-
tained by the different laboratories for the two bloods having
served as international references using the "European Standard-
ized Method" with the results obtained in the 1972 experiment (1)
clearly shows the significant improvement which has been achieved
through the standardization.
In Figure 3, a Youden plot of the results obtained for the
two blood samples has been made (the 10% tolerance values have
been indicated). The plot shows that in some laboratories sys-
tematic errors still exist. The possible causes are being
examined at present.
The results obtained for the ALAD activity determinations,
as expressed by the activity ratios, are compared in Figure 4
with the ratio for blood lead values on the same samples and the
results obtained for ALA in urine in the intercomparison programme
of 1972 (1). For ALAD, only 15% of the laboratories deviated
more than 10% from the average ratio, while more than 50% devi-
ated for both ALA in urine and Pb in blood.
-------
1094
X
_,
^
0
i
<
39
OC
4
o
Uj3O
25
0
o x
X
H x
© x
X 13
x
•"a
0 x
"x^/®
0^© 3 <®
fax °
7jx0 © a
H
© x^-0,872t
© /
a BX fc
X
B
25 » 35 40 49 5i> 55
SAMPLE TROTTER
ALA-D U/L
Figure 3:
Youden plot of ALAD results from the European inter-
comparison programme, December 1973/January 1974.
Plot of "high level" sample (Trotter) versus "low
level" sample (Schaller). (Magid, E. 1974).
Laboratory Technician results from lab. no. x.
Miss Trotter's results.
11
2.5-
1.5-
r»«i.
ALA-D
European Standardized Method
(1973)
Sv 1.26 _, -~- '
^"
2.5
iffiis-
Pb-B
Different Methods
(1973)
Laboratories
Laboratoriea
Laboratories
Figure 4: Comparison of ALAU, ALAD and Pb-B determinations.
-------
1095
ALA-D U/L
80
70
N.20
y«1,094x «1,08
r« 0,9535
10 20 30 40 50
Analyst: I.H.E.
60 70 80 ALA-D U/L
Figure 5: Correlation coefficient and regression line for the
relationship between the ALAD results measured by
two laboratories in the same city for the same blood
samples.
IHE: Institut d'HygiSne et d'Epid6miologie, Bruxelles.
UTIM: Unite de Toxicologie Industrielle et MSdicale,
University Catholique de Louvain, Bruxelles.
mi mi-a-L '"in '• LIT ni ir ~—-*~ —._
An additional test of the method was carried out by two
laboratories in close geographic proximity to each other; these
laboratories carried out a double ALAD analysis on the blood of
twenty individuals taken at random. The results are reported
in Figure 5. The high correlation coefficient (0.95), the slope
close to unity and the small intercept of the regression line,
are all indicative of the reliability of the method.
Possible use in population surveys
As indicated earlier, this "European Standardized Method"
has been developed as a possible bioanalytical alternative tool
to blood lead determinations in assessing small changes in envi-
ronmental exposure to lead.
-------
1096
We have made a first attempt to use this method to examine
if the reported differences in blood lead averages found by var-
ious European researchers (i.e. 8, 9, 10} are significant.
Each of the laboratories having participated in the elabo-
ration of the standardized method examined the ALAD blood acti-
vity values in groups of 50 males aged between 18 and 40, in
principle non-smokers and not engaged in manual work. The
groups were so chosen in order to obtain a minimal dispersion of
values. Simultaneously, blood samples were sent to "central
laboratories" for lead analysis and were also analysed, if pos-
sible, locally for this element. While all the ALAD activity
analysis results are available and computed, some lead results
are still lacking. The results are reported in Table 2. The
statistical analysis seems to indicate that the differences are
significant in many cases. The correlation with the blood
leads already measured was rather poor; it can only be said
that the range of ALAD values was smaller than the range of Pb
values.
ALA-D (/umol* ALA • •in'1 • 1 RBCT1) Pb - B
ml)
Laboratories
load (ABDULLA)
Hamburg (AJIGEHEB)
Geaova (BOHSIGTORE)
?«pi« (BOUDEHE)
BruxtllM (BKUAUX)
Auterdaa (HE BBUIR)
Hiaeeldorf GHELEHTER)
Eire (GRIMES)
Lawanne (GUILLHUH)
HelBinki (HEBHBERB)
Luxembourg (HOFFMAKH)
Bnixellea (LAUWERYS)
KSbenhaven (HAGID)
Glugow (MOORE)
Zllrlcb (HOSEHMUBD)
London (SATEBS)
Erlangen (SCHALLER)
Hil«no (SECCHI)
B«rlln (VAGUER)
Median
49,0
42,9
52,2
34,7
29.7
41,2
44,9
41,2
34,6
3«,6
45,0
47,5
40,1
29,8
51,5
27,3
40,5
w
Range Median
2,5/97,5 percentlles <
34-63 6
35 - 51
20-55 26,4
18 - ft? 19.4
15 - 57 10,2
29-65 12,0
28 - 61
28 - 55 8.2
23 - 57
23 - 57 23.3
33 - 65 6,8
25-69 | 22,7
25-56 '
19 - 4} 18,3
21-46 15,9
3,0- 43
34-49 •
I
Range
,5/97,5 percent! !•«
4-10
15-44
12 - 32
9-12
7-30
4-22
9-39
2-26
16- 35
10- 33
1O - 2*
Table 2- Results for ALAD and Pb-B by population studies on
identical groups in different countries.
Measurement of ALAD activity in human blood samples
using the "European Standardized Method .
-------
1097
Conclusion
One must await the complete blood lead results before def-
inite conclusions may be drawn from this population survey, but
already the narrow range of values obtained in a homogeneous
population indicates that the biological variability in ALAD
activities for a given population is not larger than the vari-
ability (biological and/or analytical) in blood lead values (11).
The reproducibility and precision of the method, its low
cost per analysis, the easy implementation by many laboratories
(no elaborated equipment required) should allow this method to
be used as a possible alternative to blood lead determination
when carrying out population surveys of exposure to lead or when
assessing a local situation with respect to a set of "biological
quality guides" (12).
Acknowledgements
In listing the laboratories having participated in this
programme the authors wish to acknowledge the fact that the ela-
boration of the "European Standardized Method" has only been
possible through their efforts.
The authors wish to thank Madame Langevin and Miss Trotter
for their technical contribution and for the coordination of
this programme. Special thanks are also due to Professors Recht
and Valentin for their advice and encouragement during this whole
study.
-------
1098
List of participating laboratories
1. Institut d1Hygiene et d'EpidSmiologie, Bruxelles,
Belgique (P. BRUAUX)
2. Unite de Toxicologie Medicale et Industrielle, University
Catholique de Louvain, Bruxelles, Belgique (R. LAUWERYS)
3. Klinish Kemish Afdeling, Bispebjerg Hospital, K^benhavn,
Danmark (E. MAGID)
4. Zentralinstitut flir Arbeitsmedizin, Hamburg, BRD (J. ANGERER)
5. Med. Institut fur Lufthygiene der Universitat Dusseldorf,
BRD (L. GHELERTER)
6. Institut fur Arbeits- und Sozialmedizin, Universitat
Erlangen-Niirnberg, BRD (K.-H. SCHALLER)
7. Institut fur Wasser-, Boden- und Lufthygiene, Berlin, BRD
(H. M. WAGNER)
8. UniversitS Paris Sud, France (C. BOUDENE)
9. Department of Pathology and Bacteriology, Regional Hospital
Galway, Ireland (H. GRIMES) '
10. Istituto di Medicina del Lavoro dell'Univer-sitS di Genova,
Italia (D. BONSIGNORE)
11. Clinica del Lavoro, Universita di Milano, Italia (G. SECCHI)
12. Institut d'HygiSne et de la Sant§ Publique, Luxembourg
(P. HOFFMAN)
13. Universiteit van Amsterdam, Nederland (A. de BRUIN)
14. University of Glasgow, Department of Materia Medica, UK
{M. R. MOORE)
15. Department of Employment, Central Reference Laboratory,
London, UK (M. H. P. SAYERS)
16. Department of Epidemiology and Biometry, Institute of
Occupational Health, Helsinki, Finland (S. HERNBERG)
17. Forskningslaboratory, Lund, Sverige (M. ABDULLA)
18. Institut Universitaire de Mgdecine Soclale et Preventive,
Lausanne, Suisse (M. GUILLEMIN)
19. Kantonspital Zurich, Schweiz (H. ROSENMUND)
-------
1099
References
1. BERLIN, A., DEL CASTILHO, P. and SMEETS, J. ; "European
Intercomparison Programmes", Proceedings International
Symposium Environmental Health Aspects of Lead - Amsterdam
1972 page 1033. Published by the Commission of the
European Communities, Luxembourg 1973.
2. BERLIN, A., LAUWERYS, R. , BUCKET, J.P., ROELS, H. ,
DEL CASTILHO, P. and SMEETS, J.j "Intercomparison Prog-
ramme on the Analysis of Lead, Cadmium and Mercury in
Biological Fluids" presented at the International Symposium
on the Recent Advances in the Assessment of the Health
Effects of Environmental Pollution; Paris 1974.
3. HERNBERG, S., NIKKANEN, J. ; "Effect of Lead in Delta-
Aminolevulinic Acid Dehydratase. A selective review".
Pracov l§k 2A, 77-83 (1972) .
4. NIKKANEN, J. , HERNBERG, S. , TOLA, S.} "Modifications of
the Delta-aminolevulinic acid dehydratase test and their
significance for assessing different intensities of lead
exposure". Work-Environment-Health, 9_, 46-52 (1972).
5. LAUWERYS, R. , Private communication.
6. BONSIGNORE, D. , CALISSANO, P. and CARTASEGNA, C.; Med.
Lavoro, 5J[, 199-205 (1965).
7. BERLIN, A. and SCHALLER K.H.? "European Standardized
Method for the Determination of Delta-Aminolevulinic Acid
Dehydratase Activity in Blood". Zeit ftir Klin. Chem. und
Klin. Biochem. , 121, 389-390 (1974).
8. LEHNERT, G. , MASTALL, H. , SZADKOWSKI, D. and SCHALLER K.H.;
"Berufliche Bleibelastung durch Autoabgase in grosstadt-
strassen. Dtsch.med.Wechr. , 95, 1097 (1970).
9. HAEGER-ARONSEN , B. ; "Effect of lead on 8-ALAD activity in
red blood cells". Arch, environ. Hlth. £3, 440 (1971).
10. VIGLIANI, E.G. and ZURLO, N. ; "Lead in blood and lead in
urine values of adults not exposed to lead living in Milan."
Report work conf . inorg. lead, Amsterdam (1968) .
11. ZIELHUIS, R.L. and VERBERK, M.M. } "Validity of Biological
Tests in Epidemiological Toxicology". Int. Arch. Arbeitsmed.
3£, 167-190 (1974) .
12. ZIELHUIS, R.L.j "Biological Quality Guide for Inorganic
Lead". Int. Arch. Arbeitsmed. 32, 103-127 (1974).
-------
HOC
DISCUSSION
ZIELHUIS (Netherlands)
The speaker suggests ALAD measurements as a possible alter-
native for Pb in blood levels in monitoring Pb exposure for
population groups. However, in population surveys the investi-
gator is often limited in his degrees of freedom to organize the
taking of blood; he has to make use of the opportunities given
to him by circumstances, e,g, one may have to take blood at
night, on Friday. Blood samples have to be investigated within
about 2 hours for measurement of ALAD, but they can be left in
the laboratory for some days in the case of measurement of Pb.
Therefore, ALAD measurement for monitoring of population groups
will meet many more logistic problems than Pb in blood measure-
ment.
BERLIN (C.E.C.)
We are aware of these logistic limitations, which are not
quite so severe since up to 5 hours can elapse between sampling
and analysis. Methods for increasing the conservation time
are still being investigated.
However, in view of the quality of the results obtained at
present for ALAD in comparison with blood lead, we feel that
the increased logistic difficulties are warranted.
-------
1101
LEAD SURVEY OF CHILDREN - ARGENTON - BOOLAROO
NEWCASTLE - AUSTRALIA
K, H, OUW AND A, BELL
Division of Occupational Health and Pollution Control - Health
Commission of New South Wales, Australia
(paper presented by S,R. Leeder, U.K.)
ABSTRACT
A survey of possible health effects due to contamination of
the environment by lead emissions from the Sulphide Corporation
Pty. Ltd.t Cockle Creek, New South Wales began in 1972 and was
continued by the Medical Branch of the Division of Occupational
Health and Pollution Control in 1973.
While the results in 1972 did not indicate that the children
in the Argenton-Boolaroo area were at any risk from abnormal
lead absorption, the survey at that time was not considered by
the Division to be adequatet as it was based predominantly upon
lead-in-urine results (n - 257) and only to a le&ser extent upon
lead-in-blood results (n = 41).
At the International Symposium on Lead in Amsterdam in 1972
the matter of environmental lead pollution was discussed, and it
was decided to adopt the recommendations set down by R. Zielhuis
in his paper "Lead Absorption and Public Health, an Appraisal of
Hazards" (as amended), that is, that 50% of the children tested
should have a lead level of 20 .ug or less per 100 ml. of blood,
98% should have 35 .ug or leas per 100 ml. of blood, and 100%
should have 40 .ug or lese per 100 ml. of blood, as adequate indi-
cations that no health hazard due to lead absorption exists.
Accordingly, a further 204 children and infants were investigated,
-------
1102
and blood lead levels determined. Haemoglobin levels uere also
checked.
The haemoglobin levels were found to be normal for age in
all children and infants tested* and results of the 204 lead-in-
blood tests showed that 200% had 40 ,ug or less per 100 ml. of
blood* 98% had 35 ,ug or less per 100 ml. of blood and 83% had
20 ,ug or less per 100 ml. of blood.
As a result of all the tests carried out in 1972 and 19?3t
no public health hazard due to abnormal absorption of lead could
be demonstrated in the vicinity of the Sulphide Corporation lead
smelter at Cockle Creek.
-------
1103
1. Introduction
Excessive lead absorption in children due to the ingestion of lead-
containing materials, such as paint flakes, has been widely reported in
the literature (Gibaon, 1892; Henderson, 1954f Freeman, 1969).
Excessive absorption due to lead in ambient air, and residence In areas
close to emission sources of lead, is less veil documented, and still a
matter of contention. This survey reports on the lead absorption of
children residing close to a large lead-smelting complex (Sulphide Corpor-
ation Pty. Ltd.,) known to be an emission source of airborne lead. 770
workers are employed by the Corporation, and current annual capacity for
the chief products is of the following orderi
Zinc metal 70*000 tons
Lead bullion 28.000 tons
100$ sulphuric acid 130.000 tons
The smelter is located at Cockle Creek near Newcastle,
New South Wales, Australia, and the townships of Argenton and
Boolaroo are situated North and South-West of the lead smelter
respectively. The residents of these townships living within a
radius of 2 km of the lead smelter, might reasonably suspect that
their health could be affected by absorption of lead from the en-
vironment. The route of such absorption might be by inhalation of
airborne lead, or by ingestion of contamined food. Children and
infants might be especially at risk, due to their predilection for
absorbing environmental lead.
A lead survey into possible effects was carried out in 1972.
The results did not indicate abnormal lead absorption However, the
1972 survey was not considered to toe adequate, as finding were based
predominantly upon lead-in-urine results (n = 257) and only to a
lesser extent upon lead-in-blood results (n = *tl).
The lead-in-blood level is now regarded as the sole valid criterion
(Steiafeld 1971).
A further survey, based entirely upon lead-in-blood tests and
haemoglobin values, was therefore conducted In 1973.
2. Method
Two hundred and four children and infants were included in the
survey. They were drawn from both Argenton and Boolaroo, in response to
an appeal to parents to submit their children for testing. The blood
lead and haemoglobin levels for each child were estimated. 50 al
-------
1104
quantities of whole blood, standards and secondary controls were sampled
by means of an Oxford Sampler, and placed in a Spinko nderooentrifuge tube.
Red cell lysis and chelation were performed using Saponin-Ammonium
Pyrrolidine Dithiocarbamate reagent. After mixing in a vortex mixer the
ohelated lead was extracted into methyl isobutyl ketone and the extract
tested in a Varian Model 6l Carbon Rod Atomizer. The unknowns were read
against the standard calibration curve* By questionnaire, information
from parents was sought to determine whether the child indulged in "pica",
or was the child of a lead-smelter worker, and the geographical location of
each child's residence.
Levels of lead in ambient air of the residential areas were obtained
by the Air Pollution Control Branch of the Division of Occupational Health
and Pollution Control.
3. Results ;
Histogram 1.
Shows the distribution of the blood lead levels in the 204 chil-
dren. As can be seen from the histogram, the frequency distribution of the
blood lead levels was UNIMODAL, with a mean of 15 /ugm/100 ml. of blood,
and a standard deviation of 5«94yugm/100 ml.
Histogram 2.
Shows the age distribution of the children in the survey. The
majority were school children in the age range of seven to twelve years
old. Twenty one were less than three years old. The youngest was a baby
aged ten months; the oldest child in the survey was fourteen years. There
was no significant correlation between blood lead levels and age (corre-
lation coefficient = 0.1-9)-
Histogram 3.
Shows the distribution of the haemoglobin estimations on the
children. No child had a haemoglobin level below 10 g per 100 ml. and
none of the children were considered to be anaemic.
Tables I - T.
Show the blood lead results for certain sub-groups from the total
survey population, who were possibly at greater risk of lead absorption,
due to their social, or geographical circumstances.
AIR POLLUTION DATA
Ambient lead in airi Measurement of ambient lead in air, in the
vicinity of the lead smelting complex at Cockle Creek was commenced on the
27 June 1972. Initial measurements (up to 21 July 1972) from 30 sample
-------
1105
rrequency
0
28
94
HISTOGRAM 1
49
N = 204
21
< 4
0-4 5-9 10-15 16-20 21-25
Blood lead in ^ug/lOO ml.
BLOOD LEAD falSTRIBUTION.
26-30 31-35 36-40
40 +
1
zz
19
HISTOGRAM 2
75
80
N = 204
7
<1 1-3.9 4-6.9 7-9.9 10-12.9 13-15
Vre in years
AQE DISTRIBUTION OF CHILDREN EXAMINED
HISTOGRAM
Frequency
2
15
57
62
37
N * 204
10
1
.0-10.9 11-11.012-12.9 13-13.9 14-14.9 15-15.? 16-16.9
b in GM/100nj[AEMOQLOBIN DISTRIBUTION
-------
1106
TABLE 1 Blood lead and lib. levels of children whose parents work
at Sulphide
N =
Child Identification
No.
39
113
123
124
150
177
159
Mean
Corporation
7
Blood Lead
13
12
14
7
17
9
19
13
Pty. Ltd.,
/u£/100tnl . Hb. tfii/KXM , .
13.6
12.3
12.4
12.4
15.4
14.3
15.4
13.5
II
Occupation of parents Ho. of Average Blood Stswlnrd
Children Lead Juc/l
-------
1107
TABLE III
Blood lec.c? and lib. levels in children with definite Histoiy
of "PICA"
N
17
Mean
Child identification I
NO. y
53
68
80
84
85
86
90
91
92
186
187
189
205
206
207
44
17
TJi'-iLE IV
History of No. of
"Pica" children
Yea 18
No 125
TABLE V
ric.ce of Ttesidenoe No. of
Children
Ar^enton 93
Boolaroo 77
Hood Lead Hb.
^g/lOOml.
23
15
25
14
10
16
25
28
29
17
11
14
14
21
18
11
19
Moan blood lead
/UG/lOOml .
18.2
14.2
Mean Blood
level -
yujsAoOml.
15.5
14.9
gm/lOOml .
11.3
12.8
12.8
14.7
13.7
13.7
12.5
13.6
13.7
12.6
11.6
12.3
12.4
12.2
12.2
12.8
12.8
Stendard
Deviation
5.8
4.9
Standard
Deviation
5.8
5.8
-------
1108
sites at various distances ranging from 275 to 2000 metres from the works
gave an average value of 9-6 yugm/m (range from 0.15 - 31.6 yugm/m ,
24 hour period samples)* Lead emission control at the works has been
improved since 1972. Prom the beginning of August 1972, 5 fixed monitor-
ing stations were chosen, and sampling was carried out on a rotational
basis in accordance with the E.P.A. Standard procedure. (E.P.I. Federal
Register). The average lead in air from August 1972was 2.2 yugm/m .
