vvEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-82-006 June 1982
Project Summary
Chronic Toxicity of Lead and
Cadmium: III. Effects of
Chronic Intoxication on the
Reproductive Function of the
Fi Generation and on the
Central Nervous System of the
F2 Generation of Rats
Z S Herman, K. Kmieciak-Kolada, R. Szkilnik, W Felmska, J. Slominska Zurek,
B. B;en, R. Winter, J. Bodziony, B. Hebrowska, K. Kammski, D. Piskorska,
J. Wyebowska, K. Ludyga, M Strokowska, J. Konecki, G Kowalczyk,
and M Wolha
Wistar rats, descended from ani-
mals used in previous toxicity studies,
were administered lead and cadmium
via drinking water and examined for
changes in locomotor activity, neuro-
chemical levels in various brain re-
gions, concentrations of heavy metals
in liverand kidney, blood components,
and reproductive ability and viability
of offspring.
Locomotor activity was seen to vary
greatly after heavy metal treatment,
but with no clear pattern of response
evident. Neurotransmitter levels also
showed wide variations in response to
heavy metal exposure, with the most
notable being increases in noradrena-
line in the hypothalamus and dopa-
mine in the striatum. Serotonin and
5-hydroxyindoleacetic acid levels
were less variable. Acetylcholinester-
ase activity in the brain was generally
depressed by cadmium and long-term
exposure to lead, while brain mono-
aminooxidase activity appeared ele-
vated by lead and depressed by
cadmium. Erythrocyte levels in the
blood, as well as free erythrocyte
porphyrin levels, were elevated for
animals of both sexes exposed to
either metal, as were hemoglobin and
hematocrit levels, but the numbers of
leukocytes were sometimes reduced
in animals given low dosages of both
metals.
Weight gains during gestation, litter
sizes, survival of offspring and aver-
age size of surviving offspring were all
depressed in groups of animals treated
with the metals, and eye-opening was
delayed in these groups.
This Project Summary was devel-
oped by EPA 's Health Effects Re-
search Laboratory, Research Triangle
Park. NC, to announce key findings of
the research project that is fully docu-
mented in a separate report of the
same title (see Project Report order-
ing information at back).
Introduction
In previous studies the toxic effects of
lead and cadmium in parent and off-
spring Wistar rats were examined (Her-
man et al, 1980, 1981) In those studies
high concentrations of lead were seen
generally to lower the noradrenalme
concentrations in all areas of the brain
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except the limbic system, to alter the
locomotor activity of the animals, and to
enhance the levels of free erythrocyte
porphyrms The present studies are a
continuation and extension of the
above-mentioned studies and were
conducted on the progeny of the ani-
mals studied previously The goals of the
current work are (1) to determine which
biochemical or physiological parame-
ters studied previously yield reproduci-
ble responses when rats are exposed to
lead or cadmium, (2) to determine if any
long-term alteration in response results
from exposure of successive genera-
tions to the heavy metals, and (3) to
assess the effect of heavy metal expo-
sure on reproductive ability
Materials and Methods
All studies were performed on Wistar
rats descended from animals used in
the previous toxicity studies. The parent
animals had been administered lead or
cadmium, or both, in low or high con-
centrations in the drinking water begin-
ning on their 40th day and pregnant
females continued on this regimen
through gestation and lactation Their
offspring (the Fi generation) were sim-
ilarly exposed beginning 30 days after
birth, again through gestation and lac-
tation, and their offspring (the F2 gener-
ation) were subjected to the same
exposure beginning on their 30th day.
