•i
United States .
Environmental Protection
Agency
Office of Water
4607
EPA815-B-97-003
December 1997
&EPA Information Pertaining to
Lead in Drinking Water
at Transient Non-
Community Water
Systems
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"Information Pertaining to Lead in Drinking Water at Transient Non-
Community Water System's"
EPA 815-B-97-003
', December 1997
This Publication Contains the Following Documents:
Agency for Toxic Substances and Disease Registry (ATSDR). 1992. Toxicological Profile for
Lead. U.S. Department of Health & Human Services, Public Health Services, Atlanta, GA. TP-
92/12, pages 9, 65, 66, 98,, and 99. ;
Giridhar, J and Isom, G:E. 1990. Interaction of Lead Acetate with Atrial Natriuretic Factor hi
Rats. Life Science, 46(8):569-576. ,
Hindmarsh, J.T. 1986. The Porphyrias: Recent Advances. Clinical Chemistry, 32(7): 1255-
1263. i
Karmakar, N., Saxena, R., and Anand, S. 1986. Histopathological Changes Induced hi Rat
Tissues by Oral Intake of Lead Acetate. Environmental Research, 41(l):23-28.
Khan, A.J., Patel, U., Rafeeq, M., Myerson, A., Kumar, K., and Evans, H.E. January 1983.
Reversible Acute Renal Failure in Lead Poisoning. Journal of Pediatrics, 102(1):147-149.
i
Nakhoul, F., Kayne, L.E., Brautbar,N., Hu,N., McDonough, A., Eggena, P., Golub, M.S.,
Berger, M., Chang, C., Jamgotchian, N., and Lee, D.B.N. 1992. Rapid Hypertensinogenic
Effect of Lead: Studies in the Spontaneously Hypertensive Rat. Toxicol In. Health, 8(l-2):89-
102.
U.S. Environmental Protection Agency. December 1995. A Survey of Lead in Drinking Water
Supplied By Transient Water Systems. Prepared by Richard P. Maas, Steven C. Patch, Diane M.
Morgan, Geoffrey M. Brown. Environmental Quality Institute, The University of North Carolina
atAshville. Technical Report #95-019, December 1995.
U.S. Environmental Protection Agency. March 20,1996. Note from Jeff Cohen to Judy
Lebowich and Connie Bosma entitled: Analysis of UNC-Ashville Survey of Lead at Transient
i
Systems. •
U.S. Environmental Protection Agency. November 3,1997. Effects From Short-Term Lead
Exposure. Health and Ecological Criteria Division, Office of Science and Technology, Office of
Water. :
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LEAD
U.S. DEPARTMENT OF HEALTH & HUMAN SERVICES
Public Health Service ;
Agency for Toxic Substances and Disease Registry
Printed on Recycled Paper
Federal Recycling Program
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2. HEALTH EFFECTS
2.1 INTRODUCTION
The primary purpose of this
interested individuals and groups with at
health effects. It contains descriptions and
epidemiological investigations.
2.2 D1SCUSS.ON OF HEALTH EFFECTS BY ROUTE OF EXPOSURE
O
sites, the information fa this section is organized Bra : »y
L'then °y
developmental, reproductive,
three iposure periods-acute
OI more)'
Of Sircar. — *r each -teand^
points fa the figures *omne no-observcleKe
levels (LOAEU) reflect
have
are intended ,0 help the users of
Si£n,ficance of the exposure £* ^^4^^
project managers concerned ™tha^ropnaeacnons to tat ^ ^^ Am_) or
on levels of exposure assoctated „'"«' ™°'^°° '^een observed. Estimates of levels postng minimal
SEW e of interest to health professionais and citizens a,,ke.
Uvels of exposure associated with the carcinogenic effects of lead are indicated fa Figure 2-2.
of exposure lev., posmfaima, ,*»
ou methods have heen established
uncertainties are associated with these
uncertainties inherent in the appljcanon of the ££*£> >™ that are delayed m development
example, acute inhalanon MRU may not ^P™"^™ hvpersensitivity reactions, asthma, or chronic
"
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65
2. HEALTH EFFECTS
An increase in systolic blood pressure was observed in rats at a very low exposure level (1 ppm lead in
the drinking water for 159 days, but the dietary and drinking water content of essential and nonessential
metals was abnormally low, and the low-contamination quarters in which the rats were housed also limited
their exposure to essential and nonessential metals (Perry and Erlanger 1978). These conditions, which
result in greater absorption of lead and effects at lower lead intakes than when the diet is less restricted
and the living quarters less isolated, may not be relevant to human exposure. Increases in blood pressure
at low exposure levels have been demonstrated in other studies. For example, rats administered 0.1 and
1.0 ppm lead acetate in drinking water beginning at weaning through 18 months of age exhibited an
approximate 14-mmHg elevation in blood pressure from 3 months (1 ppm) or 12 months (0.1 ppm)
through the entire 18 months of exposure (Perry et al. 1988). No obvious signs of toxicity were observed
in these animals.
Low-level, chronic-duration exposure of rats to lead (30 ppm lead acetate in the drinking water) for
18 months resulted in a 10-15-mmHg increase in both systolic and diastolic blood pressure without any
change in heart rate or histopathological evidence of damage to the kidney, heart, brain, aorta, or liver
(Carmignani et al. 1988a). However, the authors were able to demonstrate that lead exposure in these
animals increased their responsiveness to stimulation of a and & receptors, altered the renin-angiotensin
system, perhaps through inhibition of the renin-angiotensin converting enzyme, and altered the cyclic
, adenosine monophosphate (cAMP)-dependent contractile processes in both myocardium and vascular
myocells. Please refer to Section 2.4 for a more complete discussion of the proposed mechanisms for lead-
induced hypertension.
Cardiovascular effects other than effects on blood pressure have also been observed in laboratory animals
following ingestion of lead. Male rats given 1% (10,000 ppm) lead acetate in their drinking water from
6 to 12 weeks of age had changes in the myocardium (including myofibrillar fragmentation and separation
with edema fluid), dilation of the sarcoplasmic reticulum, and mitochondrial swelling (Asokan 1974). Blood
lead levels in these rats averaged 112 ^g/dL, versus 5 /ig/dL in controls. The highest NOAEL values and
all reliable LOAEL values for each study for cardiovascular effects are recorded in Table 2-4 and plotted
in Figure 2-2.
Gastrointestinal Effects. No studies were located regarding gastrointestinal effects in humans or animals
after oral exposure to inorganic lead. See Section 2.2.1.2 for a discussion of the gastrointestinal effects
of lead in humans after multi-route exposure.
Hematological Effects. As discussed in Section 2.2.1.2, lead has long been known to affect heme
biosynthesis by affecting the activities of several enzymes in the heme biosynthetic pathway. The
mechanisms for these effects are discussed in detail in Section 2.4. Two experimental studies of the effects
of oral exposure to lead on heme synthesis in humans were available. Two groups of five women and one
group of five men who ingested lead acetate at 0.02 mg lead/kg/day every day for 21 days experienced
decreases in erythrocyte ALAD by day 3 of lead ingestion (Stuik 1974). The decreases became maximal
by day" 14 and then remained constant through day 21. An increase in EP occurred in the women, but
not in the men, starting after 2 weeks of ingestion. Blood lead levels were approximately 15 ^ig/dL before
exposure and increased to approximately 40 ptg/dL during exposure. Increased EP was observed in five men
at a higher dosage, 0.03 mg lead/kg/day (which produced a mean blood lead level of 46 ^g/dL), starting
after 2 weeks of lead ingestion (Stuik 1974). Similar results were reported by Cools et al. (1976) for
11 men ingesting lead acetate at an initial dosage of 0.03 mg lead/kg/day, which was decreased to 0.02 mg
lead/kg/day or less as necessary to maintain a blood lead level of 40 ^g/dL; the mean pre:exposure blood
lead level was 17.2
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66
2. HEALTH EFFECTS
Intermediate-duration studies in animals indicate that adverse hematological effects (i.e., decreased
hematocrit, impaired heme synthesis) occur following oral exposure (Krasovskii et al. 1979; Overmann 1977;
Walsh and Ryden 1984). The lowest dose at which these effects are seen depends on the route of
exposure and the nature of the end point studied. For example, decreases in hematocrit were observed
in rats that received 19.2 mg lead/kg/day by gavage (Overmann 1977), but this effect was not seen until
a dose of 318 mg lead/kg/day was administered to rats in their daily diet (Walsh and Ryden 1984). Rats
that received up to 34 mg lead/kg/day as lead acetate in their drinking water exhibited no adverse effects
on hematocrit (Fowler et al. 1980; Victery et al. 1982b). However, evidence of impaired heme synthesis
(increased urinary ALA and coprophobilinogen) was observed in rats that received 0.01 mg lead/kg/day as
lead acetate in their drinking water (Krasovskii et al. 1979). A similar correlation between exposure to
lead in the drinking water as lead acetate and increased urinary ALA was observed by Fowler et al. (1980).
Increased urinary ALA and erythrocyte ZPP were also correlated with increasing doses of lead in rats
receiving 0, 1.67, or 6.35 mg lead/kg/day in their drinking water (Cory-Slechta 1990b). The increase was
observed earlier in the course of the study in older rats.
Adverse hematological effects have been noted in chronic-duration studies, as well. The effects appear to
increase in severity with increasing dose. For example, dose (blood lead)-effect information for heme
synthesis and hematological effects is available; rats and dogs were fed lead acetate in the diet for 2 years
(Azar et al. 1973). In rats, lead produced no effects at 10 ppm (blood lead level = 11.0 /zg/dL; not
elevated above controls), significant inhibition of ALAD at a50 ppm (blood lead level a!8.5 /zg/dL),
significant increase in urinary ALA at a500 ppm (blood lead level a77.8 pg/dL), and slight but significant
decreases in hemoglobin concentration and hematocrit at a 1,000 ppm (blood lead level ^98.6 ptg/dL). In
dogs, lead produced no effects at s50 ppm (blood lead level S31.5 /ig/dL), significant inhibition of ALAD
at alOO ppm (blood lead level &42.S /zg/dL), and no effect on urinary ALA, hemoglobin, or hematocrit at
'any exposure level (highest exposure level = 500 ppm, blood lead level = 75.8 fj.g/dL). Control blood lead
levels were 12.7 and 16.4 /jg/dL in the two rat groups and 15.8 /ig/dL in the dogs.
Studies in animals indicate that the effects of lead on heme synthesis occur in many tissues. Oral exposure
of rats to lead decreased liver ALAS activity (Silbergeld et al. 1982), increased spleen ALAS activity
(Silbergeld 'et al. 1982), decreased kidney ALAS activity (Fowler et al. 1980), decreased brain (Gerber et
al. 1978), liver, and spleen (Silbergeld et al. 1982) ALAD activity, and decreased kidney ferrochelatase
activity along with mitochondrial injury and disturbance of mitochondrial function (Fowler et al. 1980).
r. , The highest NOAEL values and all reliable LOAEL values for each study for hematological effects are
I recorded in Table 2-4 and plotted in Figure 2-2.
Musculoskeletal Effects. Several case reports of individuals who experienced high exposures to lead
either occupationally or through the consumption of illicit whiskey described the occurrence of a bluish-
tinged line in the gums (Eskew et al. 1961; Pagliuca et al. 1990). The etiology of this "lead line" has- not
been elucidated. Individuals having high exposures to lead have also been reported to complain of muscle
weakness, cramps, and joint pain (Holness and Nethercott 1988; Marino et al. 1989; Matte et al. 1989;
Pagliuca et al. 1990).
No studies were located regarding musculoskeletal effects in animals after oral exposure to inorganic lead.
i
Hepatic Effects. No studies were located regarding hepatic effects in humans after oral exposure to
inorganic lead. See Section 2.2.1.2 for a discussion of hepatic effects in humans following multi-route
exposure to inorganic lead.
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98
2. HEALTH EFFECTS
from the respiratory tract may be the lack of lead found at autopsy in the lung tissues of occupationally
exposed lead workers (Barry 1975) and nonoccupationally exposed subjects (Gross et al. 1975).
Organic Lead. Following a single exposure to vapors of tetraalkyl lead compounds (approximately 1 mgto3
breathed through a mouthpiece, 10-40 breaths of approximately 1 L volume) m four male subjects, 37%
«d 51% of inhaled tetraethyl and tetramethyl lead, respectively, were initially found in the respiratory
tract but a considerable percentage of these volatile compounds was lost through exhalation (Heard et al.
19791 Approximately 60-80% of the deposited tetraalkyl lead was absorbed by the lungs. In a case
report of a 22-year-old male exposed to tetramethyl lead, absorption was evident because of elevated
urinary lead levels for 4 days after exposure (Gething 1975).
Limited experimental data suggest that inhaled lead is absorbed rapidly by animals (EPA 1986a). One hour
af£ femT Wstar rats breaThed total lead concentrations of 0.01 mg lead/m* as tetraethyl lead m the
form of aerosolized leaded gasoline labeled with lead-210 (»°Pb) tracer for 30-45 minutes, lead c earance
n the lungs was 30%; the majority of the particles were 0.1-0.5 Mm in diaraeter (Boudene et a. 1977£
Immediately after nose-only breathing, of engine exhaust aerosols containing 6 mg lead/m* as lead-203
SbVlabeled tetraethyl lead for 40 or 60 minutes, 25% of the dose was accounted for m tissues other
ban the lung and gastrointestinal tract in rats (Morgan and Holmes 1978). Initially the lead content in
ungs dec eased quite rapidly; only 7.5% of the dose was retained in the lungs after 48 hours followed by
a sl?wer decline in which less than 2% of the dose remained in the lungs after a week The lung had. he
SoweTtissue lead content in rats and rhesus monkeys who inhaled 0.0215 mg lead/m3 continuously (22
hours per day) for a year (Griffin et al. 1975b).
2.3.1.2 Oral Exposure
Although there were limited data, oral absorption of lead appears to be low in humans except in children.
The extent and rate of gastrointestinal absorption are affected by fasting and the solubility of a particular
?ad 'alt in gastSc acil Male subjects were fed diets supplemented with 0.0008-0.003 mg/kgAIay^of
ead-M4 (2°4)-labeled lead nitrate for up to 124 days in which 6.5-10.9% of the tracer was absorbed
fRabinowitz et al. 1976). Absorption can be as high as 45% in adults under fasting conditions to as low
f6% S food (Chamberlain cTaL 1978). It was reported that intestinal absorption of|touI cMonde^was
3% with meals but increased to 60% when the subjects were fasted (Heard and Chamberlain 1982)
LtSSvSS variability of oral absorption is shown in the study by Heusler-Bitschy et al. (988) m winch
intestinal absorption ranged from 10% to 80% in eight fasted volunteers receiving a single exposure of
0.007 or 0.02 mg lead/kg/day in drinking water. .
There is indirect evidence from autopsies which reveal high lead content in tissues, occurring from life-time
exposure (Barry 1975). Oral intake of lead'can result from consuming lead-containing food and beverages
and from swallowing lead deposited in the upper respiratory tract after inhalation exposure (Kehoe 1987) -
In addition, the ingestion of lead in children may occur through normal mouthing activity and pica,^ which
is the ingestion of material not fit for food, such as soil, clay, ashes, paint chips, or plaster (EPA 1986a).
The primary site of lead absorption in children is the gastrointestinal tract (Hammond 1982). For dietary
lead absorption in children is approximately 50% compared with 15% gastrointestinal lead absorption
measured in adults (Chamberlain et al. 1978). When daily intake was greater than 0.005 mg lead/kg
absorption was increased from 26.2% to 41.5% in infants, 2 weeks to 2 years of age, who were exposed
for 72 hours to lead in milk and commercially prepared strained food (Ziegler et al. 1978). However, the
authors indicated that at lower lead levels there was difficulty in controlling lead intake.
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99
2. HEALTH EFFECTS
There is evidence to suggest that gastrointestinal absorption in animals is controlled by a saturation
phenomenon, as well as influenced by fasting (Aungst et al. 1981). In fasted rats, absorption was estimated
at 42% and 2% following single oral administration of 1 and 100 mg lead/kg, respectively, as lead acetate
fAunest et al 1981). In mice, absorption was 14% in fasted mice versus 7.5% in fed mice 4 hours
following an oral gavage dose of 0.003 mg lead/kg as lead acetate (Garber and Wei 1974). However, no
difference in absorption (4-5%) was observed in fasted and nonfasted mice receiving 2 mg lead/kg.
The extent of gastrointestinal absorption of lead in experimental animals is age dependent. The rat pup
absorbs 40-50 times more lead via the diet than does the adult rat (Forbes and Rema 1972; Kostial et
al 1978). In rats receiving an oral dose of 1 mL lead-212 (212Pb)-labeled tracer, absorption was
approximately 74-89% for animals 16-22 days of age, 15-42% in animals 24-32 days old, and only 16 /«
at 89 days old (Forbes and Reina 1972). A single dose of lead resulted in 52% absorption in 1-2-week-
old suckling rats compared to 0.4% in adults (Kostial et al. 1978). Age differences in absorption rate were
evident in rat pups who had slightly higher tissue levels compared to adult rats following a single gavage
dose of 1 or 1CI mg lead/kg as lead acetate (Aungst et al. 1981). Absorption was 37.9% for young monkeys
versus 264% in adults following a single radiolabeled gavage dose of 6.37 rng lead/kg as lead acetate
(Pounds et al. 1978). This age difference.may be due, in part, to dietary differences and to the presence
of an undeveloped, selective intestinal barrier to lead in the rat neonate (EPA 1986a).