4* Discussion
At the International Symposium on Lead in Amsterdam in 1972 the
matter of environmental lead pollution was discussed, and it was decided
to adopt the recommendations set down by R. Zielhuia in his paper "Lead
Absorption and Public Health, an Appraisal of Hazards" (as amended,
Zielhuis 1972) that is, that 5096 of the children tested should have a lead
level of 20 yug /100 ml. of blood, 98$ should have 35 A« or less per 100ml
of blood, and 100# should have 40 A« or less per 100ml. of blood, as
adequate Indications that no health hazard due to lead absorbtion exists.
The lead-in-blood level is now regarded as the sole valid criterion. Using
this corrected "Amsterdam" permissible percentile distribution standard,
the distributions in this survey among the children weret 8# of children
had blood lead levels of 20 yug/100ml. or leas, 9Q# had levels of 35
100ml. or leas and 100& 40 /ug/lOOml. or leas. Thus, it can be seen
that the distribution for lead in blood in this survey is within noraal
limits.
The distribution of haemoglobin levels among the children
surveyed are shown in Histogram 3. For our survey population the normal
level of haemoglobin varies from 11.8 gm/lOOml. for the child of 10 months
to 13.4 gm/100ml. for the child of 14 years. The generally agreed
definition in New South Wales of the lower limit of haemoglobin below which
anaemia is considered to be present ranges from 10 gm/lOOml. at birth
rising to 11.0 gm/100ml. at 14 years. (Hendry 1973, Lovric 1973 - Personal
communications). From Histogram 3 it can be seen that none of the
children in this survey could be regarded as anaemic.
A lead survey in Bristol, U.K., (Report of a Committee under the
Chairmanship of Sir Brian Windeyer appointed to inquire into Lead Poison-
ings at the RTZ Smelter at Avonmouth) showed that there was an increased
risk of lead absorption among lead smelters children because of lead dust
brought into the home by their fathers. Tables 1 and 11 show blood lead
and haemoglobin levels of seven children in our survey whose parents work
-------
1109
at the Sulphide Corporation Pty. Ltd. Analysis shows that there was no
significant difference in the mean blood lead levels between the group of
Sulphide workers children and the children whose parents work elsewhere
( p) 0*05). This finding is not surprising, as lead workers in the
Sulphide Corporation don clean work clothes before they start their shift,
and change back into street clothes at the end of their shift. There is
thus no reason to suspect that these workers bring lead dust into their
home environment.
Pica
Children exhibiting the pica habit are also more at risk from lead
poisoning than children without this habit. "Pica" is defined as eating
unnatural foods, including dirtt plaster and paint flakes* In the first
year of life, it takes the form of mouthing, but not ingestion, of any
object the infant can grasp* Later on, the child begins to eat non-food
substances, but the habit usually disappears when the child Is between 3
and 5 years of age. Emotionally disturbed children tend to acquire this
habit. (Chisholm, Jr. 1970)* In this survey, pica was considered present
in children of any age when the answer to the following question on the
questionnaire form was in the affirmative. "Has your child ever had the
habit of chewing or eating any non-food items such as toys, old paint,
soil etc". Children with a history of "pica" were compared with those
without a history of NpioaH. The total of respondents was 143» IS of
whom gave a history of "pica". (Tables III and IV.).
No statistically significant difference in the mean blood lead
levels was found, (p} 0.05).
As far as this survey is concerned, children with a history of "pica"
did not show an increase in lead absorption* Children, of course, often
place non-food items in their mouths, and some mothers may have given
negative answers to the "pica" question for fear of being told that they
had not trained their children correctly.
Place of residence
Comparison of possible differences in lead absorption by children
living in the townships of Argenton and Boolaroo was also carried out.
Argenton. is north of Sulphide Corporation and Boolaroo is south-west of
the industry, and both areas are approximately the same distance from the
Sulphide Corporation. The Standard Wind Analysis in the area (combined
summary 9099 and 1500 Hr. reading, 1967-1971), showed that the prevailing
-------
1110
wind drift from the Sulphide works was towards Argenton. Figures avail-
able for 1972 and 1973 show the same trend. Assuming other factors
remain equal, and that lead pollution from the Sulphide works is an
important factor in lead absorption among children, then we would expect a
greater degree of absorption in children living in Argenton than in
Boolaroo. Table 7 shows the mean blood levels of the Argenton and
Boolaroo children. No significant difference was found between the blood
lead levels in the two groups. (p>0»05).
In summary, judged by the internationally accepted criteria
(Zielhuis, 1972), none of the children surveyed showed an abnormally high
blood lead level, or low Hb. level, and none of the potentially high
exposure groups had elevated blood lead levels. The children surveyed
were not a random sample, but they did comprise about 50$ of all school-
age children in the area, and we have no reason to think that they were
unrepresentative of all children in the area* Comparison with other
studies of blood lead levels must be made with caution. Differences in
socio-economic status, age, and perhaps most importantly, in laboratory
analysis can all hinder a true comparison between sample groups. However,
the results of this survey do support the initial conclusions of an earlier
survey done at Cockle Creek in 1972« there was no excessive absorption of
lead among children examined, despite their proximity to a lead smelting
complex*
REFERENCES
Chisolm Jnr. J.J. (1970) Poisoning due to Heavy Metals. Paediatrio
Clinics of North America 17, 599.
Freeman, R. (1969) Chronic lead poisoning in childrent a review of
90 children diagnosed in Sydney, 1948-1967.
Aust. paediat. J. 5*27.
Gibson, J.L., Love W., Bardie D.., Bancroft, P., and Turner A.J.(1892).
Notes on lead-poisoning as observed among
children in Brisbane. Inter-colonial Medical
Congress of Australia. Transactions of the
Third Session, Sydney, 76.
-------
1111
Henderson, D.A. (1954)
Steinfeld J.L. (1971)
Zielhuis (1972)
A follow-up of cases of pluralism in children.
Aust. Ann. Tiled. 3*219.
Medical Aspects of childhood Lead Poisoning.
H.S.U.H.A. Health Reports. Vol.86 No.2 140-143.
Environmental health aspects of Lead. Lead
absorption and Public Health: An appraisal of
Hazards. U.S.A. Environmental Protection.
Agency Report. Amsterdam, Oct.2-6, 1972.
HARRISON (U.K.)
DISCUSSION
Was the particle size distribution of the airborne lead
measured? This would be highly relevant to pulmonary retention.
LEEDER (O.K.)
The lead particle size was not determined in that survey/
but in 1972, the Division of Occupational Health and Radiation
Control did make a study of the lead particle size, and the
conclusion was:
"the percentage by weight of particles smaller
than 5 microns varied between 74 and 39 percents
of the total with an average of 55%"
-------
1113
RECENT EPIDEMIOLQGICAL STUDIES OF ENVIRONMENTAL
LEAD OF INDUSTRIAL ORIGIN
A, E, MARTIN/ F. A, FAIRWEATHER, R. ST.J. BUXTON AND
LI LA M, ROOTS
Department of Health and Social Security, London, United Kingdom
ABSTRACT
During the past three years Local Authorities in the United
Kingdom have carried out a number of epidemiological surveys in
the vicinity of lead works. In some of these the proportion of
children and sometimes of their mothers with raised blood levels
living in the vicinity of the works was higher than expected.
The levels were highest in those living close to the works. Fur-
ther epidemiological studies showed also that the families of
lead workersf even when living some considerable distance from
the works sometimes had markedly raised blood levels. In the
latterf the source of the raised lead intake was believed to be
lead particles on the person or clothing of the workers^ notwith-
standing the provision of changing and shower rooms at the works.
In the case of families living in the immediate vicinity of the
works the respective importance of the pathways by which lead
was leaving the works could not be ascertained. High lead con-
centrations were found in the dust within and in the vicinity of
the houses and this was thought to be due in part to dust leaving
the works on the footwear of employees, on vehicles and particu-
larly on the wheels of vehicles. Some airborne or deposited
lead derived from ventilation flues and chimneys would also be
present. In one case where particularly heavy pollution was
obviously coming from the works the factory management voluntarily
agreed to the closure of the plant pending the installation of
more satisfactory filtration equipment.
-------
1114
Individuals with seriously raised blood levels were investi-
gated further and as far as possible other sources of excessive
lead ingestion were excluded. In no case was any individual
found with clinical symptoms of lead intoxication. The atten-
tion of the Medical Officers of Health throughout the country
was drawn to the importance of investigating lead pollution from
all sources. The Departments of Employment and of the Environ-
ment took immediate action to tighten pollution control and a
Code of Practice produced by the former, has now been issued
laying down methods by which pollution of industrial origin may
be minimized. The surveys indicate the need for periodic checks
to ensure the effectiveness of the anti-pollution measures adopted
by industry.
-------
1115
Introduction
Towards the end of 1971 the London Borough of Tower Hamlets reported
strong evidence of lead pollution in the neighbourhood of a large lead
works. This particular area is in the middle of London and therefore an
urban site and the contamination arose from a smelter from which the
lead was being emitted from the chimney. The pollution problem was
therefore mainly in the vicinity.
The second site which we wish to discuss is derived from an extremely
large smelter in an area which is an industrial estate surrounded by
countryside. The contamination is from several sources with high level
emission and low level emission with some take home problems, the cattle
and vegetation in the neighbourhood being involved to lesser extent.
The third area is semi-rural with a lead mill and chemical factory as
the sources. This was not such a severe problem as the two previously
mentioned but has certain particular facets of interest*
The fourth episode derived from old lead mines which exist in the area
on which a smelter has now been built. This is essentially a rural
neighbourhood and the problems which existed were derived partly from
take home lead and partly from the high lead levels in the soil from the
proximity of the mines*
The fifth site was again a London based source, the neighbourhood being
mainly docks with many industries in the area. There were houses around
which were mainly blocks of flats built before the turn of the century,
some of these being very close to the factory area. The emissions came
from a high level from the chimney and the problems were essentially
vicinity ones.
Physical Data
In surveys of this sort undertaken by a variety of different people the
data is not always as homogeneous as one would wish. We have in most
cases managed to get reasonably complete information. The first area
in our discussion has houses close around the factory the nearest being
approximately 100 meters away and we have examined in the survey the
houses up to 500 meters. Being an urban site, there is little soil
-------
1116
which is available for analysis but dust has been examined in the
gutters and other appropriate sampling places, and the figures obtained
ranged up to IJOOppm of lead. Dust was also gathered from the rooves
in the neighbourhood and some samples were up to 5,000ppm of lead* Lead
in air samples gave a mean daily concentration of 3pe/m with a maximum
2k hour figure of 28ug/m . Grass and vegetation were examined, and the
figures for grass were of the region of 500ppm lead while plane tree
leaves were found to have iSOOppm lead.
At the second site the nearest house was 2km from the smelter and
therefore from the environmental point of view the house contamination
problem was likely to be of relatively small importance. Lead in air
samples taken in the works canteen gave average values from 1.1 to 7*9
jig/m . Heavy deposits of lead were found on vegetation in the vicinity.
The outer leaves of green vegetables within one mile of the smelter
yielded 32ppm, from one to one and one half miles I6ppm and at a
distance of one and one half to two miles 8ppm.
The third site had houses 75-250 yards from the works. Dust at 150 yards
away ranged from 10,000 to 50,000ppm lead, and soil samples in the
gardens of these houses all contained less than 3»000ppn.
In the fourth situation as one would expect the soil had very high lead
values 2,000 to 45,000ppm and the lime stone dust content in the
neighbourhood ranged from 175-270ppm. Grass in this neighbourhood had
15ppm of lead. Dust in the area ranged from 300-JJ2,OOOppm lead when
obtained from the children's houses.
The last site in London had a very high dust level in the vicinity of
the factory; the levels of lead ranged from 2,000-80,000ppm; and mud
since soil is not readily obtainable in this neighbourhood ranged from
l,000-19»000ppm.
Blood Lead Values
At the. first works which had a vicinity problem, pre-sehool children were
examined, that is children under 5 years. 16 of 39 children were found to
have blood lead values of greater than the maximum acceptable level of
-------
1117
findings represent a 40 % increase in blood lead of those children who
lived between 100 and 400 meters from the factory. These findings compared
with ppe-school children living 400 to 500 meters from the factory where
11 out of 80 had blood lead values which were raised,that is 13.7$, and
only one was over 6o^J.£/100ml. Of the mothers who were examined, those
living between 100 and 400 meters from the factory 3 out of 25,that is 12$,
had raised blood lead values but in the group which lived 400-500 meters
from the factory there were none out of the 53 examined whose blood lead
findings were increased.
In the second survey which did not have a vicinity problem the nearest
houses were 1,600 metere from the factory and in consequence there were
no raised blood lead findings in those children that were examined. In
the other London borough where the children were examined, those who
lived in houses less than 500 yards from the factory 124 out of 220
children had raised blood findings, of these 8 were greater than fiOfig/
100ml.
Workers families have been found also to be at r.isk. In the first
survey the workers children were not separated from the other children
but on subsequent examination of the data it was observed that the only
2 workers' children who were sampled had higher values than their
colleagues. In the second study it was noted that the blood lead
findings of the children expressed on a frequency distribution curve
showed a shift to the right. There were actually only a small number
in this study who had blood lead levels of greater than JfOpg/lOOml.
In the third study the control group children were found to have
rather high levels, the reason for which is not known. 10 out of
93 had blood lead levels in excess of ^0/ug/lOOml, whereas of the
workers' children 7 out of 13 were also high. This difference must
be important. In the fourth study 13 out of 31 children had blood
lead values greater than 'tO^g/lOOml. These children had a mean value
for their group of 'O/wg/lOOml, while control groups which were
available had mean values of 22 and 25^/g/lOOml, respectively so that
the difference between the control values and the exposed group is
a significant one.
-------
1118
Consideration of the Results
Blood lead values are difficult to interpret in that there are problems
with sampling and with the technical analysis. If the sampling is to be
done on children and particularly where a repeat sample may be necessary
it is desirable to use a capillary sample rather than undertaking
venipuncture. On the other hand capillary samples are much more likely
to be contaminated and therefore have a higher failure rate and a more
frequent need for a second sampling than using blood from a venipuncture.
It should however be noted that where skin cleaning is undertaken prior
to obtaining a capillary sample the skin swabbing must be done with care
and must not be skimped otherwise spuriously high results may be obtained.
It is also important that the blood is free flowing and should not be
squeezed out of the ear or fingertip, The technical variations have
been noted in an inter-laboratory study which is being carried on in
the U.K. The results of this are not yet available but the
conclusion which appears to be a likely outcome is that where blood
lead estimations are being done in large numbers by a single person
using one technique, the estimates are more likely to be consistent and
reliable. Where only a few are being carried out there is less likelihood
of the consistency and reliability which are needed for such measurements.
It has been our custom not to accept a single raised blood lead as being
representative of the individual's lead profile. In each case a repeat
sample has been examined usually obtained at a clinic or hospital rather
than in the atmosphere of the home which may have a high lead in air
characteristic.
Subsequent Action
Where the repeated blood lead value has been found to be raised the
individual has been referred for a further medical opinion. In each
case the general practitioner was informed and where appropriate the
paediatrician was asked to handle the subsequent treatment and progress
of the child. In no case has any child or adult been found to have any
deleterious effects which could be attributed to the excessive
ingestion of lead*
The next point with regard to subsequent action is that of considering
from where the lead has come. It is important to exclude other sources
-------
1119
of lead such as paint from woodwork or off toys, eye make-up and so on,
and having excluded all individual sources the environmental situation
for the affected people as a whole should be considered*
Other Investigations
There are still many aspects of the lead problems which are not fully
understood;for example there is still discussion as to how far excessive
lead intake in a child can be related to hyperactivity or to an
alteration in behaviour. As a result of the first investigation which
we have been discussing an additional study was undertaken into the
intelligence, reading ability and behaviour of the group of school
children. The Weschler Intelligence Scales were used, and using school
children it was concluded that the lower levels of intelligence and
higher rates of behavioural disorder were found to be related to social
factors rather than the effects of lead*
Conclusion
Uach study had its own characteristics in the form of the different type
of lead source and the geography of the neighbourhood* We have used the
blood lead values as our criterion of excessive lead ingestion, but we
would like to have other tests which would indicate early toxic effects.
The number of blood samples was small in relation to the number of
people exposed so that the techniques used provided relatively coarse
assessments of the situation.
Acknowledgmenta t We must express our thanks to all who took part In the
organization and conduct of these surveys and in particular to Professor
Barbara Clayton, Dr W C Turner, Mr P Broughton and Dr D Barltrop, to the
Medical Officers of Health responsible for the local organization of the
surveys in their areas (Dr R Adams, Darley Dale; Dr Q K Brown, Greenwich}
Dr E II Hargreaves, Rothwell; Dr R Kind, Market Harborough; Dr J E Epsom,
Southwarkj Dr W Maughan, Beverleyj Dr P P Morgan, Chesterj Dr. R 0 Taylor,
Welwyn Garden City; Dr It * Watton, Tower Hamlets; and Dr R C Woffinden,
Bristol) to the various laboratories and to the factory managements who so
willingly cooperated.
-------
1120
REFERENCES
Department of Employment (1973) Lead : Code of Practice
for Health Precautions, London
Lansdown, R.C., Clayton, B.E., Graham, P.J., Shepherd, J., Delves, H.T.,
and Turner, W.C. (1974). Blood-lead levels, behaviour and intelligence t
a population study. The Lancet, i, 538.
DISCUSSION
EPSTEIN (U.S.A.)
The results of the reported studies, like previous studies
in children living in vicinity to smelting plants in El Paso,
Toronto, and elsewhere clearly demonstrate a correlation
between elevated blood levels and proximity to the smelting
plants.
The data presented do not support the confident assertion
that "no harmful effects had been found" in children with
elevated blood lead levels. Detection of low level lead toxiclty
required a sophisticated battery of specialized testing for
learning disabilities, behavioural anomalies, disturbances in
co-ordination and in motor nerve conductivity velocity.
The author is also requested to provide data on air lead
levels in the smelting plants studied.
MARTIN (U.K.)
The observation "no harmful effects have been found" does
not necessarily mean that no harmful effects occurred. The
surveys were carried out by local authority public health depart-
ments and any abnormality, such as a raised blood lead level in
a child, had to be referred to the child's family doctor with,
if necessary, a suggestion that he might seek the advice of a
(named) consultant paediatrician. Tl\us in the Tower Hamlets
survey all such children needing further investigation were
referred to a paediatrician at the Great Ormond Street Hospital
for Sick Children. In the Tower Hamlets survey a special inves-
tigation of intelligence and behavioural defects was undertaken
by Landsdown and his colleagues and reported in The Lancet (1974,
i, 538)
The measurement of air lead level in the works was beyond
the scope of the investigation.
-------
1121
CHARLTON (U.K.)
What examinations were undertaken to investigate the trans-
port of lead in clothing? Have the lockers in which worker's
clean clothing is stored been checked? Is there any relation-
ship between the distance workers have to travel home and the
lead transported home.
MARTIN (U.K.)
Any follow-up of employees was the responsibility of, and
was undertaken by the Department of Employment Factory Inspec-
torate and by the works doctor. Lead dusts were found in the
underclothing, socks, and on the boots of workers and also in
their cars. Changing and washing facilities in the works were
checked.
It was noted 'that some of the workers/whose families were
affected,lived some miles from the works.
DIEHL (Federal Republic of Germany)
Dr. Martin's observation that rather high lead exposure in
the vicinity of lead works is not associated with any symptoms
of lead intoxication of the population is in full agreement with
other results presented at this conference. De Graeve has re-
ported on a population group in Belgium which is exposed to
elevated lead levels in drinking water and McNeil has studied
the population of Smeltertown, Texas, which has been exposed to
high environmental lead levels for 3 generations. In no case
were indications of lead poisining observed.