Animal activity was monitored by use
of photocells connected to a cage, with
motor activity considered proportional
to the number of times light beams
were interrupted Individual rats were
placed m this acticnmeter for 24 h at a
time, and total impulses were recorded
from counters every 12 h, correspond-
ing to daytime and nighttime activity
Rats were sacrificed by cervical dislo-
cation and their brains were removed
and stored on ice The brains were dis-
sected into the following regions hypo-
thalamus, pons and medulla oblongata
(brain stem), hippocampus with nucleus
accumbens (limbic system) and stria-
tum Spectrofluorometnc analysis was
performed on each brain section to
determine the concentrations of the fol-
lowing amines noradrenalme, sero-
tonin, dopamme and 5-hydroxymdo-
leacetic acid (5-HIAA)
Blood samples were collected from
anesthetized animals by heart punc-
ture, after which the carotid and subcla-
vian arteries were perfused with 4° C
saline solution Brains were removed,
frozen in dry ice, weighed, homogen-
ized in five parts of 0 067 sodium phos-
phate buffer, and chilled The following
enzyme activities were assayed ami-
nolevulmic acid dehydratase (E.C
42.1 24), free erythrocyte porphyrms,
acetylcholmesterase, and monoammo-
oxidase
Animals of the Fi generation received
lead and/or cadmium in their drinking
water at the same concentrations
administered to the parents Levels of
peripheral blood components (erythro-
cytes, leukocytes, hemoglobin and
hematocnt) were estimated in 90- to
100-day-old animals At the same time
vaginal smears were made for females
to determine the mean time of the
estrous cycle Animals were mated in
the proestrous phase, and when vaginal
plugs were proved, the females were
housed m separate cages The offspring
of these females were the F2 generation
Parameters used to quantify the gen-
erative ability of the treated rats include
weight gams in pregnant and offspring
rats, number of pups per litter, number
of live births and survivors at the 4th
and 21st day, and the implantation and
resorption number for fetuses m fe-
males sectioned on the 20th day of preg-
nancy
Samples of liver (2g) and kidney (1g)
were taken immediately after animal
sacrifice. The tissues were dried and
burnt in a muffle furnace at 550° C for
30 mm, after which 2.5 volumes of nitric
acid were added and the samples were
evaporated and burned again This pro-
cess was repeated several times until
only a white residue remained. This
residue was dissolved in 50 ml of 0 1 N
nitric acid and water was added to yield
a final volume of 100 ml. Cadmium and
lead were complexed with ammonium
pyrrolidme dicarbonmm (APDC) and the
complex was extracted by shaking with
methylisobutyl ketone The ketone layer
was decanted and centrifuged, and the
lead and cadmium concentrations were
estimated by comparison of the atomic
absorption spectra with those of known
standard solutions For lead, the wave-
length 228 8 nm was the reference
point
Results and Discussion
Values of locomotor activity m F2 gen-
eration rats varied widely within the dif-
ferent test groups, with no consistent
pattern discernible Comparison of
changes from control groups values in
this generation and in the Fi generation
(Herman et al., 1981) also gave incon-
clusive results at least as many groups
showed opposite as similar effects
when considered pairwise, and the
values for the control groups m the two
generations also showed wide varia-
tions
Table 1 summarizes the results of
assays of noradrenalme levels in four
brain regions from 30-, 60- and 90-day-
old F2 generation rats Each entry m the
table represents an average value for
eight animals The noradrenalme con-
centrations for each brain region
remained fairly constant for the control
animals over the course of the study,
but greater variations were seen in the
noradrenalme levels for the animals
treated with the heavy metals No con-
sistent pattern of response was readily
apparent for any brain region through-
out the study, except in the hypothala-
mus, where high dosages of cadmium
plus lead resulted m enhanced norad-
renalme levels Of the 72 test groups,
nearly half showed statistically signifi-
cant changes in noradrenalme levels,
all but two being enhancement Sim-
ilarly, very large (over double) increases
of dopamme levels in the striatum were