Particle size also influences the degree of gastrointestinal absorption (EPA 1986a; Grobler et al.-1988)
An inverse relationship was found between diets containing metallic lead of particle sizes *250 /zm and
absorption in rats (Barltrop and Meek 1979). There was a 2.3-fold increase in tissue lead concentration
when animals ingested an acute dose of 37.5 mg/kg with a particle size of <38 Mm (diameter) compared
to a particle diameter of 150-250 /jm (Barltrop and Meek 1979).
2.3.1.3 Dermal Exposure
Inorganic Lead. Limited information is available regarding absorption after dermal exposure in humans.
Dermal absorption of inorganic lead compounds is reported to be much less significant than absorption
by inhalation or oral routes of exposure, because of the greatly reduced dermal absorption rate (tPA
1986a). Following skin application of 203Pb-labeled lead acetate in cosmetic preparations (0.1 mL ot a
lotion containing 6 mmol lead acetate/L or 0.1 g of a cream containing 9 mmol lead acetate/kg) to eight
male volunteers for 12 hours, absorption was S0.3%, but expected to be 0.06% during normal use of such
preparations (Moore et al. 1980). Most of the absorption took place by 12 hours of exposure.
Organic Lead. No studies were located regarding dermal absorption of inorganic lead in animals; however
tetraalkyl lead compounds have been shown to be rapidly and extensively absorbed through the skin ot
rabbits and rats (Kehoe and Thamann 1931; Laug and Kunze 1948). A 0.75-mL amount of tetraethyl lead
which was allowed to spread uniformly over an area of 25 cm2 on the abdominal skin of rabbits, resulted
in 10.6 mg of lead in the carcass at 0.5 hours and 4.41 mg at 6 hours (Kehoe and Thamann 1931).
Tetraethyl lead was reported to be absorbed by the skin of rats to a much greater extent than lead acetate,
lead oleate, and lead arsenate (Laug and Kunze 1948).
2.3.2 Distribution
Inorganic Lead Once absorbed, inorganic lead is distributed in essentially the same manner regardless of
the route of absorption (Kehoe 1987). This implies that a common lead transport system is involved.
Therefore, the distribution and body burden of absorbed lead for all routes will be discussed in one section.
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Life Sciences, Vol. 46, pp. 569-576
Printed in the U.S.A.
PeTgawon Prosa
INTERACTION OF LEAD ACETATE WITH ATRIAL NATRIURETIC FACTOR IN RATS
Jaisri Giridhar and Gary E. Isom
Department of Pharmacology and Toxicology
school of Pharmacy and Pharmacal sciences
Purdue university
West Lafayette, IN 47907.
(Received in final form December 29, 1989}
.&•
exposure alters cardiovascular 'unction and
has been implicated in ^he etiology^ of hypertension.
Therefore it was of interest to study th. short term
of lead treatment on atrial natriuretic factor
hormone which produces vascular »"»<»«» »u«*«
1
period water consumption and urino volume were
daily. At the end of the 30 day period, immunoreacfeWe
levels of AKF in hypothalamus, atria and plasma were
measured by radio immunoaesay. Lead treatment did not
atter water consumption, but significantly decreased
urine output. At all doses, lead produced a decrease in
hypothalamic content of ANF and slightly increased
atrial levels. The content of ANF in P1""18. ^|
decreased. The changes in ANF content indicate that lead
interacts with the hormonal regulation «* JJJ
cardiovascular system and these observations may relate
to the cardiovascular toxicity of this heavy metal.
The observation by deBold efc aJU CD that atrial extracts
produce potent diuretic and natriuretic properties led to the
characterization of the family of P^Ptides known as atrial
natriuretic factor (ANF) . Subsequently it has bee"
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570
Lead Acetate Interaction with ANF
Vol. 46, No. 8, 1990
Related, exposure to heavy metals can induce significant
changes in cardiovascular function. Lead has been implicated in
the pathogenesis of hypertension following both acute and chronic
exposure in animals and humans (7-11). Additionally, hypertension
has a high incidence among workers exposed to lead, possibly
related to lead-induced renal impairment (10). Several studieo
have associated a direct relationship between blood lead
concentration and blood pressure (7, 11). Also elevated blood lead
levels have been related to an increased risk of myocardial
infarction (12). Elevated ronin-angiotensin activity was detected
following long tarm exposure to lead in dogo (13) and acute and
chronic exposure in rats (14, 15) . Acute lead administration can
produce a transient decrease in plasma renin activity followed by
a prolonged elevated activity as a result of increased renin
secretion (14) . Chronic exposure to lead increased basal plasma
renin activity and renal ranin concentration (16). Also in some
high renin hypertensive patients, high blood lead levels have been
detected (16). It has been proposed that the mechanism of lead-
induced hypertension may involve the inhibition ol vascular sodium
and potassium pump, where impaired sodium and potassium
cotransport produces increased intracellular sodium content, which
as result of sodium-calcium exchange results in increased calcium
levels in vascular tissue (17, IB). Since ANF plays an important
role in blood pressure regulation, the present study addressed the
effects of lead on immunoreactive levels of ANF in plasma, heart
and bypothalamus.
Methods
Animals; Twenty male Sprague bawley rats (Harlan, Indianapolis,
IN.) .(IS0"175 g in weight) approximately 5 weeks old were randomly
divided into 5 groups of 4 animals each and housed in metabolism
cages (9 by 10.5 by 9 inches). Tap water and laboratory chow were
made available to tho animals ad 1 ibituro . The animalr. were
injected intraperitoneally with different doses of lead (0.01,
0.1, 0.5 and 1.0 rag/Kg of body weight) daily for 7 days at 900 and
2100 hrs. Control animals received normal saline. Following lead
treatment tho animals were maintained for an additional 30 days
during which water consumption and urine volume wer«i monitored
daily.
preparation of plasma extracts: At the end of the treatment period
the animals were sacrificed and the trunk blood was collected in
polystyrene tubes containing 0.5 ml of a solution containing 0.47*
sodium chloride, 1% ethylenediamine tetraacetic acid, 0.1%
bacitracin, 30 iM phenylmethylsulphonyl fluoride and 1 pK
aprotinin. Blood was immediately centrifuged at 1500 g at 4°C for
15 min. Plasma was separated and ANF was extracted, following haat
inactivation of interfering plasma proteins, as reported by linuma
et al. (19). Briefly, 0.5 ml o.f plasma was mixed with an equal
volume of 0.1 M acetic acid solution containing protease
inhibitors. The mixture was heated at 85°C for 10 min and then
centrifuged at 800 g for 10 min. The supernatant was separated and
stored at -70°C. Aliquots of 100 pi of the extracted plasma
supernatant were used for radioimmunoaseay.
Preparation of atriaj extracts; Atrial extracts were prepared
according to the method of Gutkowska et al. (20) . After
I
Vol..
dec.- j
eaclj
acic!
at :j
at -j
cent!,
for !
isol
prej
atri
radJ
meth
Radi
rccci
stanj
stan
..with[
EDTAi
I
not
deer
(0.5
trea-
ANF.
deer
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Vol. 46, No. tf, 1990
Lead Acetate Interaction with ANF
571
for ANF by RIA.
.
Izfo?0 g forY30 »in. suitably diluted and aosaycd
The hypothalami were
C
atria and* T hpothaa3nc extracts, ware roade and i »«J
ralioimmunoassay. Protein determinations -wore mada by the modified
method of Lowry (22) .
WJ.«. rat ANF(1-28). 'in order to prevent interference by lead,
EDTA (1 mM) was added to the samples.
g«-.«fcisti«»i.' analysis: The data were subjected to one-way ANOVA and
then analyzed by Duncan's multiple ranges test.
Results
At the end of the treatment period lead treated animals did
dilpla? observable signs of toxicity. Plasma ANF levels were
ea-jed in all lead treated animals as compared to control value
To^le + 0.037 ng/«H of plasma)-. Except for the 001 mg/kg
treatment group, lead produced a dose related decrease in plasma
ANF Statistically the 1.0 mg/kg dose of lead was significantly
decreased (P < Q.05) as compared to the control value (Fig. 1).
no-
not
o
u
OS
w
D.
100-
90
60
70
60
50
0.5
1.0
0.01 0.1
COSE
FIG. 1
Effect of varying doses of lead on ir-ANF levels in.
plasma, expressed as percent control ± SEM (ng/ml).
Each value is the mean of 4 animals. *significantly
different from control (P < 0.05).
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n u. y
U X I . W —'
1
572
Lead Acetace Interaction with ANF
Vol. 46. No. 8, 1990
Atrial ANF level at 0.5 ing/kg dose was significantly higher
(p < 0.01) than that of the control (SB. 359 i 23.399 fig/gro of
atrial tissue). The other doses were not significantly different
from the control value (Fig. 2).
In the hypothalaaus lead produced a dose related decrease in
ANF content as compared to control values (703.352 ± 203.937 ng/gm
of hypothalaroic tissue) and this decrease for the 0.01, 0,5 and
1.0 mg/kg was significantly different (P < 0.05) from the control
value (Fig. 3)-
400
ir-
360-
300
250
ZOO
160
100
60
0.01
0.1
0.6
1.0
I
lia*
DOSE(mgAg)
Fig. 2
Effect of varying doses of lead on ir-ANF content in
atria, expressed as percent control ± SEM (Ig/gro of
atrial tissue) . Each value is the mean of A aniTnale.
**Significantly different from control (P < 0.01).
80i
70-
60
60-
40
30
20
0-01
0.1
0.5
1.0
DOSE (mg/kg)
Fig. 3
Effect of varying doses of lead, on ir-ANF content in
hypothalamus,. expressed as percent control + SEM (ng/gm
of hypothalamic tissue) . Each value is the mean of 4
animals. *Signifieantly different from control (P <
0.05).
Vc i
CO
th !
wa j
la j
pe|
I
Tw
Gr<
EX) j
COI
LEV |
LE;
LEA
I
Wat
'ml
fir
sal
exp.
Twe;
LEAL
-------
C.L. • J. — <4 0.
~ i <4OO
i-iug uo . a f
INO . UUi
. UD
Vol. 46. No. 8, 1990
Lead Acetate Interaction with ANF
No change in the water intake was observed between the
control animals and the lead treated animals at all dose levels
throughout the experimental period (Table 1). However urine output
Evas significantly decreased in all the lead treated animals on the
•last treatment day (day 7) and at the end of the maintenance
(Table 2).
TABLE I
Twenty-four Hour wator Consumption of Control and Lead Treatment
^Groups
* Exp Group
CONTROL
LEAD (0
LEAD (0
..-1S»D (0
LEAD (1
.01
.1
.5
. 0
mg/kg)
mg/kg)
mg/kg)
mg/kg)
pav
26
29
24
28
26
.50
.00
.25
.75
.75
(01
+
±
±.
±
4-
1
2
1
3
3
.19
. 48
.55
.20
.71
pav
26
25
31
29
31
.25
.00
.75
.25
.25
(T>
±
±
±
+
+
0.
3 .
1.
2.
1.
63
34
65
14
89
Dav
23
27
27
32
35
.75
.75
.50
.00
.00
f37}
±
±
±
±
+
4.13
4 .13
2.90
2.71
5-02
Water consumption of control and lead treated groups, expressed as
ml •+• SEM (total consumption over 24 hrs) , on day 0 (day before the
first lead treatment),, day 7 [last day of treatment with either
saline (control) or load (treatment groups)] and day 37 (last
experimental day). Each value is the mean of 4 animals.
TABLE II
Twenty—Tour Hour Urine Volume of Control and Lead Treatment Groups
Dav
7 .
-g) 5,
r) 7.
r) 6.
!) 6.
00
00
5O
25
00
(0) :
-*-
±
.*.
±
±
0
o
0
2
0
.71
.71
.87
.18
.41
9
5
4
1
3
pay
.00
.25
.50
. 33
.00
(7) Dav f37J.
± 0.
± 0-
± 1.
± o.
± 1.
71
4B**
19**
24**
08
5.
0.
0.
O.
1.
00 ±
50 £.
25 ±
38 ±
25 +
0
0
0
0
0
.82
.50
.14**
.13**
.75
Exp. Group
CONTROL
LEAD '(O-01 m
LEAD (0.1 wg/kg)
LEAD (0.5 mg/kg)
LEAD (1.0 mg/kg)
Urine volumes of control and lead treated groups, expressed as ml
± SEM (total urine volume over 24 hrs), on day 0 (day before the
first treatment), day 7 [last day of treatment with cither saline
(control) or ]ead (treatment groups)] and day 37 (last
experimental day). Each value is the mean of 4 animals,
"significantly different from control (P < 0.01) on respective
days.
-------
''-§
574
Lead Acetate Interaction with ANF
Vol. 46, Mo. 8, 1990
TABLE III
Body Weight Measurements of Control and Lead Treatment Groups
Exp.
Group
CONTROL
LEAD
LEAD
LEAD
LEAD
(0
(0
(0
(1
.01
.1
.5
.0
mg/kg)
mg/kg)
mg/kg)
mg/kg)
Dav
189.
189.
191.
194.
203.
O
5
0
B
5
{01
+
+
+
+
i
6.
1.
0.
2.
5.
5
0
9
7
1
Pav f 7 > Dav
235.
227.
247.
251.
253.
5 ±
5 ±
5 ±
0 ±
5 ±
2
2
1
1
1
.7
.9
.5**
.0**
.7**
327 .
314.
347.
364.
357.
5
5
0
0
3
(37>
+
+
4-
+
-^
8.3
11.2
5.8*
7.0*
5.9*
Body weights of control and lead treated groups, expressed as
grams ± SEM, on day o (day before the first treatment) , day 7
[last day of treatment with either salino (control) or lead
(treatment groups)] and day 37 (last experimental day). Each
value is the mean of 4 animals. Significantly different from
control (P < 0.05) and significantly different from control (P
< 0.01) on respective days.
Discussion
The present study was designed to investigate the short ten
effect of lead on cardiovascular system and subsequent changes in
tho ANF system (23). Lead produced significant alterations in ANF
immune-reactivity with dose-related decreases observed in the
plasma and hypothalamus and an increase in atria. These changes
may result from the pathological effects of lead, since changes in
ANF levels have been reported in hypertension and kidney disease-,
(6, 24).
As the. Riecha-: sm of lead-induced toxicity is not completely
known, it is not clear as to how lead acts on the ANF system. Lead
may act at one or more sites involved in regulation of the
hormone. Lead may have direct actions on ANF synthesis and release
both in hypothalamus and atria. Additionally, it - may indirectly
modify ANF levels in hypothalamus and atria, by altering J-
heurotransmitter systems such as norepinephrine and dopamine (25),'.-'"'
which are known to modulate ANF levels (26). Also changes in ASF^-
levels in atria and plasma may be brought about by morphological^
and biochemical alterations of the heart, and changes in vascular
smooth muscle reactivity caused by lead (17-18, 27-29). g>
Lead-induced nephrotoxicity has been well characterized in^f
animals and humans (7, 30). Lead produces proximal tubular damage^'
and epithelial cellular necrosis (30) . These effects may lead to''
changes in sodium transport and nance resulting in changes in AN
content through impairment of kidney function and altered flaid
balance. In the present study indicies of nephrotoxicity were not
determined. However, the results suggest impaired renal function.
VoJ. •
grot
dec; •
acct
rett
bodj !
tre; |
whic
rele
can
altc
kidr
thif
cont
•Btr.i i
levc :
with
tisc
meta
1.
2.
3.
i
6.
7.
8,
10.
11.
12.
13.
14.
15.
16.
-------
i j. i_j i -t i\ t
nuy uo > 3i
nu . uu a. r .
•Vol. 46, No. 8, 1990
Lead Acetate Interaction with ANF
575
There was no change in the water intake in the lead treatment
groups as compared to controls at all dose levels throughout the
experimental period* This was accompanied by a significant
decrease in the urine output. Decreased 'urine output without
accompanying decrease in water consumption leads to fluid
retention in lead treated animals. Also significant increase in
body weights of lead treated animals is seen on day 7 (end of
treatment period) and on day 37 (end of experimental period),
which may be explained by fluid retention. This effect may be
related to Jead-induced nephrotoxicity. Expansion of fluid volume
can produce changes in ANF and may be a contributing factor for
altering the ANF system. ANF levels in related cases of impaired
kidney function are generally elevated in plasma (2$). However in
this particular case ANF levels in plasma are lower than the
control value. Hence lead may decrease the release of ANF from
atria and this would account for the observed elevated atrial ANF
levels.
In conclusion the present study indicates acute treatment
with lead acetate can produce significant alterations in select
tissue levels of ANF. The significance oC this action of the heavy
metal is presently being investigated.
6.
7.
S .
9.
10.
11 .
12.
13.
14.
15.
is.
References
A.J. nr.BOLD, H.B. BORENSTEIN, A.T. VERESS & H. SONNENBERG,
Life Sciences 20. 89-94 (1981).
H.G. CURRIE, D.M. GELLER, D.R. COLE, J.G. BOYLAN, W. YUSHENG,
S.W. HOMUERG & P. NKEDLEMAN, Science 221, 71-73 (1983).
A.J. osBOLD, Science 230. 767-770 (1985).
N. MORI1 , K. NAKAO, A. SUGAWARA, M. SAKAMOTO-, M. SUDA, W.
SHIMOKURA, Y. KISO, M. KIIIARA, Y. YAMORI & H. IHURA, Biochcm.
Biophys. Res. Commun. 127. 413-419 (1985).
S. SUIONO, K. NAKAO, N. MOR11, T. YAMADA, H. ITOH, M.
SAKAMOTO, A. SUGAWARA, Y. SAITO, G. KATSUURA & H. JMURA,"
Biochem. Biophys. Res. Commun. 13S. 728-734 (19SG).
T. IMADA, R. TAKAYANAGI & T. INAGAMI, Biochem. Biophys. Res.
Coromun. UL1, 759-765 (1985).