I am interested in this problem from the viewpoint of food
legislation. In some countries (the Federal Republic of Germany
for instance) very strict legislation is now in preparation which
sets very low limits for lead, mercury, cadmium and arsenic in
foods - with the argument that this is necessary to protect the
health of the consumer. After 4 days of this conference, I do
not have the impression that such measures are justified in
view of recent research results. I have already mentioned the
case of lead. Turning to mercury I would like to recall
Bernstein's paper at this conference indicating that a Canadian
population group consuming very much fish with mercury contents
of up to 4.4 ppm is not showing any symptoms of mercury intoxi-
cation. I was glad to hear from den Tonkelaar that no necessity
is seen in 'the Netherlands to limit fish consumption although
much fish is consumed there, and although some of the fish caught
in Dutch waters contains over 0,5 ppm mercury - the limit set
in the USA. As to cadmium, Lorke has found no toxicity of a
dirt containing 30 ppm Cd in subchronic studies with rats and
dogs. All this information indicates to me that no hasty leg-
islation is necessary to set limits for these elements in the
general food supply. I would like to hear Dr. Martin's comment
on the attitude taken by the UK authorities in this respect.
-------
1122
MARTIN (U.K.)
Detailed surveys involving many thousands of analyses have
been made of the lead, cadmium and mercury content of foods in
the UK and the results published by the Ministy of Agriculture
Fisheries and Food. These yielded information on the respective
content of individual items of the diet and on the total diet
of the average man. Britain is not a big fish-eating country
and in the case of mercury/ Investigations of heavy fish-eaters
did not show levels of mercury in the body which could be judged
potentially dangerous.
Food contamination in the UK is controlled by, for instance,
the Lead in Food Regulations or, in other cases, by measures
which prevent the sale of shellfish from polluted areas. While
the contamination of food in manufacture of as a result of spray-
ing in agriculture can be controlled, it is not practicable to
limit the natural content of foods. Thus a retailer could not
be expected to know the mercury content of the individual fish
or the cadmium content of the kidneys or liver he sells for
human consumption.
-------
1123
VARIAZIONI DELL'ATTIVITA' ALA-DEIDRATASICA ERITROCITARIA
IN RAPPORTO ALL'ETA' ED AL SESSO IN SOG6ETTI NON
PROFESSIONALMENTE ESPOSTI A PIOMBO
G, C, SECCHI/ L, ALESSIO
Clinica del Lavoro "Luigi ty£voto" della Universita di Milano,
Italia
RIASSUNTO
Lo studio dell'attivita ALA-deidratasica (ALAD) eritrocitaria
di soggetti sanif non professionalmente esposti a contatto oon
piombo ed abitanti in una grande cittd induatrializzatas ha dimo-
strato che queata attivitd enzimatica diminuisce progressivamente
oon 1'aumentave dell'etd; queato fenomeno & interpretato come
espvesaione di un progreeaivo inoremento delZ'aooumulo di piombio
nell'organismOf oon il paaaare degli anni.
Nei soggetti di sesso femminile il dearemento dell'attivitd
ALAD eritrocitaria 4 meno e-oidente e piU tardivo she nei soggetti
di eeeao maechile.
ABSTRACT
A study of ALA-dehydrataae (ALAD) activity in the red blood
oella.of healthy aubjecte who are not exposed to lead in the
oourae of their work and who live in an industrial city ahoue
that this enzyme activity diminishes gradually with age. This
phenomenon ia taken to reflect the gradual increase in the amount
of lead accumulated in the organism over the years.
In female subjects, the decrease in ALAD activity in the red
blood cells is lees evident and slower than in male subjects.
-------
1124
1. Introduzione
E' state ormai estesamente dimostrato che 1'attivita ALA-deidratasi-
ca (ALAD) eritrocitaria e elettivamente inibita dal piombo; questa inibi-
zione e particolarmente cospicua nella intossicazione conclamata da piom-
bo, ma e gia dimostrabile anche in occasione di modesti increment! dello
assorbimento di questo metallo! una parziale inibizione di questa attivi-
ta enzimatica, per esempio, e stata documentata in soggetti che vivono in
vicinanza di fonderie di piombo (Secchi e coll. [ij, [21, Nordman e coll.
[3]) e persino nella popolazione generale di citta fortemente industrially
zate (Secchi e coll, [ij, fzl , Henberg e Nikkanen MJ, Hernberg e coll.
£5] ). L1inibizione dell'ALAD eritrocitaria e percio considerata un sen-
sibile parametro, atto a documentare gli effetti sull'organismo umano del
1'inquinaraento ambientale da piombo. In questa ricerca abbiamo studiato
il comportamento dell'attivita ALAD eritrocitaria in soggetti abitanti in
una grande citta industrializzata, non esposti professionalmente a piombot
suddivisi secondo il sesso e di eta progressivamente crescente, al fine di
indagare se le variazioni di questa attivita enziraatica potessero documen-
tare un eventuale progressive accumulo di piombo nell'organismo, che gra-
dualmente aumenta con il progredire dell'eta.
2* jfateriali e metodi
La ricerca e stata condotta su 168 soggetti viventi in Milano: di es
si 89 erano di sesso maschile e 79 di sesso femminile. La casistica e
stata suddivisa per sesso e per eta e sono stati costituiti i seguenti
gruppi: sangue del funicolo ombelicale (11 maschi - 10 femraine); soggetti
di eta compresa:fra 3 e 6 anni (9 maschi - 9 femmine); fra 8 e 11 anni (15
maschi - 15 femmine); fra 12 e 15 anni (6 maschi - 7 femmine)} fra 20 e
40 anni (30 maschi - 20 femmine); fra 60 e 90 anni (18 maschi - 18 femmi-
ne). Tutti i soggetti esaminati erano apparentemente sani, non risultava-
no esposti a contatto con piombo per motivi professional! ne al momento
dell'esame ne in precedenza, non erano forti consumatori di alcoolici. II
campione di sangue per la determinazione dell'attivita ALAD eritrocitaria
(0,8 ml) e stato ottenuto in occasione di prelievi di sangue per scopi
-------
1125
medici (screening di massa, ecc,.)» L'attivita ALAD eritrocitaria e sta-
ta determinata secondo il metodo Europeo Standardizzato dalla C.C.E.(1973).
3. Risultati
I risultati sono esposti nella tabella I. L'esame del risultati di
mostra che nei primi 3 gruppi presi in considerazione (sangue del funico-
lo ombelicale, soggetti di eta compresa fra 3 e 6 anni e fra 8 e 11 anni)
1'attivita ALAD eritrocitaria si mantiene del tutto costante e che non esi^
stono differenze fra i due sessi, Nei soggetti di sesso maschile si osser
va successivamente un progressive decreraento dell'attivita ALAD eritroci-
taria con il progredire dell'eta: esso e modesto nei gruppo di soggetti
di eta compresa fra 12 e 15 anni, diviene piu manifesto nei soggetti di
eta compresa fra 20 e 40 anni, raentre nei soggetti di eta compresa fra 60
e 90 anni 1'attivita enzimatica e ridotta del 50$£ ed oltre rispetto ai va
lori osservati nell'eta infantile. Nei soggetti di sesso femminile, in
vece, i valori dell'attivita enzimatica si mantengono praticaraente a li -
velli costanti anche nei gruppi di eta compresa fra 12 e 15 anni e fra 20
e 40 anni ed un decremento di essi si osserva solo nei soggetti di eta
compresa fra i 60 e 90 anni. Anche in questo gruppo il decremento della
attivita enzimatica e certamente inferiore a quello osservato nei gruppo
corrispondente di soggetti di sesso maschile.
4. Discussione
Un'analisi statistica dei risultati ottenuti dimostra che 1'attivi-
ta ALAD eritrocitaria subisce con il progredire dell'eta una progressiva
riduzione. Questo fenomeno e chiaramente evidente nella casistica di ses_
so maschile e si manifesta gia a partire dal 3° quinquennio di vita. Es-
so sembra indicare 1'effetto di un progressive aumento dell'accumulo di
piombo nell'organismo umano, con il passare degli anni. Le ricerche di
Beasley e coll. [6j hanno, d'altronde, dimostrato che in soggetti sani
anche i livelli di piombemia si innalzano gradualmente con 1'aumentare
dell'eta (e tuttavia da rilevare che altre indagini, condotte in epoca
precedente, non avevano evidenziato questo fenomeno - Hofreuter e coll.
, Hammond [8J ). La progressiva riduzione dell'attivita ALAD eritro
-------
1126
Tabella I - VARIAZIONI DELL'ATTIVITA' ALA-DEIDRATASICA ERITROCITARIA
IN RAPPORTO ALL'ETA' ED AL SESSO IN SOGGETTI NON PROFES-
SIONALMENTE ESPOSTI A PIOMBO.
N° del soggetti
SESSO MASCHILE
ALAD
U.I./L G.R.
N° del soggetti
SESSO FEMMINILE
ALAD
U.I./L G.R.
SANGUE DEL
FUNICOLO
OMBELICALE
11
42,25
10
41,09
3-6
anni
9
45,40
9
44,46
8-11
anni
15
45,32
15
44,66
12 - 15
anni
6
38,45
7
46,53
20 - 40
anni
30
32,18
20
42,10
60-90
anni
18
20,46
18
34,84
ANALISI DELIA VARLANZA PER UN MODELLO MISTO, ESSENDO RAN-
DOMIZZATA LA VARIABILE ETA' E FISSA LA VARIABILE SESSO.
SORGENTE DELLA
VARIAZIONE
SESSO
ETA'
INTERAZIONE
SESSO- ETA'
HtRORE
SS
1621,39
6831,6?
1645,76
8099, 18
df
1
5
5
156
MS
1621,39
1366,33
329, 15
51,92
F
31,23
4,15
6,39
P
< 0,001
<0,10
<0,01
-------
1127
cLtaria con 1'aumentare dell1eta non e altrettanto evidente nella casisti
ca di sesso femminile da noi esarainata: in questa casistica il fenoraeno
e ben evidente solo a partire dalla 6° decade di vita. Ne deriva che nei
soggetti di sesso femminile,nell'eta adulta,I1attivita ALAD eritrocita-
ria risulta piu elevata che nei soggetti di sesso maschile, come d'altron
de gia constatato da Haeger Aronsen e coll. 9 , da noi stessi I 2| e da
• L J L J
Valloton e coll 110}• La casistica da noi studiata e costituita da sog-
getti abitanti nella stessa citta e pertanto si puo ritenere che tutti
i soggetti esaminati fossero esposti allo stesso assorbimento di piombo
per via inalatoria. Le differenze da noi riscontrate fra i due sessi
potrebbero pertanto essere attribuite, in via di ipotesi,ad un diverse
assorbimento di piombo per via digestiva,per different! abitudini alimen
tari fra i due sessi. Questa ipotesi non ci serabra sufficiente ad in -
terpretare i risultati ottenuti,in quanto le prime differenze fra i due
sessi si osservano gia nei 3° quinquennio di vita,quando le abitudini
alimentari non sono in pratica different! e, soprattutto,non esiste an-
cora quel maggior consume di vino e di alcoolici nei sesso maschile che,
secondo recenti dati (Moore e coll.Till , Krasner e coll.[l2J, Secchi e
Alessio I13j), potrebbe giustificare una piu spiccata inibizione della
attivita ALAD eritrocitaria* Altri fattori debbono pertanto essere in
gioco: essi potrebbero collegarsi alle emorragie periodiche del sesso
femminile,tali da determinare nella donna 1'esistenza di una popolazio-
ne eritrocitaria costituita da cellule piu giovani. E1 d'altra parte
noto come 1'attivita ALAD eritrocitaria sia particolarmente elevata nel
le cellule rosse piu giovani (Battistini e coll.j141). Questa seconda
ipotesi pu6 essere convalidata dal fatto che le prime differenze fra i
due sessi,per quanto riguarda i livelli di attivita ALAD eritrocitaria,
si osservano a partire dal 3° quinquennio di vita e che invece dalla
6° decade di vita in poi, 1'attivita ALAD eritrocitaria subisce un de-
cremento anche nei soggetti di sesso femminile.
-------
1128
5. Bibliografia
1. SBCCHI G.C., ALESSIO L. and CAMBIAGHI G.: Ricerche sull'attivi
ta ALA-deidrasica eritrocitaria di soggetti non esposti a con-
tatto professionale con piombo ed abitanti in zone rural! ed
urbane. Med. Lavoro 62, 435-450, 1971.
2. SECCHI G.C., ALESSIO L., CAMBIAGHI G. and ANDREOLETTI F.: ALA-
Dehydratase activity of erythrocytes and blood lead levels in
"critical" population groups. Proc. International Symposium
"Environmental Health Aspects of Lead", Amsterdam, October 2-6,
1972, p. 595-602.
3. NORDMAN C.H., HERNBERG S., NIKKANEN J. and RYHXNEN A.: Blood
lead levels and erythrocyte delta-aminolevulinic acid dehydratase
activity in people living around a secondary lead smelter-work.
Environ. Hit., 10, 19-25, 1973.
4. HERNBERG S., NIKKANEN J.: Enzyme Inhibition by Lead under Normal
Urban Conditions. Lancet, 1, 63-64, 1970.
5. HERNBERG S., NIKKANEN J., MELLIN G., LILUIS H.: /^ -aminolaevu-
linic Acid Dehydratase ,as a Measure of Lead Exposure. Arch. En-
viron, Health, 21, 140-145, 1970.
6. BEASLEtf W.H., JONES D.D., MEGIT A., LUTKINS S.G.: Blood Lead
levels in a Welsh rural community. Brit. Med. J. 4, 267-270,
1973.
7. HOFREUTER D.H., CATCOTT E.J., KEENAN R.G. and XINTARAS C.: The
public health significance of atmospheric lead. Arch.Environ.
Health, 3, 568-574, 1961
8.HAMMOND P.B.: Essays in Toxicology .Vol.1. Acad.Press,N.Y.1969,p. 115
9. HAEGER-ARONSEN B., ABDULLA M. and FRISTEDT B.I.: Eccets of lead
on delta-aminolevulinic acid dehydratase activity in red blood
cells. Arch. Environ. Health, 23, 440-445, 1971.
10. VALLOTON M.N., GUILLEMIN M. et LOB M.: Plombemie et activity
de la dehydratase de 1'acide delta-aminolevulinique dans une
population lausannoise. Schweis. med. Wschr., 103,547-550,1973^
-------
1129
11. MOORE M.R., BEATTIE A.D., THOMPSON G.G. and GOLBDERG A.:
Depression of delta-aminolaevulinic acid dehydratase activity
by ethanol in man and rat. Clinical Science, 40, 81-88,1971.
12. KRASNER N., MOORE M.R., THOMPSON G.G., McINTOSH W., GOLDBERG
A.: Depression of erythocyte delta-aminolaevulinic acid
dehytratase activity in alcoholics. Clinical Science (in press),
13. SECCHI C.G., ALESSIO L.: Erythrocyte ALA-dehydratase activity
in critical general population groups and in workers esposed
to lead (1974, in press,)
14. BATTISTINI V., MORROW J.J., GINSBURG D., THOMPSON G., MOORE
M.R., GOLDBERG A.: Erythocyte delta-aminolaevulinic acid
dehydrase activity in anaemia. Brit. J. Haematol. 20,177-l84>
1971.
DISCUSSIONE
HERNBERG (Finland!a)
Dispone Lei di misurazioni di piombemia per il Suo gruppo?
II ricorso a questi dati Le avrebbe forse risparmiato molte
congetture circa 1'accumulazione di piombo con 1'avanzare dell1
eta. Come Lei certamente sapra, il collega S. Tola ha djtmo*-
strato che il rapporto piombemia/ALA-D & in pratica quasi sempre
il medesimo (Work-Envir. Health 10, suppl.l, 1973).
ALESSIO (Italia)
I livelli di piombo nel sangue sono stati determinati nei
gruppi di eta compress fra 2O e 40 anni; i livelli di piombemia
sono risultati significativamente piCt elevati nei soggetti di
sesso maschile.
Per quanto riguarda gli altri gruppi di eta purtroppo non
dispongo ancora dei dati riguardanti i livelli di piombo nel
sangue.
CARNOW (U.S.A.)
Lei fa notare una differenza di sesso all'eta di 12 anni.
Ha Lei preso in considerazione il fumo quale possibile spiega-
zione di questo fenomeno?
Ev stato dimostrato che i fumatori di sigarette haono in
genere un tenore di piombo piu elevato nel sangue per effetto
dell'assorbimento dal fumo.
-------
1130
ALESSIO (Italia)
In questa ricerca non S stata valutata 1'influenza del fumo
di tabacco sull'attivita ALAD eritrocitaria.
HINE (U.S.A.)
II nostro gruppo ha studiato ±1 tenore in pioinbo nel sangue,
nei tessuti molli e nelle ossa di persone da 1 a 90 anni. I
livelli piO elevati di pioinbo nei tessuti sono stati riscontrati
nei soggetti compresi tra i 3O e i 7O anni, mentre in seguito si
§ notata una diminuzione.
L'inibizione pid elevata dell'ALAD nelle vostre serie tra
i 6O-90 anni avrebbe luogo a pid basse concentrazioni di pioinbo.
Si renderebbe pertanto necessario un altro fattore al fine di
spiegare la diminuzione del valori dell'ALAD da voi osservati.
ALESSIO (Italia)
Poiche non dispongo ancora del dati riguardanti i livelli
di pioinbo nel gruppo da Lei indicato non posso attualmente
escludere o confermare le Sua interessante ipotesi.
-------
1131
SIGNIFICATION DES INDICATEURS BIO-ANALYTIQUES DE
[.'EXPOSITION AU PLOMB AU SEIN D'UNE POPULATION NON
PROFESSIONNELLEMENT EXPOSEE
P, BRUAUX, F, CLAEYS-THOREAU, A, LAFONTAINE, M, LEGRAND,
R, DE BOECK, I. SWYNGEDOUW
avec I1aide technique de Mmes M. Milissen-Vande Riviere et
J. Verhoeven-Daulne.
Institut d1Hygiene et d'Epidemiologie, Bruxelles, Belgique
RESUME
Au coura d'une enquSte epidemiologique effectuee au sein
d'une population non aeleationnee et non profeaaionnellement
exposeet lea auteura ont etudi£ la signification de quelques
indicateura bio-analytiques de I 'exposition au plomb: plombemie,
ALAD et ALAU. Apr&e avoir deceit bvievement les techniques
utilis&es et discutS lea reaultata obtenus^ ils arrivent aux
conclusions suivantea:
- la mesure de 1'A.LAD, paratt un indicateur sensible de
I'exposition non pvofeeaionnelle au plomb tandis que la mesure
de I'ALAU semble devoir Stre ecavtee;
- la meaure de la plombemie reate delicate et insuffiaamment
reproductible pour Stre utilisee dda a present mais il existe
indiacutablement une correlation valable entre la plombemie et
I'activitS ALAD.
ABSTRACT
During an epidemiological survey on sections of the popula-
tion uhiah had not been specially selected and were not occupa-
-------
1132
tionally exposed to lead the authors investigated the value of
some bio-analytical •indicators of exposure to lead: lead in the
blood, ALAD and ALAU. After giving a brief description of the
techniques employed and discussing the results obtained, they
come to the following conclusions;
- ALAD measurement appears to be a sensitive indicator of
non-occupational exposure to lead, but ALAU measurement must,
it would seem, be eliminated;
- the measurement of lead in the blood remains difficult
and is not reproducible enough to be used at present, though
there is unquestionably a valid correlation between blood lead
level and ALAD activity.
-------
1133
Introduction
Bien que le saturnisme soit connu depuis la plus haute anti-
quitg, I'intSrSt des toxicologues pour le plomb s'est rSveil-
16 recemment en raison des multiples pollutions dues aux con-
ditions techniques de la vie moderne.
Les principaux indicateurs bio-analytiques actuelleraent pro-
pose's pour I1exposition non professionnelle au plomb sont la
mesure de la plombSmie, celle de 1'activite1 de la dShydratase
de 1'acide deltaaminole'vulinique dans le sang (ALAD) et celle
de I'excrStion de 1'acide deltaaminole'vulinique dans les
urines (ALAU). Les probl&mes techniques pose's par le dosage
de la plombeinie ne sont pas encore entiSrement r6solus
(Berlin et coll.) (1) particulierement en ce qui concerne les
faibles teneurs que 1'on peut rencontrer dans les expositions
a la pollution saturnine de 1'environnement.