noted m some treated groups (Table 1),
although here the assay uncertainties
were rather large
The levels of serotonin and 5-hy-
droxymodoleacetic acid (5-HIAA) from
the same four brain regions are listed m
Tables 2 and 3, respectively The range
of variation in the levels of these two
neurotransmitters and the number of
groups exhibiting significant differ-
ences from the control groups were less
than for the dopamme assay
Table 4 lists the results of the biochem-
ical assays on groups of F2 generation
rats given cadmium or lead, or both, and
sacrificed after 30, 60 or 90 days With
but one exception, the ammolevulmic
acid dehydratase activity in the blood
appeared unaffected by administration
of the metals Acetylcholmesterase
activity m brain tissue appeared gener-
ally depressed by cadmium and by
longer exposures to lead Brain mono-
ammooxidase activities varied greatly
for different test groups and appeared
highly elevated by long-term exposure
to lead, reduced by long-term exposure
to high cadmium concentrations, and
unpredictable after exposure to both
metals The most notable and consist-
ent change was m the levels of free
erythrocyte porphyrms, where expo-
sure to lead or lead plus cadmium uni-
formly resulted in large increases m
porphyrin levels
Rats of the F! generation treated with
lead and cadmium for 90 to 100 days
-------
Table 1. Noradrenaline /NA) And Dopamme (DA) Levels In Various Brain Regions Of F2 Generation Rats After Exposure To Lead
Or Cadmium. Each Entry Represents An Average For Eight Animals Assayed, In Units of fjg/g Of Tissue
Part of
Brain
NA in
Hypothalamus
NA in Pons
t- Medulla
Oblongata
30
NA in Limbic
System
NA in Striatum
DA m
Straitum
Age
{days)
30
60
90
30
60
90
60
90
30
60
90
30
60
90
Metals Administered (ppm)
Control
2.27
2.50
232
067
0.66
0.71
0.48
051
051
022
022
0.19
599
622
6 17
Pb(5)
348
2.37
3 77
1 06
065
083
0.62
045
073
025
0.27
032
72 77
7 13
8.93
Pb(50)
206
3.00
281
070
0.68
081
055
043
074
023
037
0 19
709
733
7.51
CdfO. 1)
248
254
1 45
080
0 70
055
050
050
0.42
031
0 13
0.12
14.33
899
650
Cdf5)
4 17
259
303
1 18
0 75
O78
059
054
067
0 17
026
0.32
1093
9 72
7623
Pbf5)+CdfO 1)
332
2.44
224
086
067
061
065
049
050
020
030
0 11
865
4 14
6 58
Pbf50)^Cdf5)
3 18
3 10
295
088
0 75
077
063
055
066
047
021
0.35
1081
654
1006
Table 2. Serotonin Levels ffjg/g Of Tissue) From Various Brain Regions For F2 Generation Rats Treated With Lead And Cadmium
Each Entry Represents The Average For Eight Animals Assayed
Part of
Brain
Hypothalamus
Pons +
Medulla Oblongata
Limbic System
Straitum
Age
(days)
30
60
90
30
60
90
30
60
90
30
60
90
Metals Administered (ppm)
Control
1.53
1 55
7.77
0.95
0.86
1.05
0.85
0 71
087
093
097
092
Pb(5)
1 64
1 40
1 95
1 38
095
1 15
1 01
1 18
056
1 10
096
095
Pb(50)
1 27
1 57
1.43
093
096
1 11
1 03
1 05
069
1 01
094
068
CdfO 1)
1 30
1 87
1 99
096
091
084
090
1 13
0 79
096
090
064
Cd(5)
1 63
1 40
1 57
1 41
097
087
044
1 21
074
1 23
1 22
090
Pb(5)> CdfO 1)
1 70
1 39
2.12
1 31
1 01
1 14
078
1 22
083
1 17
1 05
082
PbfSO) t Cd(5)
1 60
1 48
1 46
1 24
088
082
076
067
048
1 04
1 06
1 00
TableS. 5-Hydroxymdoleacetic Acid Levels f/jg/g Of Tissue) From Various Brain Regions Of F? Generation Flats Treated With
Lead And Cadmium Each Entry Represents The Average For Eight Animals Assayed
Part of
Brain
Hypothalamus
Pons + Medulla
Oblongata
Limbic System
Striatum
Age
(days)
30
60
90
30
60
90
30
60
90
30
60
90
Metals Administered (ppm)
Control
1.61
1 63
1 57
064
065
0.65
0.71
0.69
070
0.81
0.83
083
Pb(5)
7.72
7 66
1 35
093
059
078
048
0.41
076
074
081
0.95
Pb(50)
1 21
1 65
1 77
050
0.85
077
083
044
0.79
0 72
087
087
CdfO 1)
1 74
1 99
1 81
067
0.59
0.64
0 71
0.39
076
058
076
089
Cd/5)
1.56
1 49
1 67
0.90
0.61
0.52
061
0.46
0 71
0.79
093
094
Pbf5) i CdfO 1)
1 41
1 30
1 40
088
085
053
058
046
052
071
093
082
Pb(50)
-------
were sacrificed and their blood was
assayed for hemoglobin levels and mor-
phological elements. Most of the male
groups exhibited slight increases in
number of erythrocytes as a result of
metal exposure; this increase was more
pronounced in females The hemo-
globin levels and hematocrit values for
the various groups roughly followed the
erythrocyte levels, but the number of
leukocytes were in the same cases
reduced in the treated animals, most
notably in the groups given low levels of
both cadmium and lead