S.T. WEISS, A. HONUZ, A. STEIN, D. SPARROW & F.E. SPEIZER,
Amer. J. Epidemiol. 17.3. 8OO-808 (1986).
D.G. BEEVERS, E. ERSKINE, M. ROBERTSON, A.D. BEATTIE, A.
GOLDBERG, B.C. CAMPBELL, M.R. MOORE & V.M. HAWTHORNE, Lancet
II, 3-3 (1976).
W. VICTERY, A.J. WAN^R, J.M. SHULAK, P. SCHOEPS & S. JULIUS,
J, Lab. Clin. Med. 99. 354-362 (1982).
V. BATUMAN, K. LANDY, J.K. MAESAKA & R.P. WEDEEN, New
Eng. J. Med. 309, 17-21 (1983).
W.R. HARLAN, J. R. IANDTS, R-L. SCHMOUDER, N.G. GOLDSTEIN &
L.C. 11ARLAN, J. Amer. Med. Assoc. 2-^3.t 530-534 (1985).
J.L. PIRKLE, J. SCHWARTZ, J.R. LANDIS & W.R. HARLAN, Am. J.
Epidemiol. 121, 246-258 (1985).
B.P. FINE, T. VETRANO, J. SKURNICK & A. TY, Toxicol . Appl.
Pharmacol. 93, 388-393 (1988).
D.R. MOUW, A.J. VANDER, J. COX & N. FLEISCHER, Toxicol. Appl.
Pharmacol. 46, 435-447 (1978).
N. FT.EICHER, D.R. MOUW & A.J. VANDER, J. . Lab. Clin. Mod. 95,
759-77O, (1980).
A.J.VANDER, Environ. -Health Perspec.. 7_8, 77-83 (1988).
-------
nuun i mix K.C.O i I K. i ELL • i ~*4 iu~ J3u~ m Jo
u u q u o_
INU . u u i r . u 3
57f.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
' 30.
Lead Acetate Interaction with AUK
Vol. 46, No. 8, 1990
Tl
E. WEILER, F. KHALIL-MANESH & H. GONICK, Environ. Health
Perspec. 76. 113-115 (1988).
T. MOREAU, P. HANNAERT, G. ORSSAUD, G. HUEL, R.P. GARAY, J.R
CLAUDE, B. JUGUET, B. FESTY & J. LELLOUCH, Environ. Health
Parspec. 78, 47-51 (1988),
K. IINUMA, I. IKEDA, T. OGIHARA, H. HARA, J. SHIMA, K. KURATA
& Y. KOMAHARA, Clin. Chem. 12, 674-676 (1987) .
J. GUTKOWSKA, G. THlBAULT, P. JANUSZEWICZ, M. CANTIN & J
GENEST, Biochem. Biophys. Res. Commun. 121, 593-601 (1984).
J. GLOWINSKY & L.L. IVERSEN, J. Neurochem. H, 655-659
{ X966) .
G.L. .PETERSON', Analy. Biochem. 8_3_, 346-356 (1977)
S. NISHIYAMA, K. NAKAMURA AND y. KONISHI, Environ! Res. 40
357-364 (1986). **'
S.S. SORENSEN, H. DANIELSEN & E.B. PEDERSEN, Seand. J. Clin
Lab. Invest. 48. 347-355, (1988).
M.K. SHELLENBERGER, Neurotoxicoloqy, 5 177-212 (1984)
O. KUCHEL, W. DEBINSKI, K. RACZ, N.T.~BUU,.R. GARCIA "a.R.
CUSSON, P. LAROCHELLE, M. CANTIN & J. GENEST, Life Scs. 40
1545-1551 (1987) . ,. '
'•J- WINQU1ST, W. VICTERY & A.J.
GONICK'
Health
9i-99K°1988?"T" BARR°N & J-p' TOM- Environ. Health Porsp. 2ft,
D.P. BERNARD & C.E. BECKER, Clin. Toxicol . 2£, 3-34 (1988)..
-------
L»l|g3T ^^J^^Ji^^^'Vv^'V^^^^/--,-^
k!" 5r- -*•: itr".
^^«^T>.-S^M* .-^-'^-^fa^^^a"*' ^Hr^^'^'m.^^'.f-^^- ^^^.^ga*gg»gT5aeag»:l.-g-.--^'..
; t.«..-^
..-^-
CL1N. CHEM. 32/7, 1255-1363 (1986)
The Porphyrias: Recent Advances
J. Thomas Hindmarsh
NOTICE: This Material May
Be Protected B-, Copyight
Law {Title 17 US. CcJa)
Recent research has elucidated several of the hitherto poorly
understood steps in heme synthesis. This review describes
this metabolic pathway and pinpoints the enzymatic block-
ages in the various porphyrias. Recent advances in the
understanding of the etiology of porphyria cutanea tarda are
discussed, as are the abnormalities of porphyrin metabolism
seen in chronic renal failure and in lead poisoning. An outline
is given of the clinical and biochemical abnormalities seen in
the porphyrias. Included is an algorithm to aid in {he differen-
tial diagnosis of these diseases, and a brief review of the new
analytical techniques available for the identification and
quantification of porphyrins and their precursors in body
fluids.
Additional Key phrases: porphyria cutanea tarda • erythmpoietic
porphyria • lead poisoning • chronic renal failure • metabo-
lic pathways • heme synthesis • neiirotoxicity • hepatic iron
accumulation • alcoholism • estrogen-induced effects • her-
itable disorders • harderoporphyria
Heme Synthesis
The principal sites for heme synthesis in the human are
the hcjnapoietic tissues and the liver. The synthetic path-
way is a series of irreversible reactions, some of which occur
in the cell mitochondria and some in the cytoplasm (Figure
1). Intramitochondrially, the'reactions are mainly oxida-
tion—reduction, whereas the extramitochondrial steps are
condensation and decarboxylation. The enzyme 5-aminole-
vulinate (ALA) synthase (EC 2.3.1.37) exhibits much less
activity than the other synthetic enzymes and controls flow
through the pathway; its activity is inhibited by heme or
hemin. After release from the enzyme surface, S-aminolevu-
linic acid diffuses into the cytoplasm and, catalyzed by ALA
dehydratase (EC 4.2.1.24), two molecules condense to form
the mono-pyrrole porphobilinogen. ALA dehydratase is a
zinc-dependent metalloenzyme, and zinc partly protects this
enzyme against the adverse effects of lead in vitro CO and
possibly also in vivo (2).
The enzymes porphobilinogen (PEG) deaminase (EC
4.3.1.8) and uroporphyrinogen-in synthase (EC 4.2.1.75) act
together to polymerize four molecules of porphobilinogen to
form the cyclic tetrapyrrole, uropbrphyrinogen IE. PEG
deaminase appears to catalyze the condensation of four
PEG molecules in a symmetrical head-to-tail arrangement
to form the tetrapyrrole, hydroxymethylbilane (Figure 2).
Pyridoxal
Phosphate
Division of Biochemistry, Ottawa Civic Hospital, 1053 Carling
Ave., Ottawa, Ontario, Canada K1Y 4E9; and Departments of
Biochemistry and Pathology, University of Ottawa.
Received February 25, 1986; accepted April 7,1986.
GLYCWE » SUCCINYL COA
ALA Synth*** •«
AMINOCEVULIHIC ACID
1 I ALA Dehydratase
PORPHOeiL IMOGEN
2
HYDROXYMETHYLBILANE
Negative Feedback
Mubilion
Porphobilinogen
Deaminaae
Uroporphyrinooen-UX
Synthase
UROPOflPHYRINOGEN In
UropOfpbyrmogen 4
Decarboxylose
COPROPOHPHYRINOGEN Iff
Coproporphynnogen 5
Oxtdase „
HAROEROPORPHYHIMOGEM*
I
Coproporpnynnogen 6
Oxidase
PROTOPORPHYRINOGEN
ProtopOTphyrinogen 7
Oxidase
J
PROTOPORPHYRIN
FcfroehelaUce 8
X
F«~ — HEME
Spontaneous
UROPORPHYRINOGEN I,
Uroporphyrinogen
Decarboxytase
COPROPOflPHYRINOCEN I
Fig. 1. The pathway for heme synthesis, including sites of enzyme
insufficiency in the porphyrias
J, ALA dehydratase deficiency, tyrostnemia, toad poisoning; 2, acute intermittent
porphyria; 3, .congenital etythropoietic porphyria; 4, poiphyria cutanea tarda,
hepatoerythropoietic porphyria, toxic porphyria; 5, hereditary ccproporphyria, toad
poisoning; 6, harderoporphyria; 7. porphyria variegata; 8, protoporphyria, tead
poisoning. * = probable step
The enzyme uropofphyrinogen-in synthase then in some
way "flips" the D pyrrole to an asymmetrical arrangement
and closes the porphyrin ring (3, 4). In the absence of
uroporphyrinogen-in synthase, hydroxymethylbilane spon-
taneously cyclizes to form the I isomer of Uroporphyrinogen.
Support for this synthetic pattern comes from the observa-
• tions that uroporphyrinogen-in synthase cannot consume
PEG as substrate nor can it convert the I isomer of Uropor-
phyrinogen to the HI isomer. Ordinarily, excess uroporphyr-
inogen-in synthase is present, which greatly favors the
formation of the ffl isomer series. Its activity in human and
CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986 1255
-------
4mol
PORPHO6K.INOGEN
UROPORPHYRINOGEN HI
Fig. 2. Formation of uroporphyrinogen from porphobilinogen
A acstata: P, prcpionata
pig livers reportedly is inhibited in vitro by ferrous com-
pounds (5).
The decarboxylation of the acetate side-chains of uropor-
phyrinogen DI appears to be a sequential process proceeding
through 7-, &-, and 5-carboxyl intermediates; it is catalyzed
by the enzyme uroporphyrinogen decarboxylase (EC
4.1.1.37). The acetate side-chains of uroporphyrinogen I are
also decarboxylated, ultimately to form coproporphyrinogen
I, after which the metabolic pathway of this isomer series
proceeds no further. Uroporphyrinogen decarboxylase can
react only with uit>]5orphyrinogen as substrate and not the
corresponding porphyrin. Ferrous compounds are reported
to inhibit the enzyme in vitro (6, 7), although one study has
reported the opposite (8).
Coproporphyrinogen oxidase (EC 1.3.3.3) converts copro-
porphyrinogen HI to protoporphyrinogen, in which the pro-
pionate side-chains in the A and B pyrrole rings are oxidized
and decarboxylated to vinyl groups. This reaction probably
proceeds with the preliminary formation of harderoporphyr-
inogen, an intermediate compound with only one vinyl
group (on the A pyrrole). Protoporphyrinogen is oxidized to
protoporphyrin by protoporphyrinogen oxidase (EC 1.3.3.4).
The final step is chelation of protoporphyrin with ferrous
iron to form heme, catalyzed by the enzyme ferrochelatase
(EC 4.99.1.1). Other divalent metals (cobalt, zinc, copper)
also may be chelated with protoporphyrin.
Control of the rate of heme synthesis is achieved largely
by a negative-feedback effect of heme on the enzyme ALA
synthase. PEG deaminase also has a lower activity than the
other enzymes in the pathway and probably also exerts
some control. ALA synthase activity appears to be increased
as a compensatory effect in most, possibly all, of the porphyr-
ias, and this action is responsible for the excess production of
intermediary metabolites proximal to the enzymatic blocks'
in these diseases. Brodie et al. (9) and Moore (10) have
postulated that variability in the activity of PEG deaminase-
explains the different clinical features of the acute and
nonacute porphyries. Their thesis is that in the acute
porphyries, ALA and PEG accumulate in the tissues, where-
as this is not a feature of the nonacute porphyrias; moreover,
this accumulation in nervous tissue is probably responsible }
for the neurotoxic features of the acute disease. Because
heme synthesis is potentially suboptimal in all porphyrias,
these diseases carry the potential for an accumulation of
excess intermediary metabolites proximal to the enzymatic
block. However, in the nonacute porphyrias, PBG deami- -
nase activity appears to be increased (at least hi erythro-
cytes), thereby preventing the accumulation of porphyrin. /
precursors (ALA and PBG), whereas in the acute porphyrias •
the activity of this enzyme is normal (hereditary copropor-
phyria and porphyria variegata) or subnormal (acute inter-
mittent porphyria), and therefore excess ALA and PBG
accumulate. Although this is an attractive hypothesis, it is
not certain that heptatic PBG deaminase is similarly in- •
creased in the nonacute porphyrias, and further work is
needed before the theory can be fully accepted.
The mechanisms whereby the porphyrin precursors (ALA
and PBG) produce neurotoxicity have not been fully eluci-
dated, but clinical neuropathy is related to their accumula-
tion. Neuropathy occurs in diseases hi which ALA alone .
accumulates (ALA dehydratase deficiency); ALA or a me-
tabolite may be the responsible: agent. There have been
problems in producing comparable neurotoxic effects in vivo
in experimental animal models (11), but toxicuy may be
related to the ability of ALA to inhibit competitively the
binding of the central nervous system neurotransmitter, y-
aminobutyric acid, to synaptic membranes in brain tissue
(12,13). . .
Factors known to precipitate acute attacks in porphync
subjects include alcohol, stress, infection, starvation, hor-
monal changes, and the administration of certain drugs. It is
not completely understood how drugs precipitate acute
attacks. The mechanisms may be related to the depletion of
the mitochondria! free-heme pool by decreased heme syn-
thesis or increased utilization of hemoproteins such as
cytochrome P450, resulting in activation of cellular ALA
synthase (11).
The Porphyrias
The porphyrias traditionally are classified according to
whether the liver or the erythropoietic tissue is the main
source of excess porphyrin production (14), and this ap-
proach has some merit it is simple. The classification shown
in Table 1 is based on whether patients with the diseases
suffer acute attacks, dermatological manifestations, or both.
Any classification should include an account of the related
enzyme deficiencies.
Congenital Erythropoietic Porphyria
Patients with this rare disease suffer from mutilating
photo-induced skin lesions, are excessively hirsute, and
often have hemolytic anemia. Fluorocytes—erythrocytes
exhibiting fluorescence when activated with the appropriate
' light energy—are present in the blood, and the teeth and
bone marrow may also exhibit fluorescence'. Studies of,
erythrocytes and skin fibroblasts have shown the activity of
uroporphyrinogen-in synthase to be markedly decreased in
1256 CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986
-------
Table 1. Classification 'of the Porphyrias
Condition
Nonacute porphyrias:
Porphyrias producing cutaneous lesions
Congenital erylhropoietic
porphyria
Porphyria cutanea tarda
Toxic porphyria
Hepato-erythropoietic
porphyria
Harderoporphyria
Protoporphyria
Mode of Inheritance
Autosomal recessive
Sporadic and autosomal
dominant
Acquired
Autosomal recessive
Autosomal recessive
Autosomal dominant
Acute porphyrias:
Porphyrias producing neurological symptoms
Acute intermittent porphyria Autosoma! dominant
ALA dehydratase deficiency Autosomal recessive
Porphyrias producing both neurological and cutaneous manifestations
Variegate porphyria Autosomal dominant
Hereditary coproporphyria Autosomal dominant
Enzyme defect
Uroporphyrinogen-lll
synthase
Uroporphyrinogen
decarboxyiase
Uroporphyrinogen
decarboxyiase' -
Uroporphyrinogen
decarboxyiase
Coproporphyrinogen oxidase
Ferrochelatase
Porphobilinogen deaminase
ALA dehydratase
(porphobilinogen synthase)
Protoporphyrinogen oxidase
Coproporphyrinogen oxidase
Predominant sitefs) of
metabolic expression
Erythroid cells
Liver
Liver
Erythroid cells and
liver
?
Erythroid cells and
liver
Liver
?
Liver
Liver
this condition, resulting in overproduction of the I isomers of
Uroporphyrinogen and Coproporphyrinogen. Excessive uro-
porphyrin and coproporpnyrin (the I isomers) are found in
the urine.
Pimstone and Kushner (15-17) described an unusual case
of congenital erythropoietic porphyria in which erythrocyte
uroporphyrinogen-in synthase activity was within normal
limits but Uroporphyrinogen decarboxyiase activity was
subnormal by approximately 50%. Urinary porphyrins were
mainly the HI isomers, and fecal isocoproporphyrin was
greatly increased. Seventy percent of bone marrow normo-
blasts showed nuclear porphyrin fluorescence; liver porphy-
rins were not increased. These authors believe that two
inherited abnormalities coexist in this patient: erythropoi-
etic Uroporphyrinogen decarboxyiase deficiency and a dys-
erythropoietic anemia.
Porphyria Cutanea tarda- (PCT)
This disease is associated with reduced liver Uroporphyr-
inogen decarboxyiase activity. There appear to be two types:
a sporadic, and a rare inherited form (familial PCT). In the
latter, erythrocyte uroporphyrinogen decarboxyiase activity
is also reduced.(IS, 19). The enzymatic defect in familial
PCT is inherited as an autosomal dominant trait (20). One
study of familial PCT has reported that the amount of
immunologically reactive enzyme in erythrocytes is de-
creased along with the enzymatic activity (21).
Sporadic PCT is the commonest porphyria in North
America. Its onset is often precipitated by ethanol overin-
dulgence or, more rarely, by therapy with estrogen or use of
the contraceptive pill. However, only a small proportion of
persons exposed to these substances is affected, indicating
that other etiological factors are also operating.