Au cours de la prSsente enquete Spide'miologique, les auteurs
se sont attache's S dSgager la signification de la mesure de
1'ALAD, de I1 ALAU et celle de la plombe'mie dans I1 estimation
des risques sanitaires que comporte la pollution de 1'envi-
ronnement par le plomb.
Population examinee
La population choisie n'est pas professionnellement exposSe
au plomb et peut etre consid6re"e comme 6tant en bonne sante".
Au cours de notre enquete qui s'est gtale"e de fin 1972 3
dSbut 1974, cette population a etS examin6e deux fois.
1. Lors de la premiere sgrie d'examens, nous avons effectug
le dosage de I1ALAD chez 143 personnes et le dosage de
1'ALAU chez 140 d'entr'elles.
Nous avons classg cette population en 106 homines, 37 fem-
mes, 66 fumeurs, 69 non fumeurs ainsi que par age.
2. La deuxiSme sSrie d'exaraens a portg pour la quasi totali-
t6 sur les memes personnes rgexamine'es S environ un an
d'intervalle. Cette seconde s£rie a comport^ le dosage de
1'ALAD et de la plomb6mie et nous n1avons retenu que les
124 personnes pour lesquelles nous avons pu obtenir ces
deux mesures sur le meme Schantillon.
-------
1134
Ces 124 personnes se repartissent en 86 homines, 38 fem-
mes , 60 non fumeurs et 63 fumeurs.
Materiel et Mgthodes
d£ 1'ALAU
Pour ce qui est de I'excrStion de 1'ALAU, les auteurs ont
utilis6 la mSthode classique de DAVIS.
Dojsa£e_de JL'ALAD_
Lors de la premiere s6rie d'examens, nous avons utilise"
une technique d6rivee de celle de BONSIGNORE (2).
Lors de la deuxieme serie, nous avons suivi strictement
la methode europgenne standardis6e decrite en d6tail par
ailleurs (3). Les principales dif£6rences avec notre
technique initiale sont les suivantes :
- remplacement du TRITON X 1 00 comme agent h6molysant par
eau distillee;
- incubation a pH 6,4 au lieu de pH 7;
Afin d'6viter toute confusion dans 1'expression des r6-
sultats, nous donnons , ci-apres, la formule du calcul des
unites que nous avons utilisees :
Unites=microm6les ALA/min/L RBC-Hct
E : extinction mesurSe
Hct : hematocrite
K : facteur d'etalonnage du spectrophotoiretre = 1.099
3. Mes^u£e_d_e J:a_plLomb^iniLe
Pour ce qui est de la mesure de la plombemie, nous avons
utilise une technique d* absorption atomique sans flamme.
II serait Sgalement hors de propos d'entrer ici dans les
details de cette technique dont nous ne citerons que les
principales Stapes.
Appareil utilise1 : AA Perkin Elmer 6quip6 du four graphite
(avec tube special pour solution organique) et du
"Deuterium-Corrector" avec "Gazstop".
L'Schantillon (sang total heparine) est dilu6 6 fois dans
une solution a 0,5% de TRITON X 100. Apres hgmolyse, on
-------
1135
injecte 10 microlitres de cette dilution dans le four
graphite. On realise 1'Stalonnage interne par addition de
quantites connues d'une solution standard de plomb.
Resultats et Discussion
Afin de faciliter la comprehension et I1interpretation de nos
resultats, nous les avons pre'sente's sous forme de tableaux
et graphiques.
1. Do_sage_d«3 ,1'ALAU
Le tableau 1 resume les rSsultats de nos dosages d'ALAU.
On peut en tirer les quelques considerations suivantes :
- toutes les valeurs de 1'ALAU que nous avons dSterminees
sont basses : elles se situent toutes en-dessous de
5 mg par gr de crSatinine.
- 1'analyse statistique n'a montrg aucune correlation si-
gnificative entre les mesures d'ALAD et d'ALAU dans les
divers groupes de population conside're's.
En conclusion, il nous a paru que le dosage de 1'ALAU ne
semblait pas repr^senter un indicateur bio-analytique sen-
sible de 1'exposition non professionnelle au plomb et
c'est la raison pour laquelle nous avons abandonng cette
me sure.
TABLEAU 1.
Repartition des mesures de I'excrStion de 1'acide deltaaminolgvulinique dans les urines.
(exprime'es en mg d'acide deltaaminolfivulinique par gramme de crfiatinine).
Milligrammes d'A.L.A. par gramme de
cr6atinine
<1
1 i 2
2 a 3
3 a 4
4 a 5
Pourcentage
26 I
SO \
20,3 t
2,5 \
1,2 \
Pourcentage.
cumulg
26 1
76 \
96,3 \
98.8 \
100 1
-------
1136
Graphique 1 ALAD lere et 2eme aerie
lere serie Bl 37,*t6
2eme serie m 3
unite
Graphique 2 Plombemie
100
90
80
cumule
60
50
kO
30
20,
10
12 15
20
85
30
35
'to i»5
50
55
60
-------
1137
2. Do£a£e_d£ JL'ALAD - Comparaison entre les re"sultats obtenus
lors des deux series d'examens.
Le graphique 1 illustre cette comparaison. Rappelons qu'il
s'agit des memes personnes examinees a" environ 1 an
d1intervalle. Bien que la technique utilised n'ait pas Ste"
strictement la meme, on peut remarquer que les rSsultats
sont cependant assez proches : les moyennes ont ite" de
37,46 et de 35,7 unites, respectivement pour les premiere
et deuxifeme series d'examens.
Cependant, il faut souligner que 1'adoption de la methode
europ^enne standardised a permis d'obtenir vine moindre
dispersion des rgsultats : 90i des r^sultats obtenus sont
compris entre 19,5 et 48 unite's alors que dans la premiSre
sSrie, ils s'Stalaient de 10 5 57 unite's.
Nous avons 6galement compare1 les valeurs individuelles
obtenues £ 1 an d'intervalle : le coefficient de corre"-
lation est de + 0,56. (valeur limite + 0.28 pour p=0.01).
On pourrait done penser, bien qu'il soit nScessaire de le
confirmer par des experiences ultSrieures, que la valeur
ALAD est assez stable chez un meme individu.
Ajoutons que lors d'un essai realise" chez 6 volontaires
de notre laboratoire, le coefficient de variation de la
mesure ALAD, determined une fois par semaine pendant 4
semaines cons6cutives, a &t6 de 6,29$.
3. Me_sures_ de_La £l^mb_gmie_
Le graphique 2 r6sume les rgsultats de nos 124 d6termina-
tions.
Remarquons que :
- 60$ des plombSmies se situent en-dessous de 20yg. $
- 90$ en-dessous de 31yg $
- 98$ en-dessous de 40ug $.
Ces valeurs se rapprochent assez fort des valeurs guides
propose'es par ZIELHUIS (4) . On pourrait en conclure que
la population que nous avons examin6e ne semble pas etre
expos6e 9 un risque appreciable au point de vue exposition
au plomb.
-------
1138
Le graphique 3 illustre bien la difference entre ces
2 groupes : les femmes ont une activite" ALAD nettement
plus elevee que les hommes. Nous avons utilise le test t
pour ^valuer le degr6 de signification de la difference
entre les 2 moyennes. Le calcul de t donne : 3.061 : la
valeur limite de t au niveau 0.01 etant 2,62, nous pou-
vons en deduire que la difference est significative.
5. £ompjireii^on de£ mesures^ ALAD_eiitr_e_fuine_u_rs_et^ non frjmeurs
Le graphique 4 montre la difference entre ces 2 groupes :
les fumeurs ont manifestement une activite ALAD plus bas-
se. Le test t dont le calcul donne la valeur 3.275 indi-
que egalement une difference significative entre les 2
moyennes .
6. Ac£ivi^e_de .I'ALAI^en .rap_pojt_ £ve_c_l_^ag_e_
L'analyse statistique a montre qu'il existait une diminu-
tion progressive de I1 ALAD avec 1'age. Cette diminution
s'est manifestee dans les deux series d'examens mais elle
n'est significative qu'au niveau p=0.05 : les coefficients
de correlations calculus ont $t& respectivement pour les
premiere et deuxieme series de -0,19 et -0,186 (valeur
limite pour p O.OS=-0,17).
Le tableau 2 nous montre ces correlations entre les di-
vers groupes. L'exaroen de ce tableau nous permet les con-
siderations suivantes :
a. Pour les mesures d'ALAD, nous retrouvons les differences
signalees entre les hommes et les femmes d'une part,
les fumeurs et le s non fumeurs d'autre part.
Corollairement, les plombSmies sont plus elev6es chez
les hommes que chez les femmes et chez les fumeurs que
chez les non fumeurs. Cependant, I1 analyse statistique
montre que ces differences sont moins significatives
que pour les mesures de 1'ALAD. Ceci est dO 3 une dis-
persion relativement plus grande pour les mesures de
plombemie.
-------
1139
Graphique 3 ALAD hommes-femmes
100
96
80
70
60-
50
30. i
20
10
cufflule
homines m 5^.13
.population total*
« 55,70
femmes m 39.25
unite
12 15 20 25 30 35 ^0
50
55 60 65
Graphique k ALAD fumeurs - non fumeurs
100-
% cumule
50
»
30
20
fumeurs m 33»26
population totale
• 55,70
non fumeurs
• 38,32
unite
40
II
-a
-------
1140
TABLEAU 2.
Correlation entre Ics moyennes ALAD et Plombemie dans les divers groupes de population.
Population
totale
Homines
Femraes
Fumeurs
Kon fumeurs
Nombre de
sujets
124
86
38
63
60
ALAD
Unites
35,70
I
34,13
39,25
33,26
38,32
Plorobemie
vg I
20,69
21 ,74
18,31
22,17
19,23
Coefficient
de corrfila-
tion
r
-0.39
-0.40
-0.26
-0.51
-0.21
Valeur
limite
de r
pour
p-0.01
-0.23
-0.28
-0.41
-0.32
-0.33
Pour les mesures d'ALAD et de plomb6mie, on observe
une correlation negative. Cette correlation est signi-
ficative au niveau p=0.01 pour la totalit6 de la
population examin6e, pour le groupe des homines et ce-
lui des fumeurs. Elle est moins significative pour les
autres groupes.
L'examen du tableau 2 montre clairement que nous n'a-
vons une correlation significative entre ALAD et plombe'-
mie que lorsque la moyenne ALAD du groupe consid6r£ est
infe"rieure 3 la moyenne g6ne>ale et lorsque la moyen-
ne de la plombemie est par contre supgrieure 3 la moyen-
ne g^ndrale. Les difficulty's techniques de la mesure de
la plombe'mie, surtout pour les valeurs basses, pour-
raient expliquer ce fait.
La mesure de 1'ALAD nous parait done etre un indicateur
bio-analytique valable et sensible de 1'exposition sa-
turnine d'une population non professionnellement expos€e
au plomb, surtout que 1'on dispose actuallement d'une
mSthode bien standardised permettant de comparer les
r^sultats obtenus dans divers pays.
-------
1141
Conclusions ge"ne"rales
Bien que cette enquete ait 6t6 tres limited et qu'elle doive
etre 6tendue 3 d'autres groupes de population, notamment 9
des groupes critiques, il nous semble que 1'on puisse en tirer
les quelques conclusions provisoires suivantes :
1. Dans l'e"tat actuel de la pollution de 1'environnement par
le plomb et pour la population que nous avons examinee, il
ne parait pas y avoir de risque sanitaire appreciable si
1'on se re"f
-------
1142
REFERENCES
1 . BERLIN et coll.
European Intel-comparison Programmes
Proceedings Inter. Symp.
Amsterdam 2-6/10/1972 - EURO 5004 DBF. p.1033
2. BONSIGNORE D., S. coll.
Un semplice metodo per la determinazione della 6-amino-
levulinico-deidratasi nel sangue - Med. Lavoro.
56, 199(1965)
3. The European Standardized Method for the determination of
ALAD activity in Blood - Doc. CCE 2318/2/73e
4. ZIELHUIS (11 et 12/12/1972)
Biological Quality Standards for Inorganic lead
Working document - CCE Luxembourg.
DISCUSSION
HERNBERG (Finlande)
Je voudrais fairs deux observations au sujet de votre
interessante 6tude: premierement, en 1972,11168 collSgues MM. Tola,
Nikkanon et moi-meme avons Studifi une population d1environ
1.5OO personnes. Nous avions trouvfi qu'en normalisant vis-a-vis
de la plombemie,ni I1age, ni le tabagisme n'avaient pratiquement
d'effet significatif sur ALA-D. De plus nous avons constatS que
le rapport PbB ALA-D £tait le meme pour une exposition profession*
nelle nouvelle, pour un taux constant d1exposition et aussi long-
temps aprSs la fin de 1'exposition. Ainsi 1'ALA-D reflate la
teneur en plomb du sang et les differences que vous avez trouvSes
peuvent tr&s probablement s'expliquer par des PbB difffirents.
Les r^sultats auxquels je me suis r6f6r6 ont 6t6 publics il y a
un an dans Work-Environment-Health (Tola, S., Wk-Environ. Hlth
1O, suppl. I/ 1973). En second lieu je pense que la raison pour
laquelle vos valeurs t gtaient plus significatives pour ALA-D
que pour PbB est que vous ne devriez pas utiliser la statistlque
t pour la distribution log. normale d'ALA-D. Si, au lieu de
cela, vous aviez calculi le logarithme des valeurs ALA-D, je
pense que le test t aurait donnfi le meme rfisultat que pour PbB,
J'estime done que 1'explication de la divergence apparente est
simplement que vous avez employ6 une mfithode statistique inade-
quate .
-------
1143
BRUAUX (Belgique)
1. Pour ce qui est de la premiere observation du Dr. Hernberg,
je suis d'accord avec lui sur le fait que les differences que
nous avons observSes dans les mesures de l'ALA-D suivant le sexe,
1'age, le tabagisme sont associees a des differences dans la
plombemie. Le tableau 2 montre d'ailleurs clairement la corre-
lation negative existant entre les valeurs ALA-D et celles de la
plombemie. Ce que nous avons voulu montrer dans ce travail
c'est que la mesure del'ALA-D est plus sensible et plus precise
que celle de la plombemie. Ceci explique que les differences
statistiques entre les differents groupes soient plus significa-
tives pour les mesures de l ALA-D que pour celles de la plombemie.
2. Quant a la deuxieme observation, je puis dire que le test
t a ete applique sur les valeurs logarithmiques de 1"ALA-D ce qui
rend, je pense, 1'application de ce test valable pour evaluer
la difference entre les moyennes.
-------
1145
DIAMETRE ERYTHROCITAIRE MOYEN CHEZ LES ADULTES
HABITANT UNE VILLE AVEC UNE ATMOSPHERE POLLUEE
PAR LE PLOMB INORGANIQUE DE SOURCE INDUSTRIELLE
N, W, GHELBERG, E, BORDAS
Institut de Sant6 Publique et de Recherches M£dicales, Cluj,
Roumanie
RESUME
Noua nous aommea proposes d*examiner le comportement du dia-
metre erythrocytaire moyen (DEM; ,u) ches lee adulte8 d'une ville
dont lfatmosphere eat polluee - ville P - pat* du plomb inorganique
de provenance industrielle. 359 sujets non exposes profession-
nellement au plomb (groupe P) ont &t& examines aomparativement a
deux groupes temoina: le groups Tt aomprenant 125 temoina, et
le groupe C - de oontrdle - oomprenant 100 ouvriere professionnel-
lement exposes au plomb ma-is sans symptbmea oliniques.
Chez le groupe P la valeur moyenne (x + s) du DEM etait de
?.?6 + 0.64, alora que anez lea sujets du groupe Tt la valeur
moyenne etait 7.06 + 0.49. C'eat chez le groupe C que la moyenne
etait la plus elevee (8.00 + 0.24). Lea differences ont &t&
significative a tp<0i01).
Lea aspects dea courbea Price-Jones obtenua pour lea trois
groupes aont caracteriaes par la categorie dea dimensions d. pour-
centage de frequence maximale,par la baiaae du peak, un glissement
vera la droite et la presence d'une tratne a droite3 allant de T
vers Pf plus marquee pour C.
Lea faita ci-deaaus demontrent que I'influence prolongee du
contact avea le plomb environnant determine une tendance vers la
-------
1146
macrocytose chez la population adulte exempte de signea cliniquea.
On considere que I'etude du DEM pourrait constituer une me —
tJiode de screening pour 1e diagnostic de groupss en cas d1'expo-
sition au plomb chez dea groupes de population habitant des zones
urbaines avec des degres different^ de pollution par le plomb.
ABSTRACT
We decided to examine the behaviour of the mean erythrocyte
diameter (MED; ,u) in adults living in a town - town P - whoee
atmosphere is polluted by inorganic lead of industrial origin.
359 subjects not occupationally exposed to lead (group P) were
examined and results compared with two control groups: group T3
comprising 115 subjects and group C> comprising 100 workers oc-
cupationally exposed to Iead3 but showing no clinical symptoms.
In group P the mean value (x + e) of the MED was 7.76 +_ 0.64,
whereas in group T subjects the mean value was 7,06 + 0.49. The
mean was highest (8.00 + 0.24) in group C. The differences were
significant (p<.0.01).
The Price-Jones curves obtained for the three gorups are
characterized by size category at the maximum percentage frequ-
ency ,, by a drop in the peak levelt a displacement towards the
right and a gradual lengthening of the curve to the right, from
T towards P3 more marked in the case of C,
The above data show that prolonged contact with lead in the
environment tends to cause macvocytosis in an adult population
showing no clinical symptoms.
It is thought that the study of the MED might be used as a
screening method for group diagnosis in cases of exposure to
lead of population groups living in urban areas with various
degrees of lead pollution.
-------
1U7
1. INTRODUCTION
En partant de la nScessite et de 1'actualite de certains test
de screening h valeur de diagnostic de groupe dans 1'Evaluation de
1'influence du plomb de 1'atmosphere de certaines villes BUT la popu-
lation, nous nous sommes proposes 1*etude du comportement du diametre
erythrocytaire moyen (DEK), respectivement la courbe Price-Jones. Nous
avons porte notre choix sur ce parametro de laboratoire clinique pour
les raisons suivantes:
a) il peut reflechir 1'affaiblissement d'une fonction physiologique
vital e, I'hematopoi&se, qu'une exposition professionnelle au plomb
peut, comme on le Bait, affecter;
b) on a propose (l) de considerer que I1alteration significative de
I'hematopolese correspondrait au niveau III de pollution de I1atmos-
phere des villes, conformement a la classification de 1'O.M.S. (2);
c) dans le cas des intoxications au plomb, la plupart des auteurs (3-8
etc.) enoncent 1'existence d'une anisocytose a microcytosej d'autres,
moins nombreux (9-12) plaident par contre pour I1existence d'un cer-
tain degre de macrocytose* Albahari et collab. (13) soutient qu'il
n'y a ni megalocytose ni microcytose, tandis que de Bruin (8) signals
1'accroissement du nombre des erythroblastes et quelquefois celui des
m<5galo - et macroblastes dans la moelle osseuse. Nous n1 avons cependant
pas rencontre de donnees coiicernant la population du milieu urbain
faiblement pollue au plomb, et en 1'absence des signes cliniques;
d) les resultats de nos travaux ant^rieurs (14-16} Ghelberg et collab.
donn^e en coura de publication), ou nous avons etudie certains para-
metres de laboratoire utiles dans 1*appreciation des troubles du pro-
cessus h£matopo'ietique chez dee groupes d'enfants et d1 adultes habitant
une ville dans I1atmosphere de laquelle le plomb represente la nocivite
potentielle prinoipale, plaident pour 1'utilifiation de 1'indicateur DEM;
e) la determination DEM presente des avantages aussi bien du point de vue
du materiel biologique a examiner, que de celui de I1 exam en proprement
dit et peut @tre pratique^ dans les conditions d'un test de screening.