The weight gams of the females dur-
ing gestation, litter size, survival of off-
spring and average size of surviving
offspring all were reduced in test
groups administered lead or cadmium,
and all of these changes were more
severe in the groups receiving both
metals simultaneously. The appear-
ance of postural reflexes was relatively
invariant m all groups, but the appear-
ance of eye opening was delayed by at
least a day in all groups administered
metals
F2 generation animals given both
metals weighed about 8% less on aver-
age than the control animals after their
first 90 days Table 5 presents the
results from the survey of Fi generation
female rats sectioned on the 20th day of
pregnancy The average number of
fetuses not implanted but resorbed
increased with exposure to lead and
cadmium The mean number of corpora
lutea per female remained unaffected
by exposure to the metals
For most test groups, the concentra-
tion of metals in the liver was highest
when the animals were 60 days old and
substantially reduced at 90 days The
metal concentration in the kidney gen-
erally stayed constant or increased dur-
ing the course of exposure, except in the
case of simultaneous exposure to both
metals In most cases concentrations in
tissue from Fi generation animals were
equal or slightly higher than those from
corresponding F2 generation groups Lit-
tle difference in cadmium accumulation
was noted between the low- and high-
dose groups in each generation Large
differences, but no clear pattern, were
observed between groups given single
metals and those given equal concen-
trations plus a second metal
References
Herman, Z S , Kmieciak-Kolada, R
Szkilmk, R Brus, L Ludyga.R Winter,
J Jonek, J. Konecki, J Kusz, J Bod-
ziony, B Hebrowska.K Kamimski, M
Ostrowska, J Wyrebowska and J
Laskey (1980) Chronic Toxicity of
Lead and Cadmium I Changes in the
Central Nervous System of the Paren-
tal Generation of Rats After Chronic
Intoxication with Lead and Cadmium
EPA-600/1-80-012.
Herman, Z S , K Kmieciak-Kolada, R
Szkilmk, R Brus, R Winter, J Bod-
ziony, B Hebrowska, K Kammski, D
Piskorska and J Wyrebowska (1981)
Chronic Toxicity of Lead and Cad-
mium II Changes in Central Nervous
System of the Fi Generation of Rats
After Chronic Intoxication with Lead
and Cadmium EPA-600/S1-81-013
<
Table 4. Biochemical Assays For F2 Generation Rats Administered Lead Or Cadmium Each Entry Represents The Average For
Ten Animals Assayed
Assay
Blood ALA
Dehydratase
(/jmo/e/h//)
Brain Acetyl-
cholmesterase
t/jmo/e/m/n/g)
Brain Mono-
ammooxidase
f/jmo/e/h/gj
Age
(day si
30
60
90
30
60
90
30
60
90
Metals Administered (ppm)
Control
85
48
53
72
89
94
m
SO
104
Pb(5)
147
33
33
7.4
1 6
42
171
108
425
PbfSOJ
37
27
21
72
70
35
73
126
413
CdfO. 1)
1 9
7.1
4.4
65
589
185
Cd(5}
36
1 4
50
386
32
26
Pb(5)±Cd(0 1)
47
44
29
5 1
36
5.0
38
103
461
PbfSOJ Cd(5)
85
48
22
85
9.2
6.2
323
552
172
Free Erythrocyte
30
190
411
348
359
3OO
Porphynns
(ug/0
60
90
197
126
317
295
317
295
343
612
374
317
TableS. Survey Of F, Generation Female Rats Sectioned On The 20th Day Of Pregnancy. Values Are Averages For 15 Animals
Per Group
Metals Administered (ppm)
Parameter
Mean Number
of Fetuses
Mean Number
of Resorption
Mean Number of
Corpora Lutea
Control
10.1
04
11 3
Pb(5)
79
1 2
103
Pb(50j
9 1
1 1
11 5
CdfO 1)
80
1 0
10.5
Cd(5)
90
1 2
120
Pb(5j+Cd(0 1)
77
1.3
104
Pb(50)+ Cd{5)
77
20
11 3
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Z. S. Herman, K. Kmieciak-Kolada, R. Szkilnik, I/I/. Felmska, J. Slominska Zurek,
B. Bien, ft. Winter, J. Bodziony, B. Hebrowska, K. Kaminski, D. Piskorska, J.
Wyebowska, K. Ludyga, M. Strokowska, J. Konecki, G. Kowalczyk, and M.
Wo/ha are with the Silesian Medical Academy, Katowice, Poland.
John Laskey is the EPA Project Officer (see below).
The complete report, entitled "Chronic Toxicity of Lead and Cadmium: III. Effects
of Chronic Intoxication on the Reproductive Function of the f 1 Generation and
on the Central Nervous System of the F2 Generation of Rats," {Order No. PB
82-198 409; Cost: $6.00, subject to change} will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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Fees Paid
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Agency
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Penalty for Private Use $300
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