• The role of iron in the causation of PCT is confusing and
controversial. Most patients with active PCT have increased
hepatic iron stores; hepatic siderosis was present in 80% of
the patients in the study of Grossman et al. (22). The
increase in hepatic iron varies from a modest twofold
increase to concentrations similar to those seen in idiopathic
hemochromatosis and appears in many cases to be the
consequence of increased intestinal absorption (23). The iron
is especially prominent in the hepatic parenchymal cells and
the portal tract macrophages, less so in the Kupfier's cells
(23). Decreasing hepatic iron stores by repeated phlebotomy
produces clinical and biochemical remission of the disease—
amelioration of the symptoms and signs and a decrease in
urinary uroporphyrin excretion (24). Also, Sweeney et al.
(25) have shown that mice rendered iron-deficient by repeat-
ed bleedings are protected against the porphyrinogenic
effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, iron
plays a "permissive" rather than simply a causal role in
PCT, because even though alcoholics commonly have in-
creased hepatic iron stores (26), the etiology of which is
unclear, this finding is more common than PCT is in this
group. PCT is also uncommon in patients with idiopathic
hemochromatosis. Another finding irreconcilable with a
simple causal role for iron is that, although phlebotomy
produces clinical improvement in patients with PCT, it does
not increase the low activity of hepatic uroporphyrinogen
decarboxyiase in these patients (27). Hepatic siderosis does
not appear to be limited to the ethanol-induced variety of
PCT, because it was reported in at least one patient with
estrogen-associated PCT (28). Thus iron overload cannot be
attributed solely to alcoholism. How often hepatic iron
stores are completely normal in active PCT is not clear, but
it probably is uncommon. Hepatic iron accumulation was
not seen in the study of Elder et al. (29) of rats rendered
porphyric by hexachlorobenzene, so increased iron loads are
not essential for porphyria development, at least in their
model. The protective effect of phlebotomy in a similar
mouse model has already been mentioned (25).
The etiology of PCT has recently been reviewed by
Pimstone (30) and Sweeney (31); clearly, it is multifactorial.
The key problem is failure to decarboxylate uroporphyrino-
gen adequately. Uroporphyrinogen decarboxyiase activities
are decreased by about 50% in livers of patients with
sporadic PCT and in the livers and erythrocytes in familial
PCT.
In familial PCT there is a clear pattern of autosomal
dominant inheritance of the enzymatic defect, and Sassa et
al. (32) have shown that the immunoreactive enzyme con-
tent of erythrocytes, at least, is similarly decreased. This
CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986 1257
-------
disease, then, is associated with an inherited deficiency of
uroporphyrinogen decarboxylase activity and synthesis.
However, the disease is not clinically expressed in all family
members who inherit the enzyme deficiency, so other factors
such as increased hepatic iron stores and alcoholism must
contribute to the impairment of uroporphyrinogen decarbox-
ylation. . . .
The etiology of sporadic PCT is more complex. Diminished
hepatic uroporphyrinogen decarboxylase activity does not
recover, even though the clinical state improves when iron
stores are reduced by phlebotomy, so these patients also
seem to have an irremedial enzyme defect. This may also be
inherited, but the pattern of inheritance is not clear. As was
the case in familial PCT, the enzyme deficiency in sporadic
PCT does not produce clinical disease unless the patients
also have hepatic siderosis and (or) have some other disease-
precipitating factor such as alcoholism or take estrogens.
An interesting hypothesis, supported by HLA marker
studies, has been advanced by Kushner et al. (33). They
propose that, in sporadic PCT, a defect in uroporphyrinogen
decarboxylase is inherited as a recessive trait. To develop
the disease, the patient must be homozygous for the enzyme
defect and also inherit one allele, perhaps on a different
chromosome—i.e., be heterozygous—for the autospmal re-
cessive disease, idiopathic hemochromatosis. Hepatic sidero-
sis, perhaps the consequence of the recessive state for
hemochromatosis, has a further inhibitory effect upon the
already compromising enzyme (33, 34>. It is not clear
whether alcohol and estrogens aggravate the enzyme insuf-
ficiency by further inhibiting the enzyme directly or by
promoting further hepatic iron deposition. All these factors
combine to produce clinical disease from a relatively innocu-
ous enzyme deficiency. The complex inheritance pattern
explains why sporadic PCT does not appear in several
members of a family; the autosomal dominant inheritance of
the enzyme deficiency in familial PCT explains why it does.
The complex interactions of the etiological factors in PCT
have also recently been reviewed by Mukerji et al. (34).
They suggest that iron may further compromise uropor-
phyrinogen decarboxylation, in two ways. Firstly, ferrous
iron directly inhibits uroporphyrinogen decarboxylase by
combining with sulfhydryl groups on the enzyme. Secondly,
in the presence of oxygen, binding of ferrous iron to cellular
sulfhydryl groups promotes the generation of superoxide
radical anions, which might further inhibit enzyme activity
and (or) oxidize porphyrinogens to porphyrins that cannot be
acted upon by the enzyme.
The symptomatology of PCT is very variable but the
patient usually shows some cutaneous blistering and fragil-
ity in the light-exposed areas, along with some hirsutism.
Diagnosis is usually made from a combination of a classical
clinical picture and increased urinary porphyrins, with
uroporphyrin exceeding coproporphyrin. It is sometimes
difficult to distinguish patients with PCT from those with
porphyria variegata, as there may be no clear history of
inheritance in the latter disease. In porphyria variegata the
urinary excretion of coproporphyrin usually exceeds that of
uroporphyrin, but in the phase of recovery from an acute
attack, urine uroporphyrin may exceed coproporphyrin,
perhaps because of nonenzymatic conversion of porphyrin
precursors to uroporphyrin. If there is no clear history of
ethanol abuse, I usually use thin-layer chromatography to
look for the presence of grossly increased amounts of isoco-
proporphyrin in the feces of patients suspected of having
PCT (35).
Isocoproporphyrin is formed in excess in PCT, probably by
the following mechanism (36). Uroporphyrinogen decarbox- _
ylation occurs in a step-wise manner, starting with the
acetate group of the D pyrrole (Figure 3). Because flow
through this pathway is impeded, owing to impaired uropor-
phyrinogen decarboxylase activity, 7-, 6-, and 5-carboxylic
porphyrins accumulate. Heptacarboxylic porphyrin appears
in excess in the urine and can be used as a biochemical
marker for PCT. Isocoproporphyrin is a porphyrin with four
carboxylic groups in which one propionate group has under-
gone oxidative decarboxylation before the last acetate side-
chain has been decarboxylated. Its formation in excess in
PCT is probably the consequence of the action of copropor-
phyrinogen oxidase on the excess pentacarboxylic porphyr-
incgen. This enzyme converts the propionate group of the A
pyrrole of pentacarboxylic porphyrinogen in the liver to a. ,
vinyl group. The resulting compound (dehydroisocopropor-
phyrinogen) is then excreted into the feces, where fecal
bacteria reduce the vinyl group to an ethyl group. This
pathway is insignificant in normal healthy persons (37).
A PCT-like syndrome is occasionally seen in chronic renal
disease, particularly in patients who are undergoing regular
hemodialysis (38). In some cases, results of porphyrin stud-
ies have been found to be within normal limits, and the
disease has been called "pseudoporphyria"; some cases of
pseudoporphyria have baen attributed to use of the drugs
nalodixic acid, tetracycline, and furosemide. Other studies
have reported distinct abnormalities in porphyrin metabo-
lism, including increased porphyrins 'in plasma and urine
and diminished activity of uroporphyrinogen decarboxylase
OEHTOHOISOCOP90POSPHYRINOGEM
Fig. 3. Formation of coproporphyrinogen and isocoproporphyrin from
uroporphyrinogen
—<-, normal pathway; —>, pathway in porphyria cutanea tarda. A, acetate;-P,
praptonate: V. vinyl; M, methyl. Compounds in which R = hydroxyettiyt
(CH3CHOH) and desethyi (H) are also formed
1258 CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986
-------
in erythrocytes. Interpretation of the biochemical indicators
of porphyrin metabolism is difficult in renal failure, when
urinaiy excretion of porphyrins may be impaired and por-
phyrin concentrations in plasma correspondingly increased.
Day et al. (39,40) have recently reviewed porphyrin metab-
olism in chronic renal disease. Their studies show that
uroporphyrin concentrations in plasma and urine are com-
monly supranormal in chronic renal failure, whereas values
for coproporphyrin in urine and plasma are often subnor-
mal. Plasma uroporphyrin concentrations in two of their
three patients with renal failure and bullous skin lesions
were similar to those reported for patients with PCT from
other causes. However, the plasma uroporphyrin concentra-
tion in the third patient with skin lesions, although in-
creased, was similar to those seen in some dialysis subjects
without skin lesions. Poh-Fitzpatrick et al. (41) have also
reported overlap of plasma uroporphyrin concentrations in
patients with chronic renal failure, with or without skin
lesions.
Day and Bales (39) reported that the increased uropor-
phyrin in chronic renal disease was the EH isomer, in
contrast to sporadic and familial PCT, in which the I isomer
is usually found in excess. Uroporphyrinogen did not pass
throrgh the dialysis membrane, probably because of its
binding to a plasma protein. Another surprising finding in
their study (39) was the markedly decreased coproporphyrin
concentrations in urine and plasma, which, associated with
decreased fecal porphyrins, led them to conclude that the
kidney might be a major source of coproporphyrin. In
support of this thesis, they reported a patient with porphyria
variegata who underwent renal transplantation .for renal
failure (40). The patient showed no accumulation of plasma
coproporphyrin before transplantation, despite cessation of
urinary copniporphyrin'excretion; marked coproporphyrin-
uria occurred after transplantation. Why a kidney from a
presumably nonpoFphyric donor would produce excess co-
proporphyrin when transplanted into a pt^.hyric patient
remains to be explained, as do many of the other features of
this interesting hypothesis. Etiological factors contributing
to the increased prevalence-of PCT in chronic renal disease
might include the iron overload sometimes seen in these
patients as a consequence of therapy with iron and repeated
blood transfusions (42). Aluminum is known to inhibit some
enzymes in the heme synthetic pathway and, because it
appears to be retained in some patients with chronic renal
failure receiving aluminum hydroxide therapy, it may play
some part as an etiological factor (43).
Toxic Porphyria
This disease resembles PCT. Hepatic uroporphyrinogen
decarboxylase concentrations decrease in response to expo-
sure to a toxin such as hexachlorobenzene or 2,3,7,8-tetra-
chiorodibenzo-p-dioxin. However, the prevalence of porphyr-
ia in persons exposed to these toxins is much higher than in
ethanol-.and estrogen-associated PCT, and the mechanism
presumably is a more direct effect of the toxin on the
enzyme. Elder and Sheppard (44) have shown, in a rat-liver
model, that the immunoreactive uroporphyrinogen decar-
boxylase content remained unchanged, even though catalyt-
ic activity was inhibited by prior administration of 2,3,7,8-
tetrachlorodibenzo-p-dioxin and hexachlorobenzene.
Hepatoerythropoietic Porphyria
Hepatoerythropoietic porphyria clinically resembles con-
' genital erythropoietic porphyria, presenting in childhood
with severe photo-induced skin damage with inflammation
and scarring associated with increased zinc protoporphyrin
in erythrocytes and acetate-substituted porphyrins in the
plasma, urine, and feces. The principal porphyrin in feces is
isocoproporphyrin (45-47). Erythrocyte uroporphyrinogen
decarboxylase concentrations are less than 10% of normal,
and it is thought that this disease is a homozygous form of
familial PCT. Some authors (21) have reported that immu-
nological measurements of erythrocyte uroporphyrinogen
decarboxyiase are correspondingly decreased, whereas oth-
ers (32) have reported them to be within normal limits. This
could be due to genetic heterogeneity. The mechanism of
how zinc protoporphyrin accumulates in erythrocytes is
complex and has been discussed by Elder et al. (45) and Lim
•and Poh-Fitzpatrick (47).
Harderoporphyria
Few cases of this interesting disease have been reported.
It presents with jaundice and hemolytic anemia at birth (48,
49), followed in one case by the development of mild
photosensitivity at age 11 years. Large amounts of copropor-
phyrin are excreted in the urine and feces, but the pattern of
fecal porphyrin is atypical, the major fraction being hardero-
porphyrin (>60%; normally <20%). Coprop_orphvrinogen
oxidase activity in the lymphocytes of the patients was 10%
of control values, suggesting a homozygous state. The en-
zyme exhibited about 50% of normal activity in both par-
ents. In harderpporphyrin a vinyl group replaces the propic-
nate group in the A pyrrole ring only. In the normal subject, •
the propionate groups in the A and B pyrroles probably are
converted to vinyl groups in a stepwise manner, oxidation of
the group on the A ring preceding that on the B ring;
apparently this process is incomplete in harderoporphyria.
Protoporphyria
This disease usually presents in childhood with light-
induced erythema or urticaria, which is often mild. These
patients have an increased prevalence of gallstones. Recent-
ly it has been shown that, hi long-standing cases, there are
abundant porphyrin deposits in the liver, and liver damage
and failure may occur (50). The excess erythrocyte protopor-
phyrin found in this disease is a free unchelated form rather
than zinc protoporphyrin. The enzymatic defect appears to
be a deficiency of ferrochelatase (51). Diagnosis rests on the
combination of a typical clinical picture and the demonstra-
tion of increased erythrocyte protoporphyrin concentrations.
Urinary porphyrin excretion is within normal limits unless
hepatic cirrhosis impedes biliary porphyrin excretion suffi-
ciently to increase urinary porphyrin. Two cases of a variant
of protoporphyria described by Heilmeyer (52) were clinical-
ly similar to protoporphyria except that th? erythrocyte
porphyrin was mostly coproporphyrin. No fjrther cases
have been reported. '
Erythrocyte zinc protoporphyrin is increased in lead poi-
soning and some anemias, including those due to iron
deficiency, excessive blood loss, and chronic disease. When
insufficient iron is available, or it cannot be readily chelated
to protoporphyrin, small amounts of zinc protoporphyrin are
formed and circulate as nonfunctioning hemoglobin (53).
Acute Intermittent Porphyria
Acute attacks of abdominal pain and neuropsychiatric
manifestations are the main clinical features of acute inter-
mittent porphyria; dermatological lesions do not occur.
CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986 1259
-------
Common features of the acute attacks are: acute abdominal
pain (in 95%); vomiting (60%); constipation (60%); neuropa-
thy (60%); tachycardia (60%); hypertension (40%); and men-
tal changes (50%) (54). About 5% of attacks that are severe
enough to require hospitalization end fatally, and repeated
attacks in the same patient are common, so the mortality
rate is around 15 to 20% (14). Residual paralysis, hyperten-
sion, and renal failure are long-term sequelae. Acute at-
tacks are more common in females, more frequently during
the second and third decades and are rare before puberty.
Attacks are commonly precipitated by drugs, particularly
barbiturates and sulfonamides, but many other drugs are
hazardous (11,55). Because of the wide variety of drugs that
are potentially hazardous to these patients, it is more
advisable to provide the patient with a list of drugs known
to be safe rather than a list of the hazardous compounds.
Endocrine factors influence the course of the disease, and
premenstrual attacks are common. Alcohol, infection, and
fasting are other important precipitants. In an acute attack
the diagnosis is made by the association of a typical clinical
picture together with increased PEG and ALA in the urine.
Diagnosis between attacks is more difficult because occa-
sionally the amount of PEG in the urine is normal (56). If
this is so and the clinical story is convincing, I usually
proceed to measure erythrocyte'PBG deaminase.
The disease is associated with a partial deficiency of the
enzyme PEG deaminase in several tissues. The enzyme
deficiency is inherited as an autosomal dominant trait, but
only about 10% of the patients who inherit the enzyme
deficiency suffer attacks of the disease (54), so other etiologi-
cal factors are involved. It is important to identify potential
or latent cases in relatives of patients with the disease, by
measuring PEG deaminase in erythrocytes. The amount of
enzyme present in circulating erythrocytes is probably
related to the age and differentiation of the cells, and
therefore measurements are unreliable in patients with a
high reticulocyte, count for any reason and in newborns (57).
There is overlap between the normal and abnormal refer-
ence intervals, which limits the usefulness of the test (58).
Anderson et al. (59) have proposed that clinical expression
of the disease is related to impairment of Sa-reduction of
steroid hormones by the liver. Glucose (60) and hematin (61)
are partially effective in treating the acute disease, probably
by repression of ALA synthetase.
ALA Dehydratase Deficiency
This can occur as a primary inherited disorder in which
the enzyme concentration in erythrocytes is less than 3% of
normal; it presents as an acute porphyria (62). Bird et al.
(63) have described a family with 22% to 41% of the normal
concentrations of this enzyme in their erythrocytes; they
were asymptomatic. Inheritance of this latter defect was
autosomal dominant and may represent the heterozygous
form of the disease described by Doss et al. (62). ALA
dehydratase (PEG synthase) deficiency may also be a conse-
1 quence of lead poisoning. This deficiency may also be seen in
tyrosinosis, where the abnormal metabolite succinyl ace-
tone, present in excess, competes with ALA (a structural
analog) for attachment to the active site of the enzyme, and
patients suffer from acute porphyric symptoms (64).
Porphyria Variegata
The clinical features of porphyria variegata are variable
and include acute attacks and light-induced cutaneous
lesions. The disease does not usually present before puberty.
The site of the enzyme defect has been argued, but Brenner
and Bloomer (65) have demonstrated that protoporphyrino-'
gen oxidase activity in fibroblasts is 50% of normal in
patients with the disease. Inheritance of the enzyme defect
does not necessarily mean that the patient will develop the
disease; steroid 5a-reduction is commonly impaired in those
in whom the disease has appeared (54). Urinary excretion of
porphyrin precursors and porphyrins commonly increases
during an acute attack but is usually normal between
attacks. Fecal protoporphyrin and, to a lesser extent, copro-
porphyrin are invariably increased in patients who have
developed the disease, even asymptomatic ones. A homozy-
gous variety of protoporphyrin oxidase deficiency may exist
(66).