-------
1148
2. MATERIEL ET METHODS
En accord avec certaines donn^es de la litt^rature (l?)i nous avons
juge1 utile d'effectuer lea analyses sur des groupee plus restreints, mais
de maniere repetSe (1971-1973)• Nous avons examine au total:
- 115 adultes temoins - groupe T - de villes exemptes d'une pollu-
tion specifique au plomb ( < l.o ftg Pb/mc air/24 heures);
- 359 adultes non-exposes professionnellement au plomb, mais habi-
tant une ville dont 1'atmosphere est polluee par des entreprises
de metallurgie non-ferreuse (0.91-22.89 fig Pb/mc air/24 heurea,
avec une moyenne de 3*98; Ghelberg N.H. Radulescu N.D.,
Mihalka St. - donnees en cours de publication) - groupe P; et
- un groupe de contrfile - C - de la mime ville que le groupe P,
mais travaillant dans des entreprises de metallurgie non-ferreuse
(a la fonte, I1agglomeration et le raffinage du plomb) et exempts
de BymptCmes cliniques au moment du prelevement de sang*
Nous avons determine DEM (en ft ) de 100 hematics pour chaque sujet,
en utilisant de minces frottis de sang peripherique a coloration May -
Griinwald - Giemsa; on a aussi trace les courbes Price - Jones par
groupes (T,P,C).
f
3. RESULTATS
Les resultats obtenus sur les groupes de oujets examines, exprimes
sous forme de moyennes arithmetiques et leur paramitres, figurent dans
le tableau I*
II ressort du tableau I que les moyennes DE» vont en croissant,
de T (7.06 + 0.49) a P (7«76 * 0.64) puis a C ou la moyenne est la plus
elevtSe (8.00 + 0.24). Les differences des moyennes DEM entre les groupes
sont significatives (test "t"; p<0,05). Pour une vue plus suggestive BUT
les valours du tableau I, contparativement avec les valeurs moyennes indi-
viduelles considerees comme normales (6), nous pr4sentons la figure 1.
Dans la figure 2, nous donnons les courbes de type Price - Jones
pour les 3 categories de sujets: T, P et C.
-------
1149
TABLEAU I
LE PIASTRE KOYEN ERYTHROCYTAIRE (DEM) CHEZ LES GROUPES
BE SUJETS fen u )
Le groupe '
T -
T -
T —
T -
T -
Total
P -
P -
P -
P -
Total
P -
Total
C
1/1971
2/1971
3/1972
4/1972
5/1973
T/1971-1973
1/1971
2/1972
3/1972
4/1972
P/1972
5/1973
P/1971-1973
/ 1973
n
52
10
11
16
26
115
100
53
37
53
143
116
359
100
3=SS =
7
7
6
7
7
7
7
7
7
7
7
7
7
8
X
=J=S3I = r
.08
.15
.88
.02
.08
.06
.69
.75
.88
.83
.81
.74
.76
.00
s
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
54
38
49
49
12
49
71
57
65
57
59
63
64
24
X
±±= = = ±£
7.
7.
6.
7.
7.
6.
7.
7.
7.
7.
7.
7.
7.
7.
±t.-
06
13
68
00
07
98
67
73
86
67
73
72
70
98
- 7
- 7
- 6
- 7
- 7
- 7
- 7
- 7
- 7
- 7
- 7
- 7
- 7
-8
.10
.17
.90
.04
.08
.14
.71
.77
.90
.99
.89
.76
.82
.02
Observations '
T « sujets sans exposition professionnelle au Pb et qui habitent
des villes dont la teneur en plomb de 1*atmosphere est de
< 1.0^i^24 heures - groupe terooin;
P = sujets sans exposition professionnelle au Pb et qui habitent
dans une ville a atmosphere polluee;
0.29 - 12.22 yug Pb/mc air/24 heures.
C = sujets de la ville P et qui travaillent en outre en milieu
pollue au Pb; groupe de contrSle.
-------
1150
T T
y^
6,5
v,o
das qroupas -
pp pJ3 c
1971-1975
G coupe s da
so
5 1
*
~jat&x
e.68
ps^ S|
II
£$• Jf^vT^-^p1 J,
u
1
a
nor molaa
Obw") Fbur T.P.C ^-oir lobleoul
Fij°qire 1 : Diametre erithrocytaire moyen en fonction de
1'intensite de I1exposition au plomb.
50
(courbe don qroup«
do lo Wolila' das au-L
chaz Ics s
nt la i^roupa.aoit lOO'N)
I
3 «illea,«jroup«T,t«moirm ( N- 115)
villa P«xj« P,populol1o^(N-3v)9)
so ;
20 ,
«0
a
^
villa f, groi.pc. C.Ouvnara ( N-(OO)
noo -fccrausa. (Pb)
5,0
4
l*tT t t
XT xp xc
1
(0.5
Figure 2 : Gourbes Price - Jones des groupes des differentes
categories de sujets.
-------
1151
On peut voir dans la figure 2 le comportement de la courlbe T, qui
correspond presque parfaitement aux donnees de la litterature, mais qui
differe categoriquement des deux autres groupes - P et C - toutes deux
deplacees vers la droite et avec une tralne a droite. Cette me"thode,
celle de la courbe Price - Jones, permet done elle aussi de voir I1instal-
lation d'un certain degre d'anisocytose avec I1augmentation de la fre-
quence des macrocytes, qui est d'autant plus accentuee que 1*exposition
au plomb a ete plus intense.
Pour souligner certaines relations entre la grandeur DEM et les
conditions de I1exposition au plomb, nous pre"senton8 quelquee details
concernant le groups C (Ghelberg N.W. et collab. - donnees en cours de
publication). Ce groupe, qui a corapris 100 sujets a moyenne de vie de
39.4 - 7*6 ans, avec une anciennete moyenne d1exposition au plomb de
14.5 - 6.8 ans et une moyenne DEM de 8.00 - 0,24 u, nous 1'avons divise
en sous-groupes: qu'ils etaient ou non des fumeursj en fonction de
1'anciennete du travail en milieu a plomb; et en fonction du lieu de tra-
vail. Nous avons calcule les valeurs moyennes du DEM d'apres les subdi-
visions mentionnees. On a constate des differences significatives
(p < 0,05) de DEM entre lee fumeurs (8.04 - 0.25) et les non-fumeurs
(7.94 1 0.20) et entre les ouvriers de la fonderie (8.07 - 0,25), par
rapport a ceux de I'agglomerage (7»91 - 0.20).
On a calcule" les coefficients de correlation totals entre certains
caracteres du groupe C. II en est resulte un rapport direct» liniaire,
entre 1'Sge et 1'anciennete dans le travail (r = 0.470; p < 0,01; ce qui
etait previsible), mais aussi entre 1'age et le DEM (r « 0.211; p < 0.05)
et respectivement entre I1anciennete en milieu a plomb et le DEW
(r = 0.198; p = 0.05).
4. CONCLUSIONS
L'etude du diamfctre erythrocytaire moyen (DEM) sur ies groupes de
population a degree differents d1exposition au plomb, en tant que polluant
de 1'environnement (pollution urbaine et celle du milieu de travail) nous
a perm is les constatations suivantes:
-------
1152
1. Chez les groupea d'adultes du milieu urbain faiblement pollue
au plomb de provenance industrielle, le DEM £tait plus clave1
que chez nous propres temoins, d^notant une anisocytose a
macrocytoae moderee.
2. Chez les ouvriers professionnellement exposes au plomb, mais
sans symptOmes cliniques, la frequence des macrocytes £tait
d'autant plus marquee.
3. La determination DEM et les courbes de type Price - Jones par
groupes de sujets, se sent averes utiles dans le diagnostic
de groups de I1influence du plomb d'environneraent sur les
groupes de population.
Nous conclurons done, que 1'indicateur etudi<§ se presente comme
utile dans son application en tant que test de screening pour 1'appre-
ciation de I1influence du plomb sur 1'organisme.
REFERENCES
(l) GOLDSMITH, J.R. "Epidemiological Bases for Possible Air Quality
Criteria for Lead", APCA Journal. 9* 714
(2) WORLD HEALTH ORGANISATION, Atmospheric Pollutants. Technical
Report Series No. 2?1 (1964).
(3) FABRB, L. Lee on de toxlcologie. vol.X., Herman et Cie Ed. ,
Paris, Prance (1945)«
(4) DEROBERT, L. , Intoxications et maladies professionnelles. Ed.
Medical es Plamarion, Paris, Prance, (1954).
(5) PATTY, P.A., Industrial Hygiene and Toxicology, Vol. Ill,
Interscience Publisher, New-York, London, U.S.A. England (1962).
(6) ALTERAS, I., et collab. Metodele laboratorului clinic. Ed.
Medicala, Bucureqti, R.S. Romania (1964).
(7) CADARIU, Gh. , BARHAT, B. , (sous la redaction de) Igiena mun-
cii. Ed. Medicala, Bucure^ti, R.S. Romania (196?).
-------
1153
(8) BRUIN, A., de "Certain Biological Effects of Lead Upon the
Animal Organism", Arch.Bnviron.Health. 23. 249 (1971)
(9) SAITA, G., PIOCCHI, P., CATTANEO, E "Diametro e volume globulare,
indice di sferocitosi, indice di saturazione nell'anemia saturninay
La Medicina del Lavoro. 43. 99 (1952)
(10) HASAN, J., HERNBERG, S., "Interactions of Inorganic Lead with
Human Red Blood Cells", Werk-Environment-Health. 2. 26.(1966).
(11) PILAT| L., GAVRILESCU, N., (sous la redaction de), Bolile profes-
sionale, Ed. Medicala, Bucuresti, R.S. Romania (1966).
(12) KUPETZ, G., W., "Beitrag zur Fehldiagnose bei beruflicher Blei-
intoxikation, Zsch.arztl. Fortbild.. 67. 1031 fl973).
(13) ALBAHARI, C,, et collab. "La nocivite1 hematologique du plomb",
Nouvelle Revue Francaise dl|Hematologie. 5. 689 (1965)*
(14) GHELBERG N.W., BRETTBR, E., COSTIN, L., CHITUL, E., "CercetSri cu
privire la apari^ia corpusculilor Heinz sub influenta concentra-
•^iilor mici de plumb din aerul atmosferic", Igiena fBuoarest). 15.
209 (1966).
(15) GHELBERG, N.W., COSTIN, I., TOMUS, R., NAGY, S., TORNER, G.,
MAJOR E.t BAKI, L.f "Modificari sanguine la popula^ia adulta In
functie de poluarea atmosferei comunale cu concentra^ii variabile
de plumb", Clu.iul Medical (Clu.i. R.S. Romania). 44f 447 (1971)*
(16) GHELBRRG, N.W., NAGY, S., BODOR, E., TOMUS, R.t PLIERSICA, Z.,
BALCMIRI, P., VLAD, I.O., SECELEAN, L.f "Unele modificari biochi-
mice In condi^iile polu&rii mediului cu plumb", Igiena (BucurestjJ.
21. 521 (1972)
(17) FINKLEA, J., P., HAMMER, M.D., HINNEES, T.A., PHIKERTON, C.,
Human Pollutant Burden^ ACH Symposium Volume, Pall, U.S.A. (1971)•
-------
1155
PREVALENCE OF SUBCLINICAL LEAD EXPOSURE IN 761
ASYMPTOMATIC SCHOOL CHILDREN: DENTINE LEAD LEVELS
AS A RETROSPECTIVE MARKER
HERBERT L, NEEDLEMAN+ AND IRVING M. SHAPIRO**
+ Children's Hospital Medical Center, Harvard Medical School,
Boston, Mass., USA
++ Center for Oral Health Research, University of Pennsylvania,
Philadelphia, Pa., USA
ABSTRACT
Dentine lead levels were measured from shed deciduous teeth
of 761 Philadelphia schoolchildren with no prior history of lead
poisoning and residing in two school districts, one considered
high risk for lead exposure, and one considered low risk. Black
children in public schools from areas of deteriorated housing had
marked elevations of dentine lead (mean of 198 ,ug per gram, in
174 children), with 20 percent of the children having levels in
the range associated with toxicity. White children from newer
housing had the lowest levels (mean of 41.7 ,ug per gram, 304
children), but a group of white children from intact housing
living near and attending school adjacent to a major lead pro-
cessor also had elevations of dentine lead (mean of 136 .ug per
gram, 71 determinations). Lead exposure as defined by dentine
lead levels is more serious and widespread than previously ack-
nowledged, and extends to groups other than those traditionally
accepted as at risk.
-------
1156
Recent attempts to study the effects of low level lead exposure have
produced conflicting conclusions. While Burde (l) has reported increased
incidence of perceptual-motor dysfunction, and behavior disorder, and
David (2) reported a higher incidence of increased lead storage in some
hyperactive children, the studies of Kotok (3), Albert, et al, (4), and
Lansdowne, et al, (5), purported to find no relationship between lesser lead
exposure and psychological function. As discussed in the paper by McNeil and
Ptasnik on "Evaluation of Long-term Effects of Elevated Blood Lead Concen-
trations in Asymptomatic Children" presented at this symposium and during
the panel on "The Scientific Data Base Required for Decisions to Protect
Human Health" two reports of neuropsychological sequelae in high lead children
from Smeltertown, Texas, USA draw opposing conclusions as to effects bf
exposure. These differences may be due in part to problems in experimental
design and insensitive psychological outcome measures.
To have validity, a study of the neuropsychological effects of low level
lead exposure should meet the following criteria:
(1) It should employ a reliable and valid index of exposure to lead.
(2) It should utilize neuropsychologic instruments sensitive enough
to detect subtle impairment, and broad enough to sample performances in at
least these areas: perceptual motor, linguistic, and attentional behaviors.
(3) It should identify and scale other factors known to affect neuro-
psychiatric development which act to confound the effect of lead. Among
these are birth status, nutritional history, early rearing quality, and
significant illnesses in the subject's history.
(4) It should obtain its subjects in a sampling procedure free of bias.
(5) It should study a sample large enough to allow stratification of
other variables that may confound with the effect of lead.
To employ sensitive neuropsychologic instruments at their current
stage of development requires the study of children at least six years of
age. If exposure has occurred earlier in the life of the child, blood
-------
1157
lead concentrations cannot be considered reliable indices of exposure,
since they may by this time have returned toward normal. Lead is deposited
in bony tissue. The deciduous tooth offers a painless, spontaneous,
universal biopsy of bony tissue in children after their sixth year.
In 1962 Altshuler, et al, (6), demonstrated elevations in whole tooth
lead in both fatal and non-fatal cases of lead poisoning. Our initial
study of lead content of whole teeth from asymptomatic children from the
"lead belt" of urban Philadelphia showed that urban children had nearly
five times the concentration (mean-51.1 Jug/g, N-69) observed in their
suburban counterparts (mean»11.0 jug/g, N»40) (7).
Subsequent studies (8,9) showed that lead is concentrated in the
secondary dentine adjacent to the pulp, a tissue continuously laid down
during the life of the tooth.
When we compared dentine lead levels from nine children who recovered
from lead poisoning to healthy children from suburban Boston and Iceland,
we found striking differences (10). The mean concentrations were: for
lead poisoned children, 601 ± 225 pg/g> for Boston children, 84 ± 56 ;ig/g,
and for Icelandic children, 35 i 29.8 jjg/g. The lowest level observed in
a child with overt lead poisoning was 292 jaq/g.
Strehlow's studies in the baboon have confirmed that tooth storage
of lead is dose related, permanent, and unaffected by chelation (11).
We will present data today that indicates that the study of lead levels
in the circumpulpal dentine of shed deciduous tooth provides a reliable
index of past exposure, and offers a tool to study large numbers of
"normal" children attending school. In addition, our data demonstrate
a higher prevalence of exposure than heretofore reported, and also show
that children attending school near a lead processor have significant
elevations in tooth lead levels.
-------
1158
METHODS
Two Philadelphia school districts, one considered high risk and the
other low risk for lead exposure, were identified and entered into the
study in 1971. The districts were chosen on the basis of yield, in previ-
ous years, of known cases of plumbism.
Characteristics of the Districts
The western half of District 5 is considered within the acknowledged
"lead belt" of the city, from which many of the cases of frank plumbism
are reported. The population is predominantly black. Although the
population is mobile, it tends to remain within the lead belt. The houses
are older than 40 years, and many are in a severe state of deterioration.
The eastern half of District 5, which is highly industrialized, ends at
the Delaware River, and its population is predominantly white. The row
houses in this area are old, but in generally good repair. Diagnosed
lead poisoning is rare from this sector.
District 8 is the area into which Philadelphia expanded after World
War II. The houses are therefore newer, and in generally good repair.
The ethnic constitution is predominantly white. In this district,
diagnosed lead poisoning is extremely rare.
From each participating school, every child in first grade was given
a letter to his or her parents informing them of the nature of the study.
When a tooth was presented, the teacher was asked to examine the child's
mouth for a fresh socket.
Biochemical Methods
Biochemical methodology is described more fully elsewhere (12). whole
teeth were embedded in self-curing acrylic. A 600 u slice was cut through
the center of the tooth. Under a dissecting microscope, the circumpulpal
zone of dentine was inserted into a vice to a depth of 300 u. The free
area of the slice was then chiseled away. The circumpulpal dentine,
-------
1159
measuring 600 by 300 ju, was dried at 60°C and weighed. After dissolving
in 0.1 ml of 70 percent HClO/j, the samples were analyzed by Anodic
Stripping Voltammetry (Environmental Sciences Associates).
From six public schools and three parochial schools in District 5,
and three public and one parochial schools in District 8, samples of
interior dust, playground dirt and gutter dirt were obtained. Two
schools in suburban Philadelphia away from major traffic patterns or
industry were similarly sampled.
Results
Striking differences in lead dentine levels between students in the
two school districts were found (Table 1). The highest levels were found
in the public schools of District 5, where the student population is
predominantly black, but the white parochial schools in District 5 also
had elevated dentine lead levels. Children in one parochial school, St.
A.1s had marked elevation of tooth lead levels (mean of 136 fig per gram,
71 children) with some individual values among the highest measured in
this study.
Figure I shows the cumulative frequency distribution of dentine lead
levels for all public school students, and for parochial students attending
St. J.'s in District 8 and St. A.'s in District 5. While only 3% of the
public and 6.6% of the parochial students of District 8 had dentine levels
greater than 100 ug/g, 66% of the black public school students and
43% of the white students of St. A.'s had levels in excess of 100 pg/g.
Nineteen percent of the black students from District 5 had dentine levels
in the range associated with frank lead poisoning. Eight percent of
St. A.'s students also exceeded 300 fig/g. Table II shows the substantial
elevations of lead in dust and dirt in and near the schools of District 5.
-------
1160
Table I
Concentration of Lead in Dentine According to School
School
N
Mean Dentine Lead
(uq/q) + SEH
District 8
T,H.
B.
R.B.P.
W t H* X*«
St. J. #
District 5
N. ft
V. »
St. A. *
P.T.
P.L.D.
J.R.L.
G.C.
J.E.
J.F.
114
59
69
62
76
40
51
71
34
19
46
33
29
58
42+2.8
40±2.9
37+2.6
48+3.6
46±2.8
66i5.6
92±7.8
136+15.6
120+21.2
131+24.7
208+29.0
188±30.2
169+27.5
191+19.7
ft • Parochial Schools
Discussion^
These data portray a prevalence of increased body burden of lead in
groups considered at risk greater than heretofore reported, and show that
-------
1161
*«
turner *
runic
ti. J
• nine? i
0-- - - - -o runic
ft. *
M IM !•• »•• «•• »•••
.IINflNI lllkt CONCINttATIOM <••/•)
Figure I
Legend: Cumulative frequency distribution of children from District 5 and
8 according to dentine lead level.
-------
1162
Table II
qistribution of Children According to
Dentine Lead Levels by School District and Pace
Range of Lead Concentrations (uq/g)
0-50 51-100 101-200 201-300
H £ 1*> £ (V)- N J*L H 1*1
301-400 400
N
-------
1163
other groups of children than ordinarily acknowledged have elevated
body lead burdens. While 7-12% of high risk groups previously studied
are reported to have blood lead levels in the range associated with
hazard ( ~-60ug/g) (13), our data show that 66% of black children have
elevated dentine lead levels, with 19% in the range found in clinical
lead poisoning.