McColl et al. (67) have described a family of acute •
porphyries in which some family members have porphyrin
excretion patterns typical of acute intermittent porphyria,
some had patterns typical of porphyria variegata, and 'Still
others had an intermediate pattern. Only abdominal and
neurological features were present in affected patients; none
experienced photo-induced skin lesions. All had subnormal
activities of PEG deaminase in erythrocytes arid of protopor-
phyrinogen oxidase in leukocytes.
Hereditary Coproporphyria -
Clinical features of this disease include acute neurovis-
ceral attacks and photo-induced cutaneous lesions. Sympto-
matic disease does not usually appear before puberty and is
commonly the consequence of drug therapy. Porphyrin
precursors are commonly increased in the urine during an
acute neurovisceral attack, as is fecal coproporphyrin. Con-
centrations of all can be normal between attapks (68). The-
biochemical defect appears to 'be a partial deficiency of
coproporphyrinogen oxidase activity (50% of normal) in the
tissues (69). Homozygous coproporphyria has been reported
(70).
The Effect of Lead on Heme Synthesis
Although there has been much argument about the effects
of low concentrations of lead on mental development, overt
symptoms of lead poisoning do not usually appear until the
concentration of lead in the blood reaches 600-800 fig/L
(2.90-3.85 ^tmol/L). Lead appears to inhibit several steps in
the heme synthetic pathway and ALA, coproporphyrin, and
zinc protoporphyrin are produced in excess in the frankly
lead-poisoned patient. Inhibition of ALA dehydratase activi-
ty can be demonstrated at blood lead concentrations as low
as 100-150 /ig/L (0.50-0.70 yomol/L) (71). However, urinary
ALA and coproporphyrin excretion do not increase until the
lead concentration in blood reaches 400 /xg/L (1.90 ^mol/L).
Piomelli et al. (72) have recently reported that erythrocyte
zinc protoporphyrin increases when the blood lead concen-
tration reaches 150-180 /j.gfL (0.70-0.85 jumol/L). The labo-
ratory evaluation of lead toxicity has recently been reviewed
by Fell (73).
Procedure in Investigation of a Suspected Case
of Porphyria
This has recently been reviewed by With (74),. Moore (75),
and Hindmarsh (76). My approach is outlined in Table 2.
The investigator should determine if the patient has had
any symptoms of acute porphyria, a history of skin lesions,
and if there is a family history of the disease. If the patient is
1260 CLINICAL CHEMISTRY, Vol. 32, No. 7,1986
-------
Presentation
Abdominal pain
(with or without
skin lesions)
Te»t
Screen for
increased
urinary PEG
Table 2. Investigation of Suspected Porphyria
Result Further action
Negative Excludes acute porphyria as cause of abdominal pain. However, if
you suspect patient is between attacks, quantify urinary PEG and
ALA. If increased, proceed as for positive screen; if normal, mea-
sure fecal porphyrins to exclude VP and HC. If you still suspect
AIP, measure erythrocyte PEG deamihase.
Positive Confirm by urine PEG and ALA quantification. If clinical picture
clearly that of AIP, stop here. If not, measure erythrocyte PEG de-
aminase and fecal copro- and protoporphyrin to exclude AIP and
differentiate VP, HC.
Normal Excludes protoporphyria: if strong clinical suspicion, exclude other
cutaneous porpnyrias by testing urine and feces.
Increased Protoporphyria confirmed by excluding zinc protoporphyrin.
Negative Examine feces and erythrocytes for excess porphyrin. If both nor-
mal, porphyria unlikely.
Positive Determine porphyrin profile of urine and feces to differentiate PCT,
VP, ana riC. Measure erythrocyte porphyrin if CEP suspected.
AIP. acute intermittent porphyria; VP, porphyria variegate; HC, hereditary coproporphyria; CEP, congenital erytnroooietic porphyria; PCT, porphyria cutanea
tarda.
Reproduced by kind permission of Clinical Biochemistry.
Sun-induced
urticaria or
erythema
Skin lesions:
erosions ± bullae,
hirsutism,
pigmentation
Measure
erythrocyte
protoporphyrin
concn
Measure
urinary
porphyrins
suspected of currently having an acute attack of porphyria
(severe acute abdominal pain, or neuropsychiatric manifes-
tations, or both), the investigation is easy, because results of
the Watson-Schwartz urine test will be positive for PEG.
Many drugs, including chlorpromazine, produce false-posi-
tive reactions to this test, however, and I usually proceed
directly to quantification of PEG and ALA in urine by
column chroinatography. If values for these are normal, the
patient cannot be having an acute attack of porphyria.
If the patient is between acute attacks, however, it may
not be possible to establish a diagnosis of "acute porphyria
between attacks." Values for PEG and ALA in urine are
commonly normal between attacks in porphyria variegata
and hereditary coproporphyria and occasionally are normal
in acute intermittent porphyria. In porphyria variegata,
fecal protoporphyrin excretion, will be above normal,, even
between attacks. Hereditary coproporphyria is very rare;
values for fecal coproporphyrin are usually increased be-
tween attacks, but may be normal. In acute intermittent
porphyria, values for erythrocyte PEG deaminase will be
decreased. .,
If the patient has a history of skin lesions, the investigator
should determine whether these were the urticarial and (or)
erythematous lesions of protoporphyria or the bullae and
scars of congenital erythropoietic porphyria, porphyria cu-
tanea tarda, porphyria variegata, or hereditary copropor-
. phyria. A diagnosis of protoporphyria is made from the
association of a typical clinical picture-with increased proto-
porphyrin concentrations in the erythrocytes. PCT can
usually be 'diagnosed on the basis of a clinical history of
alcohol overindulgence, use of the contraceptive pill, and
increased urinary porphyrins, with uroporphyrin exceeding
coproporphyrin. If there is no history of precipitating factors,
it may be necessary to identify excessive isocoproporphyrin
excretion in feces by thin-layer chromatography. Porphyria
variegata and hereditary coproporphyria are differentiated
by demonstrating increased concentrations of protoporphy-
rin or coproporphyrin. (respectively) in feces. Congenital
erythropoietic porphyria is very rare and is diagnosed by its
typical clinical presentation and biochemical features.
Analytical Methods for Porphyrins and Porphyrin
Precursors
Methods of analysis for porphyrins and porphyrin precur-
sors have recently been reviewed by Moore (75) and Bissel
(77). Normal ranges are method-dependent. Although sol-
vent-extraction methods can be used for the investigation of
most cases of porphyria, they lack specificity, and "high-
pressure" liquid-partition chromatography of porphyrins is
preferable for those laboratories with this facility, ion-
exchange chromatography remains the method of choice for
PEG and ALA.
"High-pressure" h'quid-chromatographic methods for por-
phyrin analysis have evolved in several steps. Early work-
ers used ultraviolet-visible or fluorometric detectors with
isocratic or gradient elution of methyl esters on a normal-
phase silica column (78). This gives acceptable results, but
esterification may be incomplete and possibly may alter the
porphyrin pattern (79). The use of a reversed-phase column
obviates the need to form esters, and gradient elution gives
good and fast separations (80). Eiboflavin interference is a
problem if an ultraviolet-visible detector is used; however, a
fluorescence detector gives more accurate separations, with
good sensitivity.
Porphyrin metabolism has fascinated chemists since the
turn of the century and new porphyries continue to be
described. An interesting new development is the use of
porphyrins for the localization and photodestruction of tu-
mors (81).
I am grateful to Dr. S. L. Perkins, Dr. A. Sorisky, Dr. T. G. Jones,
Professor S. French, and Mrs. N. Meagher for advice and help
during the preparation of this review, and to Miss M. Linton for
typing the manuscript.
References
1. Tsukamoto I, Yoshiiiaga T, Sano S. The role of zinc with special
reference to the essential thiol groups in S-aminolevulinic dehydra-
tase of bovine liver. Biochim Biophys Acta 1979;570:167-78.
2. Cerklewski FL, Forbes KM. Influence of dietary zinc on lead
bMritity in the rat. J Nutr 1976;106:689-96.
CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986 1261
-------
3. Battersby AR, Fookes CJR, Matcham GWJ, McDonald E. Bio-
synthesis of the pigments of life: formation of the macrocycle
[Review]. Nature (London) 1980;285:17-21.
4. Anderson PM. Desnick BJ. PorphobiUnogen dearn^e: meth-
ods and principles of the enzymatic assay. Enzyme 1982;28:146-o7.
5. Kushner JP, Lee GR, Nacht S. The role of iron in the pathogene-
sis of porphyria cutanea tarda. J Clin Invest 1972;51:3044-S1.
6. Kushner JP, Steinmuller DP, Lee GR. The role of iron in the
pathogenesis of porphyria cutanea tarda. J Clin Invest
1975:56:661-7.
7. Pimstone NR, Mukerji SK. Effect of iron on the activity of
partiaUy purified rat liver uroporphyrinogen decarboxylase lAb-
stract). Hepatology 1982;2:147.
8. Blekkenhorst GH, Bales L, Pimstone NR. Activation of uropor-
phyrinogen decarboxylase by ferrous iron in porphyna cutanea
tarda. S Afr Med J 1979;56:918-20.
9 BrodieMJ, Moore MR, Goldberg A. Enzyme abnormalities in the
porphyrias [Review]. Lancet 1977-^:699-701.
10. Moore MR. The biochemistry of the porphyrias [Review]. Clin
Hematol 1980:9:227-52.
11 Moore MR, Disler PB. Drug-induction of the porphyrias [Re-
view]. Adv Drug React Ac Pois Rev 1983:2:149-89.
12. Brennan MJW, CantriU RC, Kramer S. Effect of 5-aminolevu-
linic acid on GABA receptor binding in synaptic piasma mem-
branes. Int J Biochem 1980;12:833-5.
13. Bonkowsky HL, Schady W. Neurological manifestotions of
acute porphyria [Review]. Semin Liver Dis 1982;2:108-24.
14 Elder GH. Clinical disorders of porphyrin metabolism [Sympo-
sium]. Ann Royal Coll Phys Surg Can 1984:17:605-7.
15. Pimstone NR, Kushner JP, Pryor M, Blekkenhorst GH, Mu-
kerji SK, Gandhi S. Congenital erythxopoietic porphyna^oone
marrow expression of famiUal porphyria cutanea tarda [AbstractJ.
Hepatology 1981;1:536.
16. Kushner JP, Pimstone NR, Kjeldsberg CR, Pryor MA, Huntley
A. Congenital erythropoietic porphyria, diminished activity ot
uroporphyrinogen decarboxylase and dyserythropoiesis. • Blood
1982:59:725-37. .
17. Mukerji SK, Pimstone NR, Gandhi SN, Tan KT Biochemical
diagnosis and monitoring therapeutic modulation of disease activi-
ty Si an unusual case of congenital erythropoietic porphyna, Uin
Chem 1985:31:1946-51.
18. Elder GH, Sheppard DM, de Salamanca RE, Olmos A. Identifi-
cation of two types of porphyria cutanea tarda by measurement of
erythrocyte uroporphyrinogen decarboxylase. Clin Sci 1980;o8:477-
84.
19. de Verneuil H, Aitken- G, Nordmann Y. Familial and sporadic
porphyria cutanea tarda, two different diseases. Hum Genet
1978:44:145-51.
20. Kushner JP. Barbuto AJ, Lee GR. An inherited enzymatic
defect in porphyria cutanea tarda: decreased uroporphyrinogen
dwatoxySSvity. J Clin Invest 1976;58:1089-97.
21. de Verneuil H, Beaumont C, Deybach JC, Nordmann Y, Sfar Z,
Kastally R. Enzymatic and immunological studies of uroporphyrin-
ogen deWboxylase in familial porphyria ^f.^^,^
toerythropoietic porphyria. Am J Hum Genet 1984:36.613-22.
22. Grossman ME, Bickers DR, Poh-Fitzpatrick MB, Deleo VA,
Harber LC. Porphyria cutanea ^a, clinical featoes and labora-
tory findings in 40 patients. Am J Med 1979;67:277-86.
23. Tumbull A, Baker H, Vernon-Roberts B, Magnus IA. Iron
metabolism in porphyria cutanea tarda and in erythropoietic proto-
porphyria. Q J Med 1973;42:341-55.
24. Ippen H. Treatment of porphyria cutanea tarda by phlebotomy.
Semin Hematol 1977;14:253-9.
25. Sweeney GD, Jones KG, Cole FM, Basford D, Krestynski F.
IronDeficiency prevents liver toxicity of 2,3,7,8-tetrachlorodibenzo-
p^ioxin. Science 1979:204:332-5.
26. Pimstone NR, French SW. Alcoholic liver disease [Review].
Med Clin North Am 1984;68:39-56.
27 Felsher BF, Carpio NM. Engleking DW, Nunn AT. Decreased
hepatic uroporph>-rinogen decarboxylase activity in porphyna cu-
tanea tarda. N Engl J Med 1982;306:766-9.
28. Haberman HF, Rosenberg F, Menon IA. Porphyria cutanea
tarda: comparison of cases precipitated by alcohol and estrogens. •
Can Med Assoc J 1975;113:653-5.
29 Elder GH Evans JO, Matlin SA. The effect of porphyrogenic
compound, hexachlorobenzene, on the activity °f hepatic uropor-
phyrinogen decarboxylase in the rat. CUn Sa Mol Med 1976;51.71-
.80.
30. Pimstone NR. Porphyria cutanea tarda [Review]. Semin Liv
Dis 1982:2:132-42.
31 Sweeney GD. Porphyria cutanea tarda or the uroporphyrinogen
decarboxylase deficiency diseases [Review]. Clin Biochem
1986;19:3-15.
32 Sassa S, de Verneuil H, Anderson KE, Kappas A. Purification
and properties of human erythrocyte uroporphyrinogen decarboxyl-
ase- immunological demonstration of the enzyme defect in porpnyr-
ia cutanea tarda. Trans Assoc Am Phys 1983;96:65-75.
33. Kushner JP, Edwards CQ, Dadone MM, Skolnick MH. Hetero-
zygosity for HLA-linked hemochromatosis as a likely cause of tbe
hepatic siderosis associated with sporadic porphyria cutanea tarda.
Gastroenterology 1985;88:1232-8.
34 Mukeiji SK, Pimstone NR, Burns M. Dual mechanism of
inhibition of rat liver uroporphyrinogen decarboxylase activity by
ferrous iron: its potential role in the genesis of porphyna cutanea
tarda. Gastroenterology 1984;87:1248-54.
35 Elder GH. Differentiation of porphyria cutanea tarda sympto-
matica from other types of porphyria by measurement of isocoprc-
porphyrin in faeces. J Clin Pathol 1975;28:601-7.
36 Elder GH. Porphyrin metabolism in porphyria cutanea tarda
[Review]. Semin Hematol 1977;14:227-42.
37. Elder GH. Enzymatic defects in porphyria: an overview [Re-
view]. Semin liver Dis 1982;2:87-99.
38 Harber LC, Bickers DR. Porphyria and pseudoporphyria [Re-
view]. J Invest Dermatol 1984;82:207-9.
39. Day RS, Bales L. Porphyrins in chronic renal.|ailure. Nephron
1980;26:90-5. . / '
40. Day RS, Bales L, 'Dialer PB. Porphyrias and the kidney
[Review]. Nephron 1981;28:261-7.
41. Poh-Fitzpatrick MB, Sosin AE, Bemis J. Porphyrin levels in
plasma and erythrocytes of chronic hemodialysis patients. J Am
Acad Dermatol 1982;7:100-4.
42. Cook JD. Clinical evaluation of iron deficiency [Review]. Semin
Hematol 1982:11:6-18.
43 Goldsman CI, Taylor JS. Porphyria cutanea tarda and bullous
dermatoses associated with chronic renal failure [Review]. Cleve-
land CUn Q 1983:50:151-61.
44 Elder GH, Sheppard DM. Immunoreactive uroporphyrinogen
decarboxylase is unchanged in porphyria caused by TCDD and
hexachlorobenzene. Biochem Biophys Res Common 1982;109:lld-
20.
45 Elder GH, Smith SG, Herrero C, et al. Hepatoerythropoietic
porphyria: a new uroporphyrinogen decarboxylase defect or homo-
zygous porphyria cutanea tarda? Lancet 1981;i:916-9.
46. Lazaro P, de Salamanca RE, Elder GH, Villaseca ML, Chinarro
S Jaqueti G. Is hepatoerythropoietic porphyria a homozygous form
of porphyria cutanea tarda? Inheritance of uroporphyrinogen decar-
boxylase deficiency in a Spanish family. Br J Dermatoi
1984:110:613-7. .
47 lira HW, Poh-Fitzpatrick MB. Hepatoerythropoietic porphyna:
a variant of childhood-onset porphyria cutanea tarda. J Am Acad .
Dermatol 1984:11:1103-11.
48 Nordmann Y, Grandchamp B, de Verneuil H, Phung L, Car-
tigny B, Fontaine G. Harderoporphyria: a variant hereditary copro-
porphyria. J CUn Invest 1983;72:1139-49.
49 Doss M, von Tiepermann R, Kopp W. Harderoporphyrin copro-
porphyria [Letter]. Lancet 1984;i:292.
50. Eales L. Liver involvement in erythropoietic protoporphyria
(EP). Int J Biochem 1980;12:9lo-23.
51. Bloomer JR. Protoporphyria [Review]. Semin Liver Dis
1982:2:143-^3.
1262 CLINICAL CHEMISTRY, Vol. 32. No. 7, 1986
-------
52. Heilmeyer L. The erythropoietic porphyrias. Acta Hematol
1964:31:137-49. ' '
53. Labb<§ RF, Finch CA, Smith NJ, Doan RN, SoodSK, Madan N.
Erythrocyte protoporphyrin/heme ratio in the assessment of iron
status. Clin Chem 1979;2o:87-92.