The elevated lead levels in the white parochial school children
attending St. A.'s were unexpected, since their homes were in generally
good repair. Located one city block away from the playground of St. A.'s
is a. major manufacturer of paints and lead stearates for plastic fabrica-
tions. This finding is consistent with the effects of residence near
stationary sources of lead reported by numerous observers (5,14,15), and
supports the statement of the National Academy of Science's Task Force
on Lead: "The swallowing of lead contaminated dusts may well account in
large part for the higher mean blood lead content in urban children, and
the rather large fraction whose blood lead content falls in the range of
40-60 ug/100 ml" (16). Our data show that children in intimate proximity
to lead processors may experience severe enough exposure to raise their
body levels into the range associated with toxicity.
In addition to industrial sources of lead, the entire area of District
5 is subject to extremely heavy automobile traffic. The dust lead levels
in the western part of the District, taken at some distance from the lead
factories, but yielding concentrations of lead in the same range, suggest
that automobile emissions were important sources of dust and dirt lead.
This unexpected finding of elevated dentine lead levels in children
in housing of good repair, but who live and attend school close to a
major lead processor, as well as the anticipated finding of elevations
in children who live in deteriorated housing, suggests that both lead in
-------
1164
Table III
Lead in Individual Environmental Samples
Lead Concentration (ug/g)
School
pistrict 8
T.H.
R.B.P.
W.H.L.
St. J.
District 5
V.
St. A.
P.T.
P.L.D.
J .R.I/.
G.C.
J.E.
J.F.
Suburban
H.
L.
Interior Dust
635
293
939
388
2838
929
3074
4947
1889
3782
15680
2066
5327
3388
3411
1206
3666
4416
3206
1854
1517
946
579
277
Playground Dirt
403
444
424
421
8683
—
• •»
761
493
2578
533
3252
983
17256
w
1207
88
67
38
118
Gutter Dirt
2626
270
1605 .
1528
3031
8201 '
6340
2392
»•»
2729
280
4332
•»«•
1515
537
1359
834
550
714
-------
1165
paint and airborne lead are sources, and that children living in deterio-
rated housing are in fact being exposed to both sources.
This method of dentine lead analysis now provides a means to conduct
large scale retrospective cohort studies in older children considered
asymptomatic for lead. If sensitive measures of a broad band of neuro-
psychological functions are then employed, and other important variables
related to development are scaled and treated by statistical techniques
appropriate and powerful enough to segregate the effect of lead, the
long debated question of low level lead effects should not prove perma-
nently refractory to inquiry.
References
1. Burde, Brigitte de la, and Choate, M. S. Does asymptomatic lead
exposure in children have latent sequelae? J. Pediat. 81;1088-1091,
1972.
2. David, 0., Clark, J., and Voeller, K. Lead and hyperactivity. Lancet
2: : 900-903, 1972.
3. Kotok, D. Development of children with elevated blood lead levels: A
controlled study. J. Pediat. 80;57, 1972.
4. Albert, R. E., Shore, R. E., Sayers, A. J., et al. Presented at NIEHS-
EPA Meeting on Low Level Lead Exposure, North Carolina, 1972.
5. Lansdownc, R. G., Clayton, B. F., Graham, P. S., et al. Blood lead
levels, behavior, and intelligence. The Lancet (London) 1_» 538, 1974.
6. Altshuler, L. F., Halah, D. B., Landing, B.: Deciduous teeth as an
index of body burden of lead. J. Pediat. 60;224, 1962.
7. Needleman, H. L., Tuncay, O. C., and Shapiro, I. M. Lead levels in
deciduous teeth of urban and suburban American children. Nature 235;
111, 1972.
8. Carroll, K. G., Needleman, H. L., Tuncay, O. C., and Shapiro, I. M.:
The distribution of lead in human deciduous teeth. Experientia 28;
434, 1972.
9. Shapiro, I. M., Needleman, H. L., and Tuncay, O. C. The lead content
of human deciduous and permanent teeth. Environmental Research 5:
467-470, 1972.
10. Needleman, H. L,, and Shapiro, I. M. Lead in deciduous teeth: A
marker of exposure in heretofore asymptomatic children. Int. Symp.
Environmental Health Aspects of Lead (D. Barth, et al, Eds.).
-------
1166
11. Strehlow, C. D. The use of deciduous teeth as indicators of lead
exposure. Unpublished doctoral dissertation, N.Y.U., 1972.
12. Shapiro, I. M., Dobkin,'B., Tuncay, O. C., and Needleman, H. L.
Lead levels in dentine and circumpulpal dentine of normal and lead
poisoned children. Clin. Chim. Acta 46;119-123, 1973.
13. Lin-fu, J. S. Undue absorption of lead among children - A new look
at an old problem. N.E.J.M. 286;702-710, 1972.
14. Mclntyre, M., and Angle, C. Air lead: Relation of lead in blood of
Black school children deficient in glucose-6-phosphate dehydrogenase.
Science 177:520, 1972.
15. Weekly Report, Center for Disease Control. Human Lead Absorption-Texas
Vol. 22, P405, 1973.
16. Lead: Airborne Lead in Perspective. National Academy of Sciences,
National Research Council. Washington, D.C. MAS, 1972.
(Study supported in part by a grant (DE-0262J) from the National
Institute of Dental Research).
DISCUSSION
GUINEE (U.S.A.)
You used the phrases "range of chemical lead poisoning"
and levels "associated with toxicity".
Are you implying that the 2O% of the children in this range.
all had some type of clinical symptoms?
NEEDLEMAN (U.S.A.)
My exact language was that "nineteen percent of the black
students ... had dentine levels in the range associated with
frank lead poisoning". We have tested 16 subjects who had been
treated for lead poisoning. The lowest dentine level was
292/ig/g; the highest was over 800,ug/g. Nineteen percent of
the'black subjects had dentine levels within that range. Some
of these "asymptomatic" children had levels greater than
800/ug/g. Eight percent of the white students also had dentine
levels in that range.
-------
1167
ALBERT (U.S.A.)
Were blood leads determined in children who had elevated
dentine lead levels and was there a correlation?
NEEDLEMAN (U.S.A.)
While there is little relationship to blood lead level at
age 8 (about two years after tooth shedding), in another study
a close relationship between blood lead level at the time of
shedding was found.
ALESSIO (Italy)
I should like to know if in the cases he has examined the
author has looked for a correlation between dentine lead con-
centration and levels of lead in the urine after chelate drugs
have been administered.
NEEDLEMAN (U.S.A.)
I am sorry but the urinary data was not available to us
though examination of hospital charts.
CARNOW (U.S.A.)
Did you examine deciduous teeth pulled out earlier than 6
years and was there any age related difference?
Comment; There was a third El Paso study - the first one carried
out by our group with the city of El Paso. Our findings paralled
those of the CDC and included anaemia, symptoms suggesting neuro-
physiologic dysfunction and so on. The statement to a previous
speaker regarding the need for removing lead from food "since
studies show no damage" must be carefully examined and such
judgments made on the basis of the total literature and not on
any single study - particularly one of variance with others which
examined the same population.
NEEDLEMAN (U.S.A.)
We did have an opportunity to mesure lead levels in some of
these teeth and could find no age related difference.
-------
1168
ZIELHUIS (Netherlands)
What is the effect of caries on Pb in tooth? What is the
intraindividual variability of Pb in different teeth in the same
child?
What is the timelag between : Pb in blood - > Pb in teeth?
NEEDLEMAN (U.S.A.)
We scaled dental caries and could find no relationship to
lead concentration. We also studied different teeth from the
same mouth and found general agreement as to lead concentration
between teeth. The critical factor appears to be the accuracy
with which the slice is taken from the central plane. I have
no data on the time relationship between blood and tooth lead
levels.
KAMINSKI (U.S.A.)
It is true that teeth, as any other calcified organ in the
body can act as a storage area for lead, but I do not believe
that one can distinguish by this method whether the elevation
of the lead content in the deciduous teeth was the result of a
continuous low level exposure to lead or as a result of infre-
quent but massive exposure to lead such as may occur in Pica.
Were there any air lead determinations made to show the
correlation to which you have referred to, that is automobile
traffic or industrial sources of lead.
NEEDLEMAN (U.S.A.)
I agree that our analysis does not discriminate between
acute high level exposure, and chronic lower level exposure.
We are working on that problem. We did not have air lead deter-
minations but the small number of dust lead determinations
(table III) shows levels as high in the western part of District
5 as those taken adjacent of the paint factory. These suggest
to me at least that another source is bringing the lead levels
in the western half to meet those near the stationary source.
The automobile and truck traffic in the western half is extremely
heavy.
McCABE (U.S.A.)
You have reported an interesting tool, i.e., tooth lead
analysis, that was used to survey a high-risk p6pulation of
children who were exposed to excess amounts of lead from their
dwelling units or from an industrial source.
-------
1169
I would like to know however, your rational for relating
any possible deliterious effect in these children to their meas-
ured levels of tooth lead.
NEEDLEMAN (U.S.A.)
We have presented prevalence exposure data today based
upon dentive lead concentration. Having identified these high
lead children, one can now measure the relationship of exposure
to deficit by traditional epidemiological techniques. These
would be essentially similar to studies employing blood lead
levels, but the advantage of using dental tissue is that one
can study older children and still have a measure of their
earlier exposure during critical periods of brain development.
-------
1171
LEAD ETHANOL AND S-AMINOLAEVULINIC ACID DEHYDRATASE
MICHAEL R, MOORE
University of Glasgow, Department of Materia Medica, Glasgow,
United Kingdom
ABSTRACT
Delta-aminolaevulinic acid (ALA) dehydratase activity is
depressed by lead and by ethanol both "in vivo" in man and ani-
mals and "in vitro". When erythrooyte ALA dehydratase activity
was measured in normal subjects and compared with blood lead
values, a highly significant negative exponential regression
curve was obtained. It was found, however, that in a group of
alcoholics* in whom blood ethanol levels wqre not measurable,
there was no relationship between erythrocyte ALA dehydratase
activity and blood lead levels, even when paired for ALA dehy-
dratase activities with the control values.
In rats, the combination of lead and ethanol administration
gave erythrocyte ALA dehydratase activities significantly greater
than the separate depressed levels of activity but significantly
lower than the control activity, "in vitro" experiments on rat
liver gave similar results.
It is suggested that these results are linked through the
cofactor reduced Glut at hi one which potentiates ALA dehydratase
activity and is depressed during lead exposure yet elevated by
ethanol administration.
-------
1172
1 . Introduction It has long been accepted that lead has a direct effect upon
Haem biosynthesis (Goldberg, 1972). In particular lead has been shown, both by
direct studies of activity and also by measurement of excreted urinary S amino-
laevulinic acid (ALA), to markedly inhibit ALA dehydratase (EC, 4,2,1, 24) the
second enzyme in the haem biosynthetlc pathway. Similarly acute ethanol
intoxication depresses the activity of ALA dehydratase. When normal subjects
consumed whisky, blood ALA dehydratase activity was depressed as blood ethanol
concentrations rose to a maximum and rose 'pari-passu' with depression of blood
ethanol levels (Moore et al, 1971). In chronic alcoholics blood ALA.
dehydrotase activity is also depressed even when blood ethanol concentrations are
zero, normal activity only being regained after about one week's abstention from
ethanol (Krasner et al, 1974).
The present studies have been designed to examine the effects of these rwo
factors on ALA dehydratase activity separately and together.
2. Materials and Methods (i) Human Studies These were carried out on a
group of alcoholics attending an outpatient clinic at Stobhill Hospital. None
were receiving any medication for their condition or for associated conditions.
(ii) Animal Studies were carried out on male Sprague Dawley rats weighing
200g. fed with diet 41, to which had been added 1 per cent lead acetate, for five
weeks. At the end of this time, two hours prior to death by cervical dislocation,
half of this group were injected with 2ml 30-per cent ethanol/soline solution;
the other half being injected with an isocaloric glucose/saline solution. In
addition, a second group of 12 animals on diet 41 without added lead acetate
were separated into two groups and injected in a similar manner. After death,
blood was collected from the carotid artery in heparinised bottles and the liver
was excised.
In all experiments blood ALA dehydratase activity was measured by the CEC
Standardised Method (1973) and tissue ALA dehydratase by the method of Moore
et al, 1971, the results being expressed in both cases as nmol ALA/mtn/mlRBC
or /g wet weight rat liver (Units ALA D). Lead levels were measured by
Graphite Furnace Atomic Absorption spectrophotometry and blood ethanol values
by gas liquid chromatography.
-------
1173
Results are expressed as Mean - Standard Deviation.
3. Results (i) Human Studies A group of 88 alcoholic patients was examined
together with a group of 22 normal volunteers. All gave blood samples with
informed consent. Blood ALA dehydratase activity, lead and ethanol values
were measured in all patients (Table 1). In all cases blood ethanol levels were
negative. In the control group, a highly significant regression line was obtained
with equation:- __, -0.582 PbB.
ALA.D = 57.2e
In the alcoholic group, there was no correlation between blood lead and ALA
dehydratase although both mean lead levels were higher and mean ALA dehydratase
activities lower (Table I). When alcoholics with greater than the lower limit of
normal for ALA D activity (25 units) were compared with the normal group, there
was no significant difference in mean ALA dehydratase activity or in mean blood
lead levels. There was however no significant correlation between these values
in the alcoholics.
TABLE 1 ALA DEHYDRATASE AND LEAD LEVELS IN ALCOHOLICS
Normals
Alcoholics
Alcoholics
(ALA.D>25)
Number
of
Patients
22
88
23
Erythrocyte
ALA.D
Activity (Units)
32.6
18.2
28.3
+
+
+
9
12
6
.1
.6*
.6
Exponential regression
Blood Lead coefficient (r)
(ug/lOOml) (ALA.D v PbB)
21
29
21
.3*
.6±
.5±
5
11
6
.2
.6*
.4
-0
-0
-0
.582
.060
.240
* P < 0.01 with respect to normal values
(ii) Animal Studies In the 'in vivo1 studies significant depressions of
erythrocyte ALA dehydratase were observed at mean blood lead concentrations
of 132 - 38 yg/lOOml whole blood and at mean blood ethanol concentrations of
194 - 41 mg/lOOml whole blood. When these were combined at similar
concentrations of 123 - 60 ug leod/lOOml whole blood and 189-34 mg ethanol
per 100 ml whole blood/ the activity of erythrocyte ALA dehydratase was
significantly elevated over the two separate depressed activities though lower
than the initial control value (Table 11).
-------
1174
TABLE 11 THE EFFECT OF LEAD AND ETHANOL ON RAT ERYTHROCYTE
ALA DEHYDRATASE
n
Group 1 ,
(Control) U
Group 2
(Ethanol) '°
Group 3 .
(Lead) '°
Group 4
(Lead + 10
Ethanol)
Blood Blood Erythrocyte ALA
Rhanol Lead Dehydratase
(mg/lOOml) (ua/100ml) (Units)
0 10.4-!" 8.3 7.26 - 3.25
194 - 41 12. 8^ 7.9 5.13 - 3.35*
0 132 * 38* 4.97 - 3.49*
189 - 34 123 - 60* 6.41 - 3.55+
Significance with respect to control:- * P< 0.001
+ P<0.05
In a subsequent experiment in vitro, with rat liver as a source of the enzyme,
it was found that ALA dehydratase activity was again depressed by both ethanol
and lead separately but elevated by these compounds together (Figure 1), although
still lower than the control value, at all times,
4. Discussion In these experiments it has been shown firstly that ALA
dehydratase activity normally a good bioanalytical measure of lead exposure,
ceases to be so in alcoholics. In addition, it has been shown that depressions of
ALA dehydratase by both lead and ethanol separately are partially reversed when
these are combined both 'in vitro' and "in vivo'. The linking factor in these
experiments seems to be the levels of reduced glutathione (GSH) in the system.
Lead is known to bind to sulphydryl groups and other workers have shown that
addition of GSH to systems measuring ALA dehydratase minimises the depression of
activity due to lead (De Barriero, 1969). Conversely, although GSH is a cofactor
for ALA dehydratase, excessive concentration of GSH lowers the activity of ALA
dehydratase (Moore et al, 1971). Ethanol oxidation substantially alters the redox
potential of the cell, and in doing so, alters the ratio of reduced to oxidised
glutathione (GSH/GSSG) in the cell. It is therefore suggested that although lead
depresses GSH levels and thus lowers ALA dehydratase activity, and ethanol raises
GSH levels and lowers activities, these two in conjunction, act in opposite
directions and lead to an effective maintenance of activity. (Fig. 2)
-------
1175
Ficmre 1 • THE EFFECT OF ETHANOL ON RAT HEPATIC ALA DEHYDRATASE
y ' ACTIVITY 'IN VITRO1 IN THE PRESENCE OF A FIXED
CONCENTRATION OF LEAD
HEPATIC ALA.D
ACTIVITY (UoHs)
40 —
35 —
NO LEAD
LEAD 1(T6 M
ETHANOL (mg/lOOml)
Figure 2 : Inter-related effets of lead and ethanol on
GSH and ALA.D activities.
MSXoA
* AMINOLAEVULINIC ACID
fOWHOI LINOGEN
HAEM
ACE1YL CoA
-------
1176
It is suggested therefore that in the use of ALA dehydratase as a bioanalytical
measure of lead exposure or as a measure of ethanol consumption in alcoholism,
cognisance be taken of the effects of ethanol and lead respectively.
5. References
(1) De Barriero, O.C., Effect of cysteine on 5-aminolaevulinate
hydrolase from liver in two cases of experimental intoxication.
Biochem.Pharmacol. ]Q_, 2267 (1969)
(2) Goldberg, A., Lead Poisoning «. Haem biosynthesis.
Brit.J.Haematol. 23,521 (1972)
(3) Krasner, N,, Moore, M.R., Thompson, G.G., Mclntosh, W. and
Goldberg, A., Depression of erythrocyte Saminolaevulinic
acid dehydratase activity in alcoholics.. Clin.Sci. and Molec .Med.
46, 415 (1974)
(4) Moore, M.R., Beattie, A.D., Thompson, G.G., Goldberg, A.,
Depression of S aminolaevulic acid dehydrase activity by ethanol
in man and rat. Clm.Sci. 40,81 (1971)
-------
1177
THE USE OF TRACER TECHNIQUES AND ENVIRONMENTAL
SOURCES FOR EVALUATION OF THE LEAD PROBLEM IN CHILDREN
GARY TER HAAR+ AND REGIME ARONOW++
+ Ethyl Corporation Research Laboratories, Detroit, Michigan,
USA
•f+ Children's Hospital of Michigan, Detroit, Michigan, USA
ABSTRACT
Many literature references cite the fact that, especially
in the United States, children living in deteriorated housing
have a problem with lead poisoning. Eating leaded paint has
been recognized as the prime cause of elevated blood leads.
Recently, there has been speculation, originally by the U.S.EPA,
that children may eat dust containing lead exhausted from auto-
mobiles and that this may contribute significantly to the child-
hood lead problem. This paper compares lead in paint on houses
with lead from automobiles as a source of lead in soil and pro-
poses a new tracer technique to evaluate whether children eat
dust and air-suspended particulate.
To determine the lead source in soilt we analyzed lead in
soil around old painted houses in the city and in rural areas
remote from traffic. The concentration and distribution of lead
around the city and rural houses was nearly identical, with the
highest concentrations near the houses. Thus, it is clear that
lead in the soil was due to paint and that lead antiknocks were
not a significant contributor.
To estimate the ingestion of dust and airborne particulate,
a naturally occurring radioactive tracer (lead-210) was used to
-------
1178
determine the relative amounts of dust and other lead-containing
materials* such as paint, eaten by the children. Analysis of
paint samples showed very low levels of lead-210, while dust
samples contained at least 100 times as much lead 210. Stable
lead and lead-210 were measured in the excreta of children sus-
pected of having elevated lead body burdens and of children li-
ving in good housing where lead poisoning is not a problem.