54. Kappas A, Sassa S, Anderson KE. The porphyrias. In: Stanbury
JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS, eds.
The metabolic basis of inherited diseases. New York McGraw Hill,
1983:1301-84.
55. Moore MR. International review of drugs in acute porphyria—
1980 [Review]. Int J Biochem 1980;12:1089-97.
56. Stein JA, Tschudy DP. Acute intermittent porphyria, a clinical
and biochemical study of 46 patients. Medicine 1970;49:1-16.
57. Nordmann Y, Grandchamp B, Grelier M, Phung N, de Vemeuil
H. Detection of intermittent acute porphyria trait in children
[Letter]. Lancet 1976;ii:201-2.
58. Bottomley SS, Bonkowsky HL, Kreimer-Birnbaum M. The
diagnosis of acute intermittent porphyria, usefulness and limit*.
tions of erythrocyte uroporphyrinogen I synthase assay. Am J Clin
Pathol 1981;76:133-9.
59. Anderson KE, Bradlow HL, Sassa S, Kappas A. Studies in
porphyria: relationship ,of the 5a-reductive metabolism of Bteroid
hormones to clinical expression of the genetic defect in acute
intermittent porphyria. Am J Med 1979;66:644-50.
60. Doss M, Verspohl F. The "glucose effect" in acute hepatic
porphyrias and in experimental porphyria. Klin Wochenschr
1981;59:727-35.
61. Pierach CA. Hematin therapy for the porphyric attack [Re-
view]. Semin Liver Dis 1982^:125-31.
62. Doss M, Schneider J, von Tiepermann R, Brandt A, A new type
of acute porphyria with porphobilinogen synthase (S-aminolevulinic
acid dehydratase) defect in the homozygous state. Clin Biochem
1982;15:52-5.
63. Bird TD, Hamemyik P, Nutter JY, Labbe RF. Inherited
deficiency of delta-aminolevulinic acid dehydratase. Am J Hum
Genet 1979;31:662-8.
64. Goldsmith LA. Tyrosinosis. Op. cit. (ref. 54):296-9.
65. Bi-enner DA, Bloomer JR. The enzymatic defect in variegate
porphyria. N Engl J Med 1980;302:765-9.
66. Elder GH. Recent advances in the identification of enzyme
deficiencies in the porphyrias [Review]. Br J Dermatol
1983:108:729-34.
67. McColl KEL, Thompson GG, Moore MR, Goldberg A. Chester
porphyria: biochemical studies of a new form of acute porphyria.
Lancet 1985;ii:796-9.
68. Andrews J, Erdjument H, Nicholson DC. Hereditary copropor-
. phyria: incidence in a large English' family. J Med Genet
1984:21:341-9.
69. Elder GH, Evans JO, Thomas N, et al. The primary enzyme
defect in hereditary coproporphyria. Lancet 1976;ii:1217-9.
70. Grandchamp B, Deybach JC, Grelier M, de Vemeuil H, Nord-
mann Y. Studies on porphyrin synthesis in fibroblasts of patients
with congenital erythropoietic porphyria and one patient with
homozygous coproporphyria. Biochim Biophys Acta 1980;629:577- -
86.
71. Farant JP, Wigfield DC. Biomonitoring lead exposure with 5-
aminolevulinate dehydratase (ALA-D) activity ratios. Int Arch
Occup Environ Health 1982;51:15-24.
72. Piomelli S, Seaman C, Zullow D, Curran A, Davidow B.
Threshold for lead damage to heme synthesis in urban children.
Proc Nail Acad Sci USA 1982;79:3335-9.
73. Fell GS. Lead toxicity: problems of definition and laboratory
evaluation [Review]. Ann Clin Biochem 1984;21:453-60.
74. With TK. Diagnostic tests for porphyria [Review]. Lab Med
1980:11:446-54.
75. Moore MR. Laboratory investigation of disturbance of porphy-
rin metabolism [Review]. Assoc Clin Pathol 1983; Broadsheet
109:1-15.
76. Hindmarsh JT. Clinical disorders of porphyrin metabolism
[Review]. Clin Biochem 1983:16:209-19.
77. Bissel DM. Laboratory evaluation of porpnyria [Review]. Semin
Liver Dis 1982^:100-7.
78. Ford RE, Ou CN, Ellefson RD. Liquid-chromatographic analy-
sis for urinary porphyrins. Clin Chem 1981^7:397-401.
79. Straka JG, Kushner JP, Burnham BF. High-performance liq-
uid chromatography of porphyrin esters. Identification of mixed
esters generated in sample preparation. Anal Biochem
1981:11:269-75.
80. Meyer HD, Jacob K, Vogt W, Knedel K. Analysis of free stool
porphyrins by high-performance liquid chromatography. J Chroma-
togr 1981:217:473-8.
81. Kessel D. Porphyrin localization: a new modality for detection
and therapy of tumors [Review]. Biochem Pharmacol 1984;
33:1389-93.
CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986 1263
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ENVIRONMENTAL RESEARCH 41, 23-28 (1986)
'*w>
** ,?**,'
Histopathoiogical Changes Induced in Rat Tissues
by Oral Intake of Lead Acetate
N. KARMAKAR,' R. SAXENA,* ANDS. ANAND!
Centre for Atmospheric and Fluid's Sciences. Indian Institute of Technology, Delhi Hauz Khas,
New Delhi 110016; 'University College of Medical Sciences, Ring Road. New Dell,,: and
tCentrefor Biomedical Engineering. Indian Institute of Technology. Delhi Ham Khax,
New Delhi 110016. India
Received July 23, 1984
In the present study an attempt has been made to observe the pathological alterations
brought about in the intestine, liver, and kidney of lead-intoxicated rats. A short-term expo-
sure to a sublethal dose of lead (44 mg/kg body wt/day) is, seen to cause conspicuous degen-
erative changes in the three tissues. The intestinal mucosal epithelium is affected wh.ch
leads to malabsorption. while in the kidney proximal tubular cells degenerate causing secre-
tion of essential materials such as glucose, amino acids, etc.. in the urine. © ivtb Acadcm.c
Press. Inc.
INTRODUCTION
Heavy metals in varying concentrations pollute our ecosystem. Lead is one of
the heavy metals commonly encountered in the environment. Lead accumulates
in the body of man and animals, through water, food, and soil, up to critical toxic
levels It further acts as a cumulative poison when taken in small doses and pro-
duces toxic effects (Harrison et al., 1971). A number of studies have been done
on the effect of lead intoxication on the ultrastructure of kidneys (Chang et al.,
1981- Beaver, 1961). However, the gut is the organ through which ingested lead is
absorbed into the body and about 10% of the ingested lead is absorbed (Rabmo-
witz et al., 1975). Further it is evident from studies of Wapnir et al. (1977) that
lead uptake by the mucosa definitely impairs the uptake of glucose, certain amino
acids, and other nutrients through the gastrointestinal mucosa. In our preliminary
•experiments, rats were exposed to 2% lead acetate (Pb ac) in food and'the blood
lead level in these rats was 37jxg/100 ml. This level is supposed to be within safe
limits, but the villi in the intestine were flattened. Hence in the present case,
experimental studies with lead-treated animals were conducted in order to deter-
mine the extent of histopathological changes produced in the tissues of the small
intestine, liver, and kidney, due to a short-term exposure to a sublethal dose of
load. The rat has been chosen as an animal model for lead intoxication and oral
administration of lead via gavage has been employed as the method of exposure.
Ligl t microscopy was performed on each organ studied and scanning.electron
micrographs were studied in the case of the intestine.
1 Present address: Department of Aeronautics. Physiological Flow Studies Unit, Imperial College.
Prince Conson Road, London SW7 2AZ. U.K.
-------
MATERIAL AND METHODS
Male and female rats of the albino Wistar strain weighing 250-300 g were used
for the experiment. They were divided into groups of five. All rats received stan-
dard pelleted laboratory diets and were given ad libitum access to drinking water
Group A served as the control. The other groups B, C, and D were exposed to Pb
acetate for varied durations of time, viz.,^15, and 30 days, respectively. The
method of exposure used was oral administration of Pb ac dissolved m distilled
water Each rat of the treatment groups B, C, and D received, via gavage^l
mg/kg body wt/day of lead as 0.053 M'lead acetate. The control rats received an
equal volume of saline. .
- Tissues (small intestine, liver, and kidney) were fixed for historical examina-
tion by light microscopy in buffered Formalin, dehydrated in graded alcohol
series, cleared in xylene, an'd embedded in paraffin wax. Six-micron-thick sec-
tions were cut and stained with hematoxylin and eosm. •
Tissue samples for scanning electron microscopy were fixed in 4% glutaralde-
hyde in 0.1 M cacodylate buffer for 18 hr at 4°C. The fixative was then washed out
of the tissue by three washes in 0.1 M cacodylate buffer and further dehydrated m
araded series of alcohol. Then they were subject to critical-point drying and fi-
'naily coated with silver. The scanning was done with Cambridge Sterecscan
S4-10.
RESULTS
During the first 2 days of lead exposure average food uptake in the lead-treated
group was 6 g per animal while that in the control group was 10 g per animal.
However later on there was no such difference. The earlier discrepancy was .
probably merely a taste problem. Further, no significant difference was observed
in the -rowth of the experimental rats from that of the control group. The histo-
pathological changes observed at different exposures are given below.
(/) Results of the Histological Study after 9 Days Exposure to Lead Acetate
(a) Small intestine. In two of five cases, mild blunting of the villi was observed.
The rest of the cases did not differ from the normal.
(b) Liver All cases showed sinusoidal dilatation and congestion and one case
showed presence of extramedullary erythropoiesis. In one case amsonucleosis
was also observed.
- (c) Kidney. All the cases were found to be normal. -
(2) Results of the Histological Study after 15 Days Exposure to Lead Acetate
(a) Small intestine. All the slides showed broadening of the mucosal villi. In
one case only, mild depletion in the number of mucus-secreting goblet cells was
seen. The broadening of the villi was confirmed in a scanning electron micrograpu
(F?b) L/ver In one case the changes were found to be within normal limits. In all
others, centrizonal dilatation and congestion had occurred and hepatocytes were
vacuolated. One case showed .ballooning degeneration of the liver. -
(c) Kidney. Changes in two cases were found to be within normal limits. In one
-------
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FIG. 1. Scanning electron micrograph of small intestine from a rat sacrificed on Day 15; arrows
show broadening of the tip of villi (x 2000). •
case, the cortical region showed focal areas of . _
Bother case there were focal areas of tubular degeneration in the medulla
sional hyaline casts were seen in the tubules.
(3) Results of the Histological Study after 30. Days Exposure to Lead Acetate
(a) Small intestine. All cases showed stunted and broadened mucosal villi. In
one of them the villus/crypt ratio had decreased and the v,H. were at the tips and
at some pies, degenerated. Yet another case showed decrease ,n the number of
S°(bWZ.S Alfcases showed centrizonal dilatation and congestion. Two of them
showed presence of extramedullary erythropoiesis (Fig. 3) as well. In one of them
focal fatty changes had also occurred in patches
(c) Kidney. Two cases depicted changes within the normal limits. Two cases
showed focal areas of extreme proximal tubular degeneration dep.cted as vac-
uoles in the medulla (Fig. 4). The rest of the tubules showed severe vacuo a
degeneration which is an indication of hypokalemia. Occasions hyaline casts
were observed.
:j ^^^^•'^'•^V^^A^^.A: DISCUSSION
'* •"':::.>'^.V:K-'i£-i The sensitivity of the active transport mechanism of the small intestinal mu-
•-•*' r?-&$f£W£.t#*'$ cosa to lead toxlcity has been reported (Wapnir et al.. 1979). Glucose mtesUna
•' * "••*- *"•'•""• " ••••'* •-•'• absorption is considerably reduced in lead-treated rats. Intestinal transport of
-------
KARMAKAR, SAXENA, AND ANAND
• no. 2. Photomicrograph of sma. intestine fronva rat sacrificed on Day 30; arrows show broaden-
ing of the lips of villi ( x UK) H and H).
some amino acids such a, lysine. glycine, and phenylalanine are also inhibited.
ThereTalo a drastic reduction of intestinal sodium absorpt.on (Wapn.r « aL,
19The histopathological changes observed in the mucosa of the small intestine «.
the present study suggest malabsorption. Thus they confirm the above findmgs
FIG. 3. Photomicrograph of liver from a rat sacrificed on Day 30; arrow shows.focus of erythro-
poiesis in the liver parenchyma ( x40Q H and E).
-------
LEAD TOXICITY AND RAT TISSUES
27
'".•*; V -,' ^-:"'l:' ^'; /;^:.:
Tro'TT^micrograph of renal medulla from a ra« sacrificed on Day 30: arrows show marked
focal tubular degeneration (x400 H and E).
hat lead causes impairment of active transport mechanisms by adversely af- ,
fecting the mucosa through which the ions, glucose, etc., are transported from
the lumen of the intestine to the serosa and blood int^Tin,i
Blair ei al (1979) suggest that the interaction between lead and the intestinal
tissue is an adsorption of lead onto the mucosal surface, probably due to its inter-
action with H2PO; present in the glycocalyx of the mucosal membrane by'enzy-
matic hydrolysis of ATP and the formation of lead phosphate. Due to the infrac-
tion of the lead ions and the mucosa, pathological changes are.caused in the epi-
''""con^picuous degenerative changes were seen in the liver which probably affect
its enzyme activities. The sinusoidal dilatation and congestion is usually a ter-
minal feature due to anoxia. It may also occur due to passive venous congestion: •
The fatty change in the liver is caused by malnutrition and can be correlated to
malabsorption. It has been studied by other workers that the lead content of the .
liver remains significantly higher than other organs because it is the mam detoxi-
!>'Lead'toxicity can induce two types of nephropathy. In the case of acute poi-
soning, as in the present study, proximal tubular morphologic changes have been
observed which cause tubular malfunctioning. Presence of hyaline casts suggests
that protein is being secreted through the tubules because premeability of base-
ment membrane of the glomerulus may have been affected. Hypokalemia leads to
vacuolar degeneration.
-------
_0 »V^1X..». V1VJ ViV, -J,1,V1_.1. K, . L. ,,^ ........ ~ ^
Thus it is seen that short-term exposure to a high, dose of lead manifests its <
effect in the form of structural damage at the cellular level.
It is also concluded that with oral administration of lead acetate the gastrointes-
tinal tract is the primary target organ and shows pathological changes much be-
fore any other organ.
Further experimental and mathematical studies involving the toxic effect of
lead on the peristaltic transport of the gut have been undertaken and the results
have been reported in a separate communication.'The effect of lead on red blood
corpuscles is also being studied.
ACKNOWLEDGMENTS
N. Karmakar expresses her gratitude to Prof. M. P. Singh for his constant encouragement
throughout this work. The technical assistance of Mr. N. C. Bhatt is acknowledged. The financial \
assistance from NCERT is also gratefully acknowledged. ;
REFERENCES
Beaver, D. L. (1961). The ultrastructure of the kidney in lead intoxication with particular reference to .
intranuclear inclusions. Amer. J. Pathol. 39, 195-208.
Blair, I. A., Coleman, I. P. L., and Hilburn, M. E. (1979). The transport of the lead calion across the
intestinal membrane. J. Physiol. 286, 343-350.
Chang, L. W., Wade, P. R., and Lee. C. W. (i981). An ultrastructural revaluation of lead induced
pathology in the kidney. Environ. Res. 26, 136-151.
Goldstein. J. I., and Yakowitz, H. (Eds.) (1975). In "Practical Scanning Electron Microscopy: Elec-
tron and Ion Microprobe Analysis." Plenum. New York/London.
Harrison, P. R., Matson. W. R., and Winchester. J. W. (1971). Annas. Environ. 5, 613. Cited in
Hasan, M. Z., Seth, T. D., and Sharma, R. (1974). Determination of cadmium, cobalt, copper.
lead, manganese, nickel, and zinc in animal feed by atomic absorption spectrophotometry. Clic-
mosphere 5,241-246.
Rabinowitz, M., Wetherill, G., and Kopple, J. (1975). Absorption, storage, and excretion of lead by
normal humans. In "Trace Substances in Environmental Health-IX" (D. D. Hemphill. Ed.), pp.
361-376. Univ. of Missouri Press, Columbia.
Wapnir, R. A., Exeni, R. A., McVicar, M.. and Lifshitz, F. (1977). Experimental lead poisoning and
intestinal transport of glucose, amino acids, and sodium. Pediatr. Res. 11, 153.
Wapnir, R. A., Moak, S. A., Lifshitz, R, and Teicherg, S. (1979). Alterations of intestinal and renal
functions in rats after intraperitoneal injections of lead acetate. J. Lab. din. Med. 94, 144—151.
-------
( itrnczl and laboratory ubsvrvalions 147
'/ .««,:t, X, U.T.-:.-..V,A ?. LvrCr *KM. t.Hcvo. uf dt^pjn
j-»i •.:;•. ^-.-i r-gi4il> A ijuiMiid-
Si.c jiutf; ;.-.' rs3c-.«-s .>;.ti p!si:.T3 Wttr:»'.»-j'.'fi. Ada Nru.-'ji
StavJ &I:J7&. JV:0'
!0, M-.-rxr::; VJ . P.-:-,.;;., N, Ti/jinvni rj. [{.cj.'i E. Bchcc>!c G.
.S'.rj.-ijcn SM. Scrcui f". Dia/.quni elimination in premature
;::id full tcnn infant:, and children. J Pcrinat Mcd 1:133
i»7:>.
II. i.jr.j:sicl A. Mcbcrs A.-Brcdcscn JJ:, Lundc PKM: Plasma
ccm-cntraiion.-. of Jidrcpum and N-dcsmcihyldinzcpam in
:ic»l>orn infants after imravrnotts, intramuscular, rectal and
oral -administration.. Ada Paediatr Scand 67:699. I97H.
Reversible acute renal failure in lead poisoning
Abdyl J. Khan, M.D., UrmiJla Palel, M.D., Mohammed Rafceq, M.D.,
Alfred Myerson, M.D., Kasum Kumar, M.D., and Hugh E. Evans, M.D.
Brooklyn. A'. Y.
Ai.TMOt:fiii Tm; an i:cis or LEAU on [ho central ner-
vous and hcmalopoictic systems dominate the clinical
picture of lend poisoning, rc-.al manifestations may occa-
sionally be present.1-3 Kidney impairment also may be a
side effect of the dictating agents used in the therapy.3'5
Renal failure, however, is rare. We describe a patient with
severe acute renal failure associated with either chelation
therapy or preexisting lead ncphropathy, or both, and
further complicated by glucose-6-phosphatc dehydrogc-
nase (G-6-PD) deficiency.
CASE REPORT
A 26-momh-old black girl was admitted lo Jewish Hospital and
Medical Center of Brooklyn with anorexia, vomiting. lethargy,
and pallor of one week's deration. Her birth history and growth
and development were normal. On rouline screening three weeks
earlier at another hospital, elevated blood lead level (97 jtg/dl
whole blood) and free crythrocyte protoporphyrin concentrations
(1.026 pg/dl crythrocytcs) were found. She was hospitalized there
and given calcium disodium cdetatc (EDTA) 150 mg and dimcr-
cnprol (BAL) 48 mg, each intramuscularly every four hours for
seven days. Urinalyscs revealed protcinuria before, during, and
after therapy. Blood urea nitrogen level was 22 mg/dl before and
after therapy (Table). Scrum crcatininc concentration v.-a; uot
measured. At a follow-up visit at that hospital two weeks later,
blood lead concentration was 32 ^g/dl whole blood. The parents
had noted anorexia and intermittent vomiting since discharge, and
progressive pallor and lethargy.
Physical examination on admission at Jewish Hospit-1 and
Medical Center revealed a pale, mildly dehydrated child with
Fnmi Iht Dtpariment of Pediatrics. Jewish Hospital and Medical
(.'filter. Sliilf University of New York. Downstate Medical Cen-
ter.
Reprint address: Abdul J. Khan. ,U.O.. Department of Pediatrics.
SSS Prospect PL Brooklyn. i\T 112J8.
normal pulse, respiration, and temperature. Blood pressure was
92/64 mm 1 Ig. Her wi;;»ht was 24.5 kg, and height 90 cm (surface
area 0.8 m!). Physical examination was normal. On admission the
complete blood count revealed hcmatocrit 22.1%, reticulocytes
0.3%, platelets 340,000/mm3, and leukocytes 20,400/mm!, with a
normal differential count. The red blood cell indices indices (mean
corpuscular volume 66 ^ and mean corpuscular hemoglobin 21
Wig) and scrum Terrain concentration 8 Ag/dl (normal 15 to 300)
indicated iron-deficiency anemia. Basophilic stippling was not
seen and a sickle cell preparation was negative. Urinalysis
revealed proteinuria (3+) and 2 to 3 red blood cells per high-
power field (Table). BUN was 113 mg/dl, and scrum crcatininc
concentration 13.4 mg/dl. In view of the severe azotcmia (renal
failure), further management included complete restriction of
protein and appropriate intravenous fluid administration. Urine
output was adequate during the firsl 24 hours (2 ml/kg/hr), and
oliguria did not occur subsequently. Renal function studies,
including creatininc clearance and fractional excretion of sodium,
were monitored serially and showed gradual improvement (Ta-
ble). Because the hematocrit level declined to 17% on day 5, a
packed red blood cell transftision was given, raising il to 30%.
Bleeding and coagulation profiles were normal except for G-6-PD
deficiency. Hemoglobin clcclrophorcsis revealed AA pattern.
Results of scrum electrolyte (Na. K. Ca, phosphorus), comple-
ment, total protein, and albumin studies were normal, i'rotcin
clcctrophorcsis and immunoglobulins (Ig'G, IgA. IgM), intrave-
nous pyciogram. renal scan, and sonogram also were normal.
Serum haptoglobin concentration was 227'mg/dl (normal 25 to
ISO mg/dl). and C-rcactive protein was -f I. The initial crythro-
cyte sedimentation rate was elevated (90 mm/hr). but gradually
returned ro normal. Blood lead level on admission was 21 Mg/dl
whole blood, and free crylhrocytc protoporphyrin concentration
was 570 Aig/dl crythrocytcs. The scrum cholesterol concentration
was elevated (290 mg/dl) on admission, but declined to 225 mg/dl
after .1 week. Blood pressure fluctuated between 92/62 and
110/70 mm Hg. The patient was discharged after 17 days. Two
weeks later, results of urinalysis were normal. BUN was 19 mc/dl.
J
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: ilt"**'
' . ri;
.
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»i3 indicates renal injury.
serum crcatininc 0.7 mg/dl. and hem;itocrit 33%. Twenty months
after discharge the patient was found to be in excellent health,
witli normal urinalysis and renal function tests.
DISCUSSION
This patient had acute renal failure with reversibility
over a 12-day interval. Various measurements of renal
function, -including urine-to-plasma ratios of creatininc
and urea and fractional excretion of sodium, indicated a
renal parenchyma! involvement or intrinsic nephropathy
rather than prerenal azotemia.6-7 Biochemical abnormali-
ties on admission also included two of three criteria of
nephrotic syndrome, namely proteinuria >40 mg/m2/hr
and serum cholesterol >250 mg/dl, but hypoalbuminemia
• was absent. Further evidence of renal parenchymal injury
included elevated acute-phase reactants (positive C-reative
protein and elevated) erythrocyte sedimentation rate).
The cause of renal injury could not be established, but
chelation therapy must have played a major role. The dose
of EDTA given to this patient was excessive, and duration
of therapy was also prolonged (recommended -dose for
asymptomatic patients with elevated blood lead level is
1,000 mg/M2/day for three to five days8; this patient was
given 1,125 mg/M2/day for'seven days). Maintenance of
adequate hydration orally or intravenously during therapy
is helpful in preventing renal injury. Fluids were adminis-
tered by mouth but not intravenously, perhaps because the
patient was not toxic, and not vomiting, during chelation
therapy. She did develop evidence of dehydration at one
point (urine specific gravity 1.035) during therapy.
Although EDTA toxicity seemed to have played a major
role, therapy with BAL might also have contributed to the
pathogenesis of renal failure by causing intravascular -
hemolysis in the presence of G-6-PD deficiency.8 The dose
per 24 hours (360 mg/M2) did not exceed the recom-
mended daily dose of 500 mg/M2, but the duration of
therapy (seven days) was longer thari recommended (five
days).8 Although the hematocrit level did fall as a result of
hemolysis, other features of acute hemolytic anemia, such
as high reticulocyte count and depletion of haptoglobin,
wee absent.
The possibility of preexisting renal injury caused by lead
itself cannot be excluded. The presence of mildly elevated
BUN (22 mg/d!) and proteinuria and pyuria prior to
treatment indicates the possibility of lead nephropathy.
The manifestations of early lead nephropathy are usually
mild and transient and include abnormalities in urinalysis
in 25% to 30%'-' and amino aciduria in 14% of patients.'
This patient's urine was not examined for amino acids.
Whether chronic lead poisoning in childhood leads to
chronic renal failure later in life is unknown. In a retro-
spective study in Australia of 401 children admitted during
1915 to 1935 for plumbism, about 25% died of renal failure
and nephritis before reaching 40 years of age.10 In 1962
another retrospective study of 165 children in the United
States did not confirm this high incidence of nephropathy;
only one patient died about 20 years later with "chronic
nephritis" of unknown cause." A kidney biopsy was
deferred in our patient in view of the rapid improve-
ment.
-------
Clinical and lal»)ralory observations 149
C.'-, , t tt —i./
•• *t*r. i ,s~.,Jf
NO
Nf;
I ? ^
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77
0?
0?
02 D
4.0
4.4
5V
Jl!.0
1.3
-) j
2.0
68
y.7
S.4
This case emphasizes the need for caution in planning a
course of therapy for childhood lead poisoning. Initial and
serial evaluations of renal function (urinalysis, BUN, and
scrum crcatimnc) are needed. The decision to include BAL
in the therapy should also be guided by the presence or
absence of G-6-PD deficiency, as BAL can cause intravas-
cular hcmolysis in the presence of such deficiency.
9
10,
II.
:. PtioclicI SM. Kapilo L. Sciiwachman H: Children with an
incrca.vxl lead burxlcn: A icrccnin^ and follow-up. JAMA
Z22:Sb2. 1972.
. Cli!.x)im JJ Jr: Aninioaciduria as » manifcsiation of renal
tubular injury in lead intoxication and a comparison with
pattern of air.niaciduria seen in other diseases. J-HeoiATR
«H17. 1962. '
. Chiiolni JJ Jr: Increased lead absorption and lead poisoning
(p!u:nbi»rn). //, Vaughan VC, McKay RJ. Behrman RE.
editors: Nt-lion text of pediatrics, cd II. Philadelphia. 1979,
\\ B Saundcrs. p 2025.
. i-'uivnun H. Pinncgan C. Lushaugh CC: Nepholoxic hazard
from uncontrolled edathamil calcium-disodium therapy.
JAMA 160:1042, 1956.
. Kcubcr MD, Bradley JE: Acute versenate nephrosisoccurrinj;
a-, a result of treatment oflcad intoxication. JAMA 174:263,
I960.
. Sclirier RW: Acute renal failure. Kidney Int 15:205, 1979.
. Ingullingvr JK, Avner E: Renal disorders. In Gracf JW, Cone
Tli, editors: Manual of pcdiatric therapeutics. Boston, 1980,
Little Brown p 205.
Chisolm JJ Jr: The use of chclating agents in the treatment of
acute and chronic lead intoxication in childhood, j PHDIATR
73:1, 1968.
Smith HD: Pediatric lead poisoning. Arch Environ Health
8:68. 1964.
Henderson DA: A follow-up of cases of plumbism in children.
Aust Ann Med 3:219, 1954. '
Tepper LB: Renal function subsequent to childhood plum-
bism. Arch Environ Health 7:82, 1962.
Lithium therapy in childhood neutropenia
Pedro A. de Alarcon, M.D., Jack Goldberg, M.D., Douglas A. Nelson, M.D., and
James A. Stockman III, M.D. Syracuse, N.Y.
NEUTROPHILIA has been observed in most patients
treated with lithium carbonate for psychiatric disorders.1
Because of this observation, lithium carbonate has been
employed in the treatment of neutropenic states, such as
those associated with rheumatoid arthritis,1 chemothera-
py,3 and congenital neutropenia.4 The benefits of this form
of therapy have been variable.
We report three children with chronic neutropenia of
childhood treated with lithium carbonate. The neuirophil
From the Departments of Pediatrics. Medicine, and Pathology. .
Upstate Medical Center, Slate University of New York.
Reprint address: Pedro A. de Atarcon. M.D.. Department of
Pediatrics. The Mary Imogenc Basset! Hospital. Coonersiown
,\'Y
response of these psticnts is evaluated in vivo and in vitro
using double-layer agar culture techniques.
CSA Colony:stimulating activity
C FU: GM Colony-forming aniss'--granulceytcs
and macrophages
SUBJECTS AND METHODS
Three patients (two boys, one girl) with chronic neutro-
pcuia, were evaluated. They were 18 months, 24 months,
and 6 years of age, respectively. The diagnosis was based
on persistent neutropenia with absolute neutrophil counts
of < 1,500 cells/^l and recurrent infections without any.
other discernible cause for the neutropenia.
Their clinical course was classified according to the
0022-3476/83/OIOI49-f04S00.40/0 © 1983 The C. V. Mosby Co.
-------
Toxicology and Industrial Health, Vol. 8, No. 112,1992 89
RAPID HYPERTENSINOGENIC EFFECT OF LEAD: STUDIES
IN THE SPONTANEOUSLY HYPERTENSIVE RAT
FARID NAKHOUL*, LAURIE H. KAYNE*, NACHMAN BRAUTBARt,
MING-SHU HU*, ALICIA MCDONOUGH+, PETER EGGENA*,
MICHAEL S. GOLUB*, MORRIS BERGER*, CHWEN-TZUEI
CHANG+, NORA JAMGOTCHIAN*, AND DAVID B. N. LEE*
* Veterans Administration Medical Center
Sepulveda and University of California School of Medicine
Los Angeles, California
t Center for Internal, Occupational and Toxicological Medicine
Los Angeles, California
^Department of Physiology and Biophysics
University of Southern California School of Medicine
Los Angeles,. California
Chronic lead exposure may cause hypertension in normotensive rats. This
hypertensinogenic effect has been attributed to perturbations in the renin-
angiotensin axis, the contractile response of the vascular smooth muscle,
or the intracellular Ca2+homeostasis as a consequence of the inhibition of
Na+-K+-ATPase activity. In this study we examined the short-term effect
of lead exposure on blood pressure, plasma renin activity, vascular con-
tractility, and renal Na+-K+-ATPase activity and abundance in the sponta-
neousfy hypertensive rat. Our data indicate that modest lead exposure
• caused blood pressure elevation within two weeks in this rat strain that is
genetically susceptible to the development of hypertension. This rapid
blood pressure-elevating effect did not appear to depend on the mecha-
nisms described in hypertension associated with more chronic lead expo-
sure listed above. This acute model provides an additional approach to the
study of lead-induced hypertension.
INTRODUCTION
Epidemiological evidence in humans and experimental studies in animals suggest that
lead exposure may cause an elevation in blood pressure (BP) (Sharp et al., 1987).
1. Address all correspondence to: Nachman Brautbar, M.D., Center for Internal, Occupational
and Toxicological Medicine, 2222 Ocean View Avenue, Suite #100, Los Angeles, CA 90057.
2. Key Words: lead, hypertension, spontaneously hypertensive rat.
3. Abbreviations: BP, blood pressure; Pb-SHR, lead-treated spontaneously hypertensive rat;
PRA, plasma renin activity; SHR, spontaneously hypertensive rat; TNS, transmural nerve stimula-
tion.
Toxicology and Industrial Health, 8:1/2, pp. 89-102
Copyright 1992 Princeton Scientific Publishing Co., Inc,
ISSN: 0748-2337
-------
90 Nakhoul et al.
Three potential pathogenetic mechanisms for this lead-induced hypertension have been
studied mainly in the rat. These include the effect of lead exposure on the activity of
S renm-angiotensin axis (Fleisher et al, 1980; Victery et al, 1982a) on vascular ten-
sion and contractility (Webb et al, 1981), and on intracellular Ca2+ homeostasis
(Blaustein 1977- Pounds, 1984; Rosen and Pound, 1989). Special interest has been
directed toward characterizing the inhibitory effect of lead on Na+-K+-ATPase actmty
?Fox et al 1991; Kramer et al, 1986; Moreau et al, 1988; Weiler et al, 1988) which
in turn, would reduce Ca2+ efflux via Ca2+-Na+ exchanger and hence cause increases
in cystolic Ca activity (Pounds, 1984).
Most of these studies measured the effect of chronic lead exposure (>3-7 months) on
the postulated pathogenic mechanisms. It is not clear whether hypertension also devel-
ops following short-term exposure, and if so, whether alterations demonstrated in
chronic lead exposure are also operative in early phases of lead-induced hypertension.
In addition virtually all studies have been conducted in rats with no genetic predisposi-
tion to hypertension. It is possible that the BP-elevating effect of lead is exacerbated or
potentiated in hosts .that are genetically susceptible to the development of hypertensioa
In the current study we examined the effect of short-term (3 and 8 weeks), modest lead
exposure (100 ppm in drinking water) on BP, plasma renin activity, vascular contrac-
tility, and renal Na+-K+-ATPase activity and abundance in the spontaneously hyper-
tensive rat (SHR).
METHODS
Animal and Treatment Schedule
Male 6- to 7-week-old SHR weighing 100-120g (Harlen-Sprague-Dawley Indiana)
were first acclimatized in hanging cages, and fed Purina rat chow ad lib (Ralston
Purina, St. Louis, MO) and distilled water for 3 days. An average weight of' 150g[was
attdned at the beginning of the study (see weight at day "0", Figures 2 and 4). They
were then transferred into individual metabolic cages. Eight ^^/f^Tnm
the same feeding and drinking schedule while 8 experimental rats had lead acetate (100
ppm) added to their drinking water for three weeks (3-wk study). In addition an eight-
week study was conducted using 7 control and 8 experimental rats (8-wk study).
Body Weight and Blood Pressure Measurements
Bodv weight was recorded twice a week, prior to the measurement of tail artery sys-
' tolicBP using a tail sphygmomanometer (Friedman and Freed, 1949). Rats were pre-
wamied for 10-15 min on a heating pad with a surface temperature of 40°C and were
then transferred to a temperature-controlled restraining cage where BP was measured
without anaesthesia. Each measurement represented the average of 5 consecutive
readings, approximately 30 seconds apart. The sensitivity of any given reading was 4
mmHg.