The "high lead" children excreted much more stable lead than the
"normal" children. Despite the differences in stable lead
excretion between the groups* excretion of lead-210 was essenti-
ally the same. These results do not support the hypothesis
that these children eat dust and air-suspended particulate.
-------
1179
1. Introduction
Lead poisoning in children, especially those under k years old, is
prevalent in areas of our larger cities where housing has deteriorated.
Historically, almost all cases of lead poisoning in children have been
attributed to eating paint chips with a high lead content /1,27-
Recently, Fine et al £%/, in a study in Illinois, found that elevated
blood lead was also common in smaller cities. In their study, they
screened children from poor housing areas and concluded that the lead
problem is not confined to large cities. The common factor between the
large and small cities was deteriorated housing.
Recently, it has been speculated that soil, dust, and air-suspended
particulates may also contribute to the lead problem in children fi\]•
This paper considers two aspects of the childhood lead problem:
1. The major sources of lead in soil around houses where children
play.
2. The extent to which children ingest dust and air-suspended
particulates.
2. Lead in Soil
Past studies have shown that deteriorating paint is an important
source of elevated lead in soil. Hardy et a^ [*J', have reported on anal-
ysis of lead in soil near a barn remote from traffic in rural Lincoln,
Massachusetts. Soil next to the barn contained 2000 ppm of lead, and the
level was 160 ppm 20 feet from the barn. Bertinuson and Clark /&J, con-
cluded that urban housing appears to be a larger contributor to elevated
lead in soil than emissions from vehicular exhaust. Fairey and Gray /"£/,
found high concentrations of lead, in soil in yards, with the highest
concentrations generally near the houses. They attributed this lead to
paint and ashes.
In our study, we sampled soil at 9 sites around each of 18 frame
houses in widely scattered urban areas of Detroit. These areas are char-
acterized by old houses that had been painted with lead-based paint,
presumably for many years. Analysis confirmed that aJLl houses were coated
with paint containing lead. Similarly, soil samples were taken at 9 sites
around each of 18 houses of brick construction. In «.n cases, these
houses had painted trim. For each urban house, surface samples were taken
at the following locations:
-------
1180
Dirt in the street gutter.
Soil between the sidewalk and curb adjacent to the curb.
Soil two feet toward the house from the front sidewalk.
Soil on each of the four sides of the house within two feet of the
house.
Soil ten feet from the house in the front and back yards.
We also sampled soil around 7 farmhouses in an area remote from
traffic located about 30 miles from the nearest city and about 50 miles
north of Detroit. Samples were taken from the surface in the same manner
as used for the urban samples. Table I is a summary of the data from this
survey.
Table I
Lead in Soil
Lead in Soil, ug Pb/g dry soil
Sampling Within 2 Feet
Location of House
Urban Frame
Urban Brick
Rural Frame
Barn*
2010
468
2529
2000
10 Feet
from House
1*36
178
609
570
Near
Sidewalk Curb
627 572
32^ 612
-
-
Gutter
966
1213
-
-
* From Hardy et al
Lead in soil within 2 feet of the urban frame houses averaged 2010
ppm, with no obvious bias for front, sides, or back. Lead in soil in the
middle of the yards averaged h% ppm, and again there was no bias toward
front or back. The distribution around the brick houses was similar, but
the lead levels were lower. The average concentration within 2 feet of
the house was U68 ppm, with 156 ppm in the front yard and 200 ppm in the
back yard. As with the frame houses, there was no evidence of higher
concentrations in the front yard compared to the back. The data for lead
in soil around the rural farmhouses are very similar to those for the
urban frame houses. The average lead concentration was 2529 ppm within
2 feet of the farmhouses and 609 ppm at 10 feet.
The comparison indicates that most of the lead in soil is due to
paint, baaed on the following reasoning. The lead in soil within 2 feet
of the frame houses in the city averages just over 2000 ppm. The lead in
soil 10 feet from these houses averages over kOO ppm and is similar in the
-------
1181
front and back yard. If vehicular traffic were a significant source of
lead, the front yard would contain more lead than the back. Since these
data at 2 feet and 10 feet from the house are similar to our data from
frame houses in rural areas and to the data of Hardy et al f^J} it is
clear that traffic is not contributing significantly to lead in the soil
in the yards of the painted frame houses.
This conclusion is supported by the data on lead in soil around the
brick houses- The lead in soil within 2 feet of these brick houses is
more than double that at 10 feet, indicating that the painted trim of the
house is the prime source. As expected, the much smaller painted surfaces
of the brick houses result in much lower lead concentrations near these
houses than those near the painted frame houses. As with the painted
frame houses, the concentrations 10 feet from the houses are similar in
the front and back yards. Here again, it is evident that traffic does
not have a significant effect. The lead in the street gutter was similar
for both brick and frame houses. Thus, all evidence points to paint as
the prime source of elevated lead in the yards, where the children would
be most likely to play,
3. Lead in Dust
The data from the first part of the study show that paint is the
major source of lead in soil around the houses where children usually
play. The role of lead in air-suspended particles and in fallout dust
must be considered separately, since lead in gasoline significantly
contributes to the lead content of air-suspended particles and fallout
dust.
To distinguish between the leaded paint a child might eat and the
contribution of lead he might receive from eating dust, it is necessary to
find a material that is present in dust but not in paint.
Lead-210, a naturally occurring radioactive isotope of lead, is a
useful tracer for this purpose. It is generated from radon, which is
present in the soil. Part of the radon escapes to the air and part stays
in the soil. The radon disintegrates with a half life of 3 days to prod-
uce lead-210, which has a half life of 22 years f&J. Because it is
present in the atmosphere, fallout dust is enriched in lead-210, while
paint has very low concentrations of lead-210. The topmost layer of soil
is depleted as the radon escapes, but rain brings the lead-210 back to
earth where the soil's topmost layer retains the isotope. Thus, the
concentration of the isotope is relatively constant with depth.
-------
1182
We found concentrations of lead-210 and stable lead in nonfood mate-
rials as shown in Table II. These results show clearly that ingestion of
paint will add little lead-210 to the daily burden, while ingestion of
dust and soil can quite readily be detected.
Table II
Lead and Lead- 210 in Nonfood Materials
(Range of Values)
Stable Lead,
Percent
1-12
1.7-3-T
0.10-0.19
0.01-0.50
0.03-0.30
Lead- 210,
pCi/g
0.005-0.07
100-600
3-39
0.2-1.3
0.7-4.6
Ratio,
pCi Lead- SlO/fJS Stable Lead
k x KT8 - 7 x 10-6
3 x 10~5 , k x 10~2
2 x 10-5 _ 4 x io-2
h x 10~5 _ i x 10-2
2 x 10"^ - 2 x ID'2
Paint Chips
Urban Fallout
Particulate
Fallout Dust
Yard Soil
Street Dirt
The concept was to use lead-210 as a tracer to determine the amount
of dust and perhaps the amount of soil eaten daily by a child. As lead
and lead-210 are absorbed poorly in the gut, an estimate of the lead and
lead-210 ingested can be made from analyses of fecal matter.
If a child has a high level of stable lead in his fecal matter and a
normal level of lead-210, we would conclude that the lead elevation is a
result of eating paint. However, if both the stable lead and lead-210
are high in the fecal matter, we would conclude that dust and soil are
contributors in addition to paint.
At Children's Hospital of Michigan, we collected urine and fecal
samples from children who were suspected of having elevated body burdens
of lead. The evidence used was one or all of the following:
1. X-ray showed radio opaque materials in the gut.
2. History of pica.
3. Elevated blood lead.
4. X-ray showed lead lines in the long bones.
Fecal and urine samples were taken from eight such children. These
children were one to three years old and all had exhibited pica tend-
encies. All stool and urine were collected separately during the first
24 hours after admission to the hospital to insure samples representative
of the child's usual environment, not that of the hospital. To provide
-------
1183
a baseline, combined stool and urine samples were taken from 10 children
of the same age level (l to 3 years) who lived in good housing in Detroit
and its suburbs where lead poisoning is not a problem. All samples were
collected during the late spring and early summer months. These samples
were analyzed for stable lead and lead-210.
Table III shows the lead and lead-210 data for the normal children
and the "high lead" children. The normal children averaged h micrograms
lead per gram dry feces, with a range of 2 to 7- Of the eight children
suspected of having elevated lead body burdens, two had fecal lead values
(k and 7 micrograms lead) within the normal range. However, the remaining
six were 1* to 400 times higher. Despite these differences in stable lead
excretion between the two groups, the groups were essentially identical
in the lead-210 content of their feces. The "high lead" children averaged
0.040 picocurie lead-210 per gram dry feces, while the normal children
averaged 0.044.
Table III
Lead and Lead- 210 in Excreta
Normal
Stable Lead,
ug/g dry
3
2
3
7
7
3
3
5
k
k
Children
Lead- 210,
pCi/g dry
0.019
0.021
0.027
0.120
0.087
O.OUl
0.026
0.028
0.044
0.024
Avg. 0. 044
Hospitalized Children
Stable Lead,
ug/K dry
19
20
18
49
4
7
to
1640
Avg.
Lead- 210,
pCi/g dry
0.046
0.018
0.021*
0.047
0.050
0.039
0.063
0.037
0.040
Statistical examination of the lead- 210 data show that they are log
normally distributed and that there is no statistical difference in the
concentration of lead-210 between the two groups. The results of this
experiment do not support the hypothesis that these young "high lead"
children eat dust.
-------
1184
An examination on the ratio of lea.d-210 to stable lead in the diet of
these children clearly shows the difference between the normal and "high lead"
children. As a baseline, the ratio in food irs about 5 x 10" to 20 x 10
pCi lead-210y^if!; lead. For the normal children, the ratio varied from 6 x 10
to 17 x 10 . For those children who were clearly intestine lead, the ratio
varied from 2 x 10 to 2 x 10 . This ratio of 10 is similar to that for
a paint containing 5 % lead and 0.05 pCi lead-210/g paint if one takes into
account that the child received some lead-210 in his food. The lead-210
data for these 18 children can be related to the amount of lead-210 that is
normally present in the diet. The normal children in this study excreted
an average of 0.67 picocurle of lead-210 per day in 15 g excreta (dry
weight). This value agrees very well with an estimation based on lead-210
data of Morse and Welford /§/ for adults. They found that adults ingested
about l.l). picocuries of lead-210 per day. Using literature estimates that
a child consumes about half the food of an adult /IO, Il7, an intake of 0.7
picocurie of lead-210 per day would be expected for a child.
4. Summary
This report has described the results of a two-part study to determine
whether lead emitted from motor vehicles contributes to the lead problem
in small children. In the first part, we determined lead in soil around
houses in urban areas and rural areas. The data from the urban areas
clearly show that the principal cause of elevated lead in the soil in the
yards is leaded paint on these houses. These data were confirmed by meas-
urements of lead in soil around farmhouses, which showed lead in soil
concentrations as high as any that have been reported in urban soil.
In the second part of the study, we determined whether children ingest
measurable amounts of particulate or dustfall. We used a naturally occur-
ring tracer, lead-210, which is present in relatively large amounts in
dust but nearly absent from paint. The results showed that these children
with pica (and other evidence of high lead intake) and normal children
excreted identical amounts of lead-210. Examination of the ratio of lead-
210 to stable lead in the feces and in the possible sources indicates that
the source of lead in these children was paint. Consequently, dust and
air-suspended particulate were not shown to be sources of lead in these
urban children.
-------
1185
APPEHDEC
Study Techniques
Soil Samples
Soil samples were collected by taking the topmost layer of soil. All
soil samples were dried at 100°C overnight. Lead was extracted with hot
dilute nitric acid and determined by atomic absorption.
Biological Samples
The samples from the normal children were collected in acid-washed
plastic containers for 2k hours by the mother.
Urine and fecal samples from children admitted to Children's Hospital
were collected separately in lead-free containers during the first 2k hours
after admission.
All fecal and urine samples were weighted and dried at 100°C. The dry
weight was recorded and the sample was taken into solution with nitric and
perchloric acid. The lead was taken into methylisobutyl ketone and anal-
yzed by atomic absorption.
Lead-210 Analysis
Only a small portion of the methylisobutyl ketone-lead solution was
used to determine lead. The remainder was oxidized with nitric acid.
After fuming three times with a few ml of HC1, the lead-210 was determined
by the method of Black /12/.
-------
1186
REFERENCES
1. CHISOLM, J. J., Jr., "Lead poisoning/1 Scientific American, 22h, 15-23
(1971).
2. CHISOLM, J. J., Jr., "Childhood lead intoxication, diagnosis, manage-
ment and prevention," Medical Times, ^8, 92-106 (1970).
3. FINE, P. R., THOMAS, C. W., SUHB, R. H., COKNBERG, R. E., and FLASHNER,
B. A., "Pediatric blood lead levels," JAMA, 221, 1^75-79 (1972).
k. EPA's Position on the Health Effects of Airborne Lead, Prepared by
Health Effects Branch, Processes and Effects Division, Office of
Research and Monitoring, U.S. Environmental Protection Agency,
Washington, B.C., November 29, 1972.
5. HARDY, H. L., CHAMBERLIN, R. L., MALOOF, C. C., BOYLEN, G. W., Jr.,
and HOWELL, M. C., "Lead as an environmental poison," Clinical Phar-
macology and Therapeutics, 12, 982-1002 (I97l).
6. BERTINUSON, J. R. and CLARK, C. S., "The contribution to lead content
of soils from urban housing," Interface, 6, 1073 (1973).
T. FAIREY, F. S. and GRAY, J. W., Ill, "Soil lead and pediatric lead
poisoning in Charleston, S.C.," The Journal of the South Carolina
Medical Association, 79-82 (l970Ti
8. PATTERSON, R. L. and LOCKHART, L. B., Jr., The Natural Radiation
Environment, Edited by J. Adams and W. Louden, The University of
Chicago Press, 383-392 (196*0.
9. MORSE, R. S. and WELFORD. G. A., "Dietary intake of 210Pb," Health
Physics, 21, 53-55 (1971%
10. KING, B. C., "Maximum daily intake of lead without excessive body lead-
burden in children," Amer. J. Pis. Child. 122, 337-3^0 (l97l).
11. BARLTROP, D., "Sources and significance of environmental lead for
children," International Symposium, Environmental Health Aspects of
Lead, Amsterdam, October 2-6, 1972.
12. BLACK, S. C., "Low-level polonium and radiolead analysis," Health
Physics, J, 87-91 (1961).
-------
VERZEICHNIS
INDEX
INDEX
INDICE
INDEX
-------
LIT
AASETH, J., 913
ADLER, M.W., 77
ALBERT, R., 1167, 2061, 2068,
2069
ALESSIO, L., 1123, 1129, 113O,
1167
ALLART-DEMUL, C. , 13O3
ALLEGRINI, M. , 1697
ALLEN, J.R., 385, 397
ALTMAN, D.G., 289
ALTSHULER, B., 2061
ALTSHOLLER, A.P., 21O9
ANDERSON, J., 1449, 1461, 1468,
1469
ANGERER, J., 1317, 1327, 1328
ARHIRII, M., 339
ARONOW, R., 1177
ARSAC, P., 603
AUBERT, M., 1613
BABCOCK, L.R., 2083
BACKHAUS, F., 2231
BAKER, F.D., 879
BARHAD, B., 333
BARQUET, A., 695
BARRATT, R.S., 1397, 1779
BARSAN, E.T., 1O73
EARTH, D.S., 1875, 1877, 1921,
1922, 1925,~19l6, 1928, 1930,
1931, 1933, 1934, 1935, 1939,
1940, 1950, 1959, 1960, 1962,
1963, 2073,
BASTENIER, H., 1303
BATES, D.V., 1007, 1967, 1978,
1979, 2001, 2003,~2T5~2
BATTI, R., 1531
BEACONSFIELD, P., 2397
BECK, E.G., 1031, 1O40, 1041
BEITZ, L., 1417, 1430
BELCHER, R., 1779
BELL, A., 1101
BENARIO, M., 2169
BENINSON, D., 845, 1878, 1924,
1927, 1934, 1935, 1941, 1949,
2297
BENSON, F.B., 423
BERGLUND, B., 119
BERGLUND, 0., 119, 142
BERLIN, A., 552, 611, 629, 693
859, 1087, 1100, 1238, 1959,
2003, 2185, 2254, 2257
BERLIN, M., 156, 491, 895, 1259
BERNA, M., 231
BERNER, A., 1729
BERNSTEIN, A.D., 1O5, 116, 117
1029, 2298 '
BERNSTEIN, D., 431
BIANCO, A., 1039
BIERSTEKER, K., 1881, 1924. 192i
1933, 1934, 1943, 1957, 1958.
1959 '
BIGNON, J., 1189, 1196, 1197
BINDER, R.E., 669
BITTEL, R., 714, 1441, 1449.
1469
BLACKBURN, C.R.B., 63
De BOECK, R., 1131
Den BOER, M.C., 1247
BOGDANOVIC, E., 2271
BONNAUD, G., 1189
BONNEFOUS, M., 51O
Underlined numerals denote authors
Arabic numerals denote participants in discussions
-------
LIII
BORDAS, E., 1145
del BORGHI, M.f 18O7
BORLAUG, N., 2397
de BORTOLI, M., 1287
BOTZENHART, K., 1757
BOUDENE, C., 6O3, 612
le BOUFFANT, L., 1645, 1651
BOUHUYS, A., 669, 675
BOUQUIAUX, J., 1239, 1298
BOURBON, P., 269
CACCURI, S., 1823
CAGNETTI, P., 1451
CALANDRA, J.C., 772
CANTON, J.H., 1479, 1489
CAPURRO, P.U., 1579
CARNOW, B.W., 45, 313, 353, 266,
368, 588, 1129, TT67
CARPENTER, L., 1729
CARPI di RISMINI, A., 2397
/^ADOfimxTO T » -> n F-
null****.., r., £« CARSTENS, L.A. , 385
BOURDEAU, Ph., 263 363, 366, "«<"—™ - «"
51O
BOUVILLE, A., 1531
BRADEN, M., 485
BRAETTER, P., 2255, 2301
•»•*» mxm»» T^ ** •* -* •* ICQ
1954
CARSTENSEN, J., 969
CARTER, M.H., 1399
del CASTILHO, P., 2185
CASULA, D., 1693
wwusj.-j.fiK, *-., ^33, *juj. CERNIK, A.A., 1207, 1221, 1237,
BRAMAN, R.S., 117, 258, 1328, 2254
1363, 137O, 1397, 1954 CERQUIGLINI-MONTERIOLO, S., 13O1
BRAVO A, H., 468, 2091, 2160, 1383
2161 CHAMBERS, P., 1O4, 259, 397, 51O,
BREIDENBACH, A.W., 751 702' 2045
.. CHANTEUR, J. , 2386
CHAPMAN, R., 193, 2O7, 2O8, 21O,
645, 658
CHARLTON, J., 112O
CHATTOPADHYAY, A., 1685
/*V»T «*T T H %« *t it M v»
0nci.i4J&HDn\,np n«n* * t JX.
BRILLE, D., 269. 356, 667
BROCKHAUS, A., 781
BROMBERG, P.A., 1989
BROOKS, A.G.F., 77,
BRUAUX, P., 1131, 1143 ^ ^
BRUCH, J., 781, 791, 1O4O, 12O6 CICOLELLAf A ~^
de BRUIN, A., 259, 911, 2O59 CIGNA - ' ' ~
BUCKET, J.P., 631, 887, 2185 clAEYS.,KOPr,11Tt/i:,—7
BULCRAIG, W.R., 209
BURGER, E.J.jr., 145, 155,
156, 157
CLAEYS-THOREAU, P., 1131
CLAUDE, J.R., 397
CLAYTON, J.W.jr., 371, 383, 383
CLBMENTE, G.F., 260, 1028, 1451,
0USTUJSVA, K. , JLUUy
BUTLER, G.C., 1431, 1884, 193O nfM '
1937, 1948, 1955, 1956, 1978 COIN, L., 7O3
BUXTON, R. St.J., 1113 COLMAN, R., 485
BYRNE, A.R., 245, 258, 259, 26O,COLOMBINI' M- •
1370
BUSH, B., 879, 885, 911, 1339
BDSTUEVA, K. , 1O09
159
-------
LIV
COLUCCI, A.V., 1043
COOPER, W.C., 555, 568, 569,
1196
COTE, R.W., 47
CROCKER, K., 2O68
CROSSMANN, G., 14O7
CRUZ, R., 1685
COCU, M., 333
DAHL, R., 2231
DAMS, E., 513, 1430
DAMS, R., 4O9
DANIEL, H., 1645, 1651
DANIEL, J.W., 877, 902, 1041
DANIELSON, L., 116, 885
DAVID, 0., 588, 1549
DAVIDOW, B., 545
DAVIES, J.E., 695
DAVOUST, P., 2384
DEAN, G., 190, 643
DEHNEN, W., 781
DELEANU.M., 1583
DELCARTE, E., 1675
DELVES, H.T., 2215, 2258
DEMUYNCK, M., 409
DENNIS, C.A.R., 1O29, 1543,
1953
DEPAUS, R., 1341
VOn DEPKA, J., 506
DERWENT, R.G., 1669
DESBORDES. J., 1199
DEVOTO, G., 1693
DIEHL, J.F., 958, 1121
DIETERICH, B.H., 2451
DIETL, F., 1853
DI FERRANTE, E., 1956
DJURIC, D., 1829
DOBIN, D.D., 1223
DOBRYSZYCKA, W., 685
DOLGNER, R., 279
DONALDSON, W.T., 1399, 1405,
1406
DONNIER, B., 1613
DONZELLI, A., 231
DUBOIS, L., 1331
DUGANDZIC, M. 2285
DUMONT, M., 2231
DUNCAN, K.P., 2175
DUNCAN, L.J., 1241
DUPUIS, P.J., 491, 1196, 1314
EDWARDS. H.W., 1277, 1285, 1286
EFTHYMIOU, M.L., 1789
EGELS, W., 1407
EISENBUD, M.f 431
ENGLISH, T.D., 401, 47O, 471,
472
EPSTEIN, S.S., 552, 569, 749,
814, 1120, 2367, 2382, 2383,
2384, 2385, 2386
van ESCH, G.J., 1O17
ESPINOSA, M.E., 2O91
EVENDIJK, J.E., 1351.
PACCHETTI, S., 1287, 1298, 1299
FAGNIART, E., 1675
FAIRWEATHER, F.A., 1113
FALK, H.L., 2331, 2348, 2349,
2350
FAVRETTO, L. , 1511
FAVRETTO GABRIELLI, L., 1511
FERRAIOLO, E.G., 18O7
-------
LV
FERRARI-BRAVO, P., 1797
FINE, P.R., 1223
FINKLEA, J., 193, 645
FISCHER, A.B., 1O31
FISHBEIN, L., 725. 749
FLOREY, C.f 289
FONDIMARE, A., 1199, 12O6
FOURNIER, E., 1433, 1439, 1789
FREDERIKSON, M., 1959
FREEMAN, G., 685, 833, 844,
1007
FRENCH, J., 193, 645
FRIBERG, L., 23O7, 2315, 2316
FRIEDMANN, J., 5O7, 51O, 511
FRIEDRICHS, K.H., 715, 723,
724
FUMAROLA, G., 1807
FURIOSI, N.J., 833
GADDO, P.P., 1287
GAFFEY, W.R., 555
GAGE, J.C., 895
GAGLIONE, P., 1287
GARDI, R., 18O7
GARDNER, D.E., 705, 713, 714,
GARIBALDI, P., 1287
GARNIER, A., 1441
GENT, M., 1263
GHELBERG, N.W., 1145, 1583
GHETTI, P.F., 1957
GIANANI, G., 1697
GIBBS, G.W., 1197, 12O6, 2271,
2296, 2297, 23O1, 2302
GIBSON, R.J.W., 289
von GIERKE, H., 1249
GIOVANNINI , I., 1797
CLAUDE, P., 1341
GLOBUS, G., 5O7
GODIN, J., 603
GOERKE, W., 37
GOLDBERG, A.M., 793, 8O3
GOLDBERG, H., 193
GOLDSMITH, J.R., 62, 155, 189,
382, 585, 675, 1889, 1923,
1935, 1936, 1942, 1945, 195O,
1956, 2156, 2165, 2179,
GOLDSTEIN, I., 1275
GONO, E., 591
GOOTJIS, P., 1316, 1951
GRAB, B., 339
GRADISKI, D., 1631, 1661
de GRAEVE, J., 523
GRAOVAC-LEPOSAVIC, L. , 1829
GRASSO, C., 861, 878
GREENLAND, R.D., 805
GREVE, P.A., 1479
GRIECO, A., 231
GRIFFIN, H.E., 23O5
GROLL-KNAPP,E., 989
GROS, R., 2225
GRUENER, N., 1O67
GRUNSPAN, M.J., 773, 20O3
GUINEE, V.F., 545, 551, 552,
553, 1166
HAAG, A., 1317
HAASE, J., 1417
HADDAD, R., 451
HAGEDORN-GOETZ, H., 2231
HAIDER, M., 475, 989, 999, 2393
HARDWICK, D.F., 961
HARKE, H.P.,1327, 1773
HARRISON, P.R., 1741
HARRISON, R.M., 1111, 1285, 1349
1370, 1405, 1783
-------
LVI
HAYES, C., 645
HAZUCHA, M., 1979, 2OO1
HEBBELINCK, D., 1303
HEM, B.f 1189
HENDERSON, P.Th., 2047
BENIN, J.P., 1651
HERNBERG, S., 568, 692, 1129,
1142, 2395
HERTZ, M.B., 1763
RICKEY, N., 658
HILLERY, P.J., 7_
HILPERT, K., 2231
BINE, C.H., 207, 260, 104O,
1130, 1937, 2029, 2298
HINTON, D.O., 1769
HISLOP, J., 959
HO, M.T., 1519
HOFMAN, B., 1017
HOGGER, D., 1247, 2157
BOLL, K., 612
HOLLAND, W.W., 39' 45
HOLM, S., 895, 902
HOLMQVIST, I, 613, 629
HORIE, Y., 2143
HOSE IN, H.R., 669
HOUCK, C.L., 879
BOWER, J., 591, 6O1
HUETER, F.G., 261, 352, 363,
364
HUTCHINSON, T.C., 1685
HOTB, F., 715
HUDNAN-SEPPALA, A., 2263
IMPENS, R., 1675
IOVENITTI, L., 1797
IRWIG, L., 289, 360
IWANKIEWICZ, S., 685
IZMEROV, N.F., 2409
JACKSON, D.L., 161, 176, 177,
178, 1956
JACOBSEN, M., 89, 211, 228, 229,
1478, 1502, 2365
JACYSZYN, K., 685
JAMIN, P., 523
JANSEN, G., 513, 999, 2O44
JEANMAIRE, L., 2225, 2252, 2256
JERVIS, R.E., 1685
JOHNSON, D.L., 1363
JOOSTING, P.E., 76, 2O05, 2029
JOST, D., 2115
JUHOS, L., 833
KAMINSKI, E.J., 551, 803, 1168
KARCHER, W., 1341, 1349
KARHAUSEN, L., 367
O'KEEFFE, A.E., 21O9, 2164, 2166
2168, 2169, 2172
KEITZ, E.L., 1241, 1247, 1248
KELLER, M.D., 47
KEVANY, J., 89
KZLPIO, J.O., 2263
KIRCHMANN, R., 1675
KJELLSTR&M, T., 62, 1221, 1939,
2197, 2252, 2254, 2257, 2299,
5303, 2328, 2365, 2383
KIAHRE, P.* 2231
KLEINMAN, M., 431
KNAUTH, P., 2O31, 2043, 2044
KNEIP, T.J., 431, 466, 473
KNELSON, J.H. , 181, 189, 190,
361, 973
KOPPLE, J.D., 847
KORICANAC, Z., 2285
KOSTA, L., 245
KOTLAREK-HAUS, S., 685, 692, 693
-------
LVII
KRACKE, W., 1853 LLOYD, W. J. , 1085
KREBS, H., 2397 LO, Fa-Chun, 879
van der KREEK, F.W., 959, 1O28, Van LOON, J., 1685, 1849
1065, 2382 LORKE, D., 817, 83O, 831
KREUZER, W., 601, 1853, 2O45, ^^ ^—
RUMMER, J., 1303, 1314, 1315, LOVE' G-J" 1P1' 51^
1316, 1339 LOWE, A.C., 2O91
KUMPF M.J., 339, 363, 365, 1957 LYNAM, D.R., 543, 791
LACOURLY, G., 1441
LAFONTAINE, A., 382, 1131, 1942,
2437
LAMBO, T.A., 11
LANESE, R.R., 4_7
LANG, R., 659
LANGER, H., 1757
LANZOLA, E., 1697
LAO, R.C., 1331, 1339, 1340,
1468
LAUER, G., 423, 2156, 2163
LAUWERYS, R., 542, 568, 631,
831, 887, 1238, 2185
LEEDER, S.R., 63, 76, 1111
LEFEVRE, M.J., 988, 2O29
LEGRAND, H., 1131
LEHNERT, G., 1317
LEHTO, V.P., 1OO7, 2315
LELLOUCH, J., 269
LENGHEL, I., 1583
LEUNG, S., 1248
LEVERE, T.E., 493, SO6
LEVY, D., 1263, 1275, 1276
LEVY, E.A., 1797
LIND, B., 2197
LINDVALL, T., 119, 1954
LINNMAN, L., 2197
MAGADUR, J.L., 1631, 1661
MAGE, D., 176, 2O7, 1275, 2O68
2156, 2158, 2162, 2164, 2165,
2168, 2170, 2174, 2176, 2178,
2179, 2181, 2315, 2364
MAGE, D.T., 2O97
MAGI, F., 857
MAGNAVAL, R., 1531
MAHEU, R., 17
MALONE, D.H., 1569
MANOJLOVIC, N., 1O31
MARCUS, A.H., 15O5
MARQUARDT, H., 16O7
MARTIN, A.B., 1113, 112O, 1121,
1122, 2305
MARTIN, J., 973
MARTIN, J.C., 1645, 1651
MATERNE, D., 631
McCABE, E.B., 1168
MCDONALD, G.C., 1491
McGUIRE, J.M., 1399
MCNEIL, J.L., 571, 584, 586,
588, 589
McNESBY, J.R., 1371, 1383
MEININGER, J., 6O3
van MEIRHAEGE, A., 2O44
MENENTI, M., 1797
MERIAN, E., 929, 1396, 2175
-------
LVIII
MERLUZZI, F., 231
MICHAELSON, I.A., 805
MILIC, S.t 1829, 2285
MILOVANOVIC, Lf., 2285
MIRE, B.f 973
MITCHELL, C.A., 669*
MITCHELL, R.I., 47, 62
NORBACK, D.H., 385
NORSETH, T., 913
NOTTEN, W.R.F., 2O47, 2O59, 206O
NURNBERG, H.W., 2231
OLEKSYK, E., 685
UitCftbXK, £., POO
nx^nr,^, «.x., ^, o* OLOFFS, P.C., 7O2, 723, 750,
MOKEMATKENGUEMBA, G., 178, 961, 1065, 1953, 2350
1941, 1951 OMENN, G.S., 1563, 1952
OREL, J.V., 2177
ORIOL, P., 269
OTT, W.R., 2O97
OUW, K.H., 1101
MOLDOVAN, N., 1583
MOLLARET, P., 1938
MONCELON, B., 1661
MONCHAUX, G., 1189
MONKMAN, J.L., 1015, 1331
MOONEY, T.F., 1637
MOORE, M.R., 535, 1171, 2252
MOORE, W. , 751
MOREAU, M. , 1315
MORGADE, C., 695
MORGAN, G.B., 2073
MORRESI, N., 231
MORRIS, S.C., 677, 683
MOSE, J.R. , 1617
MOULE, Y., 967
MRAK, E.M. , 1965
MURAYAMA, H. , 91
MUSSENDEN, R., 833
NAGDA, N.L., 2083
NANGNIOT, P., 1675
PACIGA, J., 1685
PACKHAM, R.F., 1468
PATTI, F., 2225
PEAT, J.K., £3
PECORA, L., 1823
PELECH, L., 279
PERROTEY, J., 1199
PERRY, R., 1285, 1328, 1385,
1405, 1783
PETERS, R., 2397
PETERSON, R.W. , 3,
PFANNHAUSER, W., 258, 858, 1328.
1719, 2253
PHAM, Q.T., 2O8, 470, 973, 988
PHILP. J.M.. 2350
PHILP, J.M., 2350
*1«~»l^^«~*^» f ** • *« * f m* -^f^f -mf
NANGNIOT, P., 1675 PICHE' L" 1314' 1468
NEEDLEMAN,-H.L., 584, 1155, 1166PI^ATOR, M., 830, 951, 958,
1167, 1168, 1169, 1936, 2433 y*y' •L^J" **y'
NEUBERGER, M., 989 PITTWELL, L.R., 2303
~^^~ n T\7a C* 1 *7 P O
NEWHOUSE, M., 383, 1263, 1953 FIVA' L" i^2.
NEWILL, V.A., 161 POTT' F>' —
NIEUWSTRATEN, N., 1351
POWELL, W., 833
-------
LIX
PRINZ, B., 591, 1471, 1489
PTASNIK, J.A., 571
QUAGLIARDI, A., 1287
QUINOT, E., 1519
RABINOWITZ, M., 847
RAFFONELLI, A., 695
RAINSBURY, R., 2397
RALL, D., 37
RAMACIOTTI, D., 659, 2316
REAY, J.S.S., 1669
RECHT, P., 157, 1894, 1928,
1938, 1946, 1961, 2641
REEVES, A., 724, 1637, 2385
RETHFELD, H., 1407, 1416
REUSMANN, G., 591
REUTER, L., 1O66
REY, P., 659, 667
ROBERTS, T.M., 1685
ROELS, H., 631, 887, 1237,
2185
ROGGI, C., 1697
RONDIA, D., 523, 535, 1339
ROOSKEN, A.A., 1351
ROOTS, L.M., 1113
van ROOYEN, G.I., 2382
ROSCA, Gh., 1073
ROSCA, S., 1073
ROSIVAL, L., 877, 1187, 1237
ROSSANO, A.T., 2083
ROSSI, Ai, 1823
RUBIN, R.J., 903, 911, 912
RUDEN, H., 1757
RUDOLF, W., 2115
RUTENFRANZ, J., 2031
RUTZEL, H., 2231
RYLANDER, R., 137, 477, 484,
667
SABATINI, G.C., 1697
SADOUL, P., 973
SALAZAR, S., 2O91
SANOTSKY, I.V., 10O9, 2349,
2409, 2433
SANSONI, B., 1853
SANTARONI, G.P., 1O30, 1451
SANTOLUCITO, J.A., 702, 1051,
2387, 2394
SAUERHOFF, M.W., 8O5
SAVINO, A., 931, 1749
SAYERS, M.H.P., 1207
SCASSELLATI-SFORZOLINI. G., 931,
r?49 —
SCHALLER, K.H., 1087
SCHNEIDER, T., 195V 2071, 2155,
2158, 2162, 2166, 2168, 2172,
2174, 2175, 2178, 2182
SCHILLING, R.S.F., 669
SCHLATTER, C., 723
SCHLIPKOTER, H.W., 190, 228,
369, 521, 667, 723, 771,
2328
SCHMIDT, P., 279, 354
SCHOENBERG, J.B., 669
SCHUCK, E.A., 2073, 2159, 2161,
2164, 2171, 2171, 2176, 2177,
2179, 218O, 2181, 2182
SCHULLER, P.L., 1017
SCHULZ, C.O., 903
SCHWING, R.C., 1491, 15O2, 1503,
2178, 2181
SCOPPA, M., 958
SEBASTIEN, P., 1189
SECCHI, G.C., 1123
-------
LX
SEIFERT, D., 1407
SERWER, D., 2383
EL-SEWEFY, A.Z., 1589
SHAHEEN, H.r 1589
SHAMS El-DEEN, A., 1589
SHAPIRO, I.M., 1155
SHAPIRO, M.A., 677
SHERWOOD, R.J., 471, 134O, 1954
SHUVAL, H.I., 1067
SILBERGELD, E.K., 793, 814, 1O51
SILVERMAN, A.P. , 16O1
SKJAERASEN, J. , 1233
SLATER, D.H., 1783
SHEETS, J., 1087, 2185
SMIDT, U., 1557
SMITH, R.G., 1637
SMITH, G., 1729
SORENSEN, S., 137, 477
STAKKOVIC, B., 2285
STANKOVIC, M., 2285
STANKOWSKA, K., 685
STANLEY, R., 1729
STARA, J.F., 714, 751, 771, 772,
813
STEELE, T.D., 1954, 1961, 2170
STEENSBERG, J., 472, 1OO6, I960
2432
STEPANEK, V., 1473, 1478, 15O3
STEPHAN, W.I., 1779
STERN, A.C., 2143
STEVENS, L., 1001
STEWART, H.N.M., 1669
STIDL, H.G., 989
STOEPPLER, M., 2231, 2259
STOOFF, W., 1479
STOPPS, G.J., 229
STUIK, E.J., 537
STOPFEL, M., 89, 77O, 844,
1275, 1625, 1963, 2O43
SUESS, M.J., 339
SULAIMAN, A.B., 657, 683, 1952
SUTHERLAND, L.C., 484, 485, 491
SWYNGEDOUW, I., 1131
SYROTA, M.J., 399, 457, 459,
462
SZADKOWSKI, D., 1841, 2183, 2249,
2256, 2259
SZPERLINSKI, Z., 1711
TAKABATAKE, E., 2197
TATI, M., 2251
TAYOT, J., 1199
TER HAAR, G., 8O3, 1177
THOMAS, R.S., 1331
THOMAS, T.J., 1569
THOMPSON, J.M., 1779
TOMATIS, L., 1053, 2317, 2328,
2329
TOMPKINS, E., 2197
den TONKELAAR, E.M., 1O17, 1O28,
1029, 1030
TRAKOWSKI, A.C., 2455
TREMOLIERES, J., 1902, 1921,
1927, 1944, 1950
TRUFFERT, L., 2255, 23OO
TSUCHIYA, K., 2197, 2351, 2364,
2365
TDLLIEZ, J., 921, 929
TUMASONIS, C.E., 879
TWIBELL, J.D., 1385, 1397, 1398
TYTUN, A., 545
VALENTA, P., 1416, 2231
VALJAREVIC, V., 1829
-------
LXI
VANINI, G.f 1289 YORDANOV, D., 1815
VEIL, S., 1, 25
VERBERK, M.M., 2005
ZAPHIROPOULOS, M. , 1961
VOINIER, B., 659
de ZEEUW, M., 1947
VOUK, V.B., 467, 469, 473,
1912, 1925, 1930, 1931, 1947 ZELENKO, V., 2£7
VUORI, E., 2263, 2296 ZIELHUIS, R.L., 175, 364, 537,
542, 543, 692, 104O, 110O,
1168, 2002, 2060, 2348, 2384
WALDBOTT, .G.L., 1575 ZUNIC, R., 2386
WALLER, R.E., 77, 89, 90 ZWIERS, J.H.L., 1O17
WANNAG, A., 1233, 1237, 1238
WARNER, P.O., 1001, 1OO6, 1OO7,
1008
WASSERMANN, D., 1O53
WASSERMANN, M., 103, 156, 396,
683, 702, 713, 877, 1O53,
1065, 1066, 1439, 1953
WATANABE, H., 91, 1O4
WEAVER, N.R., 369
WEBER, 0., 260, 2261, 2295, 2297,
2299, 2301, 2302, 23O3
WEICHERT, N., 1417
WEIR, F.W., 1989, 2OO1, 2OO2,
20O3, 2004
WEISS, B., 2415, 2432, 2433
WESOLOWSKI, J.J., 471, 1729
WETHERILL, G.W., 847, 857, 858,
859, 1298
WILCOX, S.L., 1241
WILLIAMS, H., 16O1
WILLIAMS, M.K., 551
WILLIAMS, R., 47_
WISSMATH, P., 1853
WOIDICH, H., 1719
WOOLCOCK, A.J., £3
WORTH, G., 1557
YANIV, S.L., 1249, 126O
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