-------
Toxicology and Industrial Health, Vol. 8, No. 112,1992 91
Blood, Urine, and Tissue Collection
Blood was collected at sacrifice at the end of 3-wk in the first study and 8-wk in the
second study. Trunk blood was collected after decapitation between 7:30 and 10:00
A.M. For plasma renin activity (PRA), blood was collected into ethylenediaminete-
traacetic acid and plasma was separated and stored at -10°C until assayed. In the 3-wk
study, blood for PRA was also collected at the end of the first week by tail-tip amputa-
tion. Serum creatinine, sodium, potassium, and lead were measured at the end of the 8-
wk study. A 24-hr urine collection was completed just prior to sacrifice in the 8-wk
study for analysis of protein, lead, calcium, and magnesium. Kidneys were also
rapidly excised and stored at -70°C pending assay for Na+-K+-ATPase activity and
abundance.
Femoral Artery Vascular Reactivity
At the end of the 8-wk study femoral arteries were rapidly isolated and cleaned of loose
fat and connective tissue. Contractile responses of 4mm arterial ring segments were
measured in vitro using a special assembly (Golub et al., 1989) modified after that of
Bevan and Osher (1972). The contractile response to transmural nerve stimulation
(TNS) was obtained following 2 hours of equilibration at 2 gm passive tension. Each
preparation was stimulated for 30-second intervals with increasing frequencies (1,2,
4> 8, 16, 24, and 40 Hz) of constant voltage pulses (17 volts, 0.5 msecs duration).
These stimulation parameters produced contractions which were totally blocked by
tetrodotoxin (10-6M, Sigma, St. .Louis, MO) confirming that smooth muscle was not
directly stimulated. A cumulative dose-response curve to exogenous norepinephrine
(1 x lO-8 to 6.5 x 10-5M) was also obtained. All vascular segments were subsequently
incubated in low potassium (0.6 mM) Krebs-bicarbonate buffer for 15 min, then
precontracted with norepinephrine (5 x 10-7M). After these contractions reached a
plateau (5 min), cumulative relaxation responses were obtained by increasing the bath
KC1 concentration in 0.2-0.4 mM increments, until no further tension changes were
obtained. At the conclusion of each experiment the arterial segment was again stabilized
in Krebs-bicarbonate buffer with normal potassium (4.7 mM), and the maximal
contraction in 100 mM KC1 was measured. Vascular segments were dried overnight at
100°C and weighed on an electronic microbalance (Cahn Model 26, Torrance, CA).
Contractile responses to TNS and norepinephrine were expressed as a percent of the
agonist's maximal response. Cumulative relaxation responses to the readdition of KC1
were expressed as a percent of the norepinephrine response in 0.6 mM KC1. Dose-
response EDso (agonist concentration which produces a 50% of maximal contraction
or relaxation) was calculated from a linear regression of 15-85% response range and
compared utilizing Student's t test.
Assay Methods
Serum creatinine, sodium, potassium, urine protein, calcium, and magnesium were
measured using standard laboratory procedures. Serum and urine lead were.measured
using atomic absorption spectrophotometry (SmithKline Bio-Science Laboratories,
-------
92 Nakhoul et al.
Van Nuys, CA). PRA was assayed as described in prior publications (Eggena et al.,
1991). Renal cortical and medullary Na+ -K> -ATPase activity and abundance were
assessed in crude microsomes prepared as described by Schmitt and McDonough
(1986). Activity was measured as ouabain-sensitive inorganic phosphate liberation
from ATP and expressed as umol phosphate per mg protein per hour (Lo et al., 1976).
Protein concentration was measured by the method of Lowry et al. (1951), using
bovine serum albumin as the standard Immunoblot analysis of Na+ -K+ -ATPase
subunit abundance was carried out using a modification of the method reported by
Renart et al. (1979). Briefly, 25 ug homogenate protein was resolved by SDS-PAGE
and electrophoretically blotted onto diazophenylthioether (DPT) paper. The DPT paper
was incubated with a combination of rat anti-a monoclonal antibody (McKl diluted
1:200, from K. Sweadner, Harvard Medical School) and anti-Bi antiserum (diluted
1:100, from R. Leverson, Yale Medical School).
Statistical Treatment of Data
All data were presented as mean±SEM. Student's t-test was used for determination of
significant difference between variables, using a standard statistics package (Winer,
1962). ' .
RESULTS
Blood Pressure and Body Weight
In the 3-wk study, both the control and the experimental SHR demonstrated steady BP
for the first 8 days (Figure 1). On days 12 and 14, systolic BP rose in the lead-treated
SHR, but not in the control SHR. Thereafter, the BP in both groups exhibited incre-
mental rises, but with the lead-treated rats displaying significantly higher systolic BP
than the control rats for all measurements. Figure 2 depicts the progressive weight gain
in the two groups of rats. No difference was observed between the control and the
lead-treated rats.
6
J,
Ed
01
D
CO
CO
Ed
01
D-,
Q
O
O
m
170-1
160-
150-
140-
130-
120-
O - O =Control Group
• - • =Lead Group
*=p<0.001.
0
4
17
20
8 12 14
DAYS
FIGURE 1. Systolic blood pressure in control and lead-treated rats in the 3-wk study.
-------
Toxicology and Industrial Health, Vol. 8, No. 112,1992 93
•i
C
c
EIGHT I
£s-
>•«
Q
O
oa
230-
220-
210-
200-
190-
180-
170-
160-
150 -
t
O O =Control Group ' I
• • =Lead Group •
/ O
T //\
k/i
/
-x*^<»-"*" II
L^^>T J 1
A
V
. •*
04 8 12 14 17 20
DAYS
FIGURE 2. Body weight of control and lead-treated rats in the 3-wk study.
jg5
~
^
t3
JD
K
Cu
Q
0
0
J
D3
190n
180-
170-
160-
150-
140-
130-
120-
^
O — O =Control Group
• — • =Lead Group T
*=p<0.001 i^^ *
* * t \/ ^•'^
* * I-'*"
* r T^^
0^^
/ 0 0 Q
T/ /i^?"9 9 ?
•^* X"
T T /o — O— o
i JL^— — ^o*^"'i i -^
o-^yi
:I
*
^i
— a
03 8111417 22263033384145 51 58
DAYS
FIGURE 3. Systolic blood pressure in control and lead-treated rats in the 8-wk study.
Figure 3 summarizes the BP changes in the second study, confirming the observation
of significantly higher BP in the lead-treated SHR versus control SHR by the end of
the second week. BP in the control SHR also rose during the third week, however, the
lead-treated SHR had higher BP throughout the remainder of the 8-wk (58 days)
study. In this study, the lead-treated group demonstrated significantly higher body
weight from day 30 through the end of the study (Figure 4).
-------
94 Nakhouletal.
Blood and Urine Chemistry
The blood level in 7 rats after 8-wk lead treatment was 5.3±3.0 (range 4-8) mcg/dl, or
2.5 (range 1.9 to 3.9) x 10-7M. The blood level in all 7 control rats was less than 2
mcg/dl, the lower detection limit. Urine lead concentration and 24-hr urine volume and
lead excretion on the last day of the 8-wk study are depicted in Figure 5. Compared to
control rats, all lead-treated'rats had higher urine lead concentration, urine volume, and
daily lead excretion. Serum concentrations of creatinine, sodium, potassium, and 24-hr
calcium and magnesium did not differ between the control and the lead-exposed rats
(Table 1>. Neither treatment group had measurable protein in the urine.
SCO-
'S 280-
00
^ 260-1
£2 240-
O O=Control Group
• • =Lead Group
*=p(0.001
1
^ 220-
0 200-
CQ
180-
160-
TXo7
T^/l
m^~ /
t Jw r\
J$ •!•
T/
< 7
n J , — , 1 — <—
-9"""i
1
03 8 111417 22 263033 3841 45 51 58
DAYS •
FIGURE 4. Body weight of control and lead-treated rats in the 8-wk study.
A
150i '
B
00
o
U^
-------
Toxicology and Industrial Health, Vol. 8, No. 1/2,1992 95
Plasma Renin Activities (PRA)
Figure 6 summarizes the PRA in the control and experiment rats in both the 3-wk and
the 8-wk studies. The 1 week measurements (Figure 6A) were based on blood col-
lected by tail-tip amputation in the 3-wk study, and the 3 week (Figure 6B) and the 8
week (Figure 6C) measurements were based on trunk blood collected at the end of each
of the two studies, respectively. PRA was not different between the control and the
lead-treated rats at all three time-points.
TABLE 1. Serum and Urine Chemistry Parameters in the 8-Wk Study
SERUM
Creat.
Cont
O.4
O.3
0.4
O.7
O.4
O.3
Mean= 0.41
SE= O.O6
mg/d!
Lead
O.3
O.5
O.3
O.3
O.3
0.3
O.33
O.O6
Na* mmol/l
Cont Lead
15O 154
148 142
1 53 1 53'
156 149
163 148
1 49 1 46
1 53 1 48
2 2
KTnmol/l
Cont Lead
6.3 6.4
8.O 8.1
8.3 8.8
8.6 7.7
'8.7 7.8"
7.3 7.9
7.8 7.7
O.4 O.3
URINE
Ca+*mg/24hr
Cont Lead
O.14 O.12
O.O8 O.1O
0.08 O.13
0.10 O.O8
O.O6 O.1O
O.14 O.16
O.OO 'O.I 5
. 0.13 O.O8
0.09 O.1 1
O.O1 O.01
Mg-"-mg/24hr
Cont
O.32
O.21
0.28
0.25
0.20
O.45
O.OO
O.45
O.27
O.O5
Lead
0.42
O.29
O.55
O.28
O.21
0.45
O.47
O.20'
O.36
O.O4
TABLE 2. Effect of Lead-Exposure on Vascular Contractility and
Relaxation In Vitro
A. CONTRACTILE RESPONSE
Procedure (N)
TNS
Maximal Response, gms
NE
KCI, (lOOmM)
Control (6)
Lead (8)
Control (7)
Lead (8)
Control (5)
Lead (6)
6.2 ± 1.1 (Hz)
7.7 ± 1.1 (Hz)
3.4 ± 0.1 (10-7M)
3.8 ±0.1 (10-7M)
1.4± 0.4
1.7 ± 0.3
3.3 ± 0.5
3.9 ± 0.5
1:8 ± 0.3
2.1 ± 0.3
B. POTASSIUM-INDUCED RELAXATION RESPONSE
Procedure EPfp. mM K+ Maximal Response, gms
Control (6)
Lead (8)
0.5 ± 0.1
0.5 ± 0.1
1.8
2.0
±0.5
±0.3
Femoral Artery Contractility
The effect of lead treatment for 8 weeks on various indices of femoral artery vascular
responsiveness are summarized in Table 2. The EDso to both TNS and norepinephrine
-------
96 Nakhoul et al.
was not different between the lead-treated and the control rats, suggesting that lead
treatment did not alter vascular sensitivity to norepinephrine, either from TNS-induced
endogenous release or from an exogenous source. The lead-treated SHR did exhibit an
apparent lower response to TNS, but the difference was significant only at 8 Hz (lead,
n=8 45±4% versus control, n=6, 55±3%, p<0.05). Maximal contractile response to
TNS, norepinephrine, or 100 MM potassium also did not differ between the lead-
treated and control vascular preparations (Table 2 A). In addition, the sensitivity (EDso)
and the maximal relaxation response to potassium-induced vascular relaxation were
also similar in the two treatment groups (Table 2B). The maximal relaxation of both
lead-treated and control groups to the readdition of potassium, expressed as a percent
of the norepinephrine response in low potassium, was similar (lead 106±3 versus con-
trol 110±4%)' Finally, there was no significant difference in the dry vessel weights
between both groups (lead-treated 0.210±0.005 versus control 0.210±0.010 mg. per 4
mm segment). 30]iwEEK T ^^
SLead
oo
c
3 WEEKS
3Ch
E
~oc
FIGURE 6. Plasma renin activity
(PRA) in control and lead-treated rats.
I WEEK. Measurements on tail blood
at the end of one week in the 3-wk
study. 3 WEEKS. Measurements on
trunk blood at the end of 3-wk study.
8 WEEKS. Measurements on trunk
blood at the end of the 8-wk study.
"ob
1O-
8 WEEKS
Renal Na+ -K+ -ATPase Abundance and Activity
Na+ -K> -ATPase a and B subunit abundance measured in crude membrane from renal
cortex and medulla'of the 3-wk study was not different between control and lead-
-------
Toxicology and Industrial Health, Vol. 8, No. 112,1992 97
treated rats (Figure 7). Renal cortical and medullary Na+ -K+ -ATPase activities, mea-
sured at 3- and 8-wk were also not different between control and lead-treated rats.
DISCUSSION
With the exception of Aviv et ail (1980), who observed BP increases after 6 weeks of
lead treatment, all other studies were conducted following 3-7 months lead exposure
(lannacone et al., 1981; Victery, 1988). These studies used normotensive rats not pre-
disposed to the development of hypertension. Our study suggests that in SHR, lead
exposure causes accelerated BP elevation within two weeks. Thus, genetic predisposi-
tion to hypertension may constitute one factor for the proposed heterogeneity in host-
susceptibility to lead toxicity (Raghvan, et al., 1980). We have no explanation for the
higher body weight in SHR exposed to lead for more than 30 days (Figure 4). This
increase in body weight did not appear to be important for the observed increase in BP
which'manifested itself between days 12 and 17 in both the 3-wk and the 8-wk study.
The increase in urine volume in the absence of weight loss suggests the possibility of
increased water consumption in the lead-treated rats.
C C Pb Pb
C C Pb Pb
FIGURE 7. Immunoblot analysis of
Na+-K+-ATPase cc and 6 subunit
abundance in renal cortex and medulla
from control (C) and lead-treated (Pb)
rats. Analysis carried out using 25ug
homogenate protein per lane, probed
with I125-labeled anti-a and anti-Bj
antibodies.
CORTEX
MEDULLA
Two other studies on the effect of lead exposure in SHR have been reported. Evis et al.
(1987) studied the-effect of chronic lead exposure (3 months) on experimentally
induced cardiac arrhythmias in post-weaning (4-week-old) SHR. They noted that at 5
weeks, SHR which drank water containing 250 or 1,000 ppm lead had marked and
significantly higher BP than SHR which drank lead-free water. No mention was made
concerning whether BP was measured prior to 5 weeks. In contrast, Wiecek et al.
(1986), studying 12-week-old (100-150g) male SHR treated for 10 weeks with 0, 0.5,
0.1, and 1.0% lead acetate in drinking water (0, 250, 2,500, and. 5,000 ppm, respec-
-------
98 Nakhoul et al.
i
lively) observed a BP-lowering effect in a dose-dependent manner. Parenthetically, the
body weight of the 12-week-old male SHR used was unusually low because the
expected weight would range between 240 and 260g.
The unanticipated observation of Wiecek et al. (1986) may be explained, in part, by the
study of Victery et al. (1982a). These latter authors noted that while 100 ppm of lead in
the drinking water led to the development of hypertension in the male rat, (Sprague-
Dawley, confirmed through personal communication), exposure to higher doses
negated the hypertensinogenic effect (Victery et al., 1982a). Although Victery et al.
(1982a) did not use SHR, our study would confirm that 100 ppm drinking water is
also "hypertensinogenic" in SHR. This observation, in combination with those of
Wiecek et al. (1986) (whose SHR received 250 ppm or higher doses of lead) would
suggest a biphasic effect of lead on BP in SHR, that is, a BP-elevating effect at 100
ppm and a BP-lowering effect at higher doses. Such a hypothesis, while dovetailing
with the observation of Victery et al. (1982a), is at variance with the observation of
Evis et al. (1987), who documented a clear BP-elevating effect in SHR given 250 and
1,000 ppm lead in the drinking water.
It may be relevant to point out that Wiecek et al. (1986) noted that "despite the absence
of elevated basal BP in surviving Pb-SHR, a high proportion of Pb-exposed SHR died
from cerebral hemorrhage." It was not clear whether the reported BP was only taken
from the surviving Pb-SHR. It is interesting to note that Evis et al. (1987) also ob-
served that at 8 weeks, 7 of the 8 high-lead-dose rats died upon warming in preparation
for BP measurements. The reason for this fatal sensitivity to heat and the direct cause
of death were not investigated.
The rapid manifestation of the hypertensinogenic effect of lead in our animals allowed
us to examine the question of whether the pathogenetic mechanisms believed to be im-
portant in sustaining the hypertension associated with chronic lead exposure are also
involved in the early phases of lead-induced hypertension. As mentioned earlier, previ-
ous studies have focused on three major pathogenic mechanisms, i.e., the effect of lead
exposure on the activity of the renin-angiotensin axis (Fleisher et al., 1980; Keiser et
al., 1983; Victery et al., 1982a), on vascular tension and contractility (Webb et al.,
1981), and on Na+ -K+ -ATPase activity and the consequent effect on intracellular
Ca2+ homeostasis (Blaustein, 1977; Kramer et al., 1986;Weiler et al., 1988).
Lead exposure has been reported to cause either an increase, no change, or a decrease
in PRA in the rat, and appears to be influenced by factors such as the dose and duration
of lead exposure and the age of the animal when exposure first began (Vander, 1988).
In one-month-old rats whose exposure to lead began in utero, PRA was increased
compared to age-matched controls (Victery et al., 1983). However, in rats in which
lead exposure was prolonged 5 to 6 months, either no change or a reduction in PRA
-------
Toxicology and Industrial Health, Vol. 8, No. 1/2,1992 99
was observed (Victery et al., 1982a). In the chronic exposure studies it was observed
||| that lead-induced hypertension was associated with low PRA (Victery et al., 1982a;
W 1982b). However, it was postulated that the hypertension may have first gone through
a high-renin phase during the earlier stages of lead exposure (Victery et al., 1982a;
1982b). In our study, measurement of PRA at 1 week and 3 weeks, a time period
;- bracketing the acceleration of hypertension in the lead-exposed rats, failed to demon-
strate such an early, high-renin phase (Figure 6, A and B), suggesting that the rapid
j: initiation of lead-induced BP elevation can occur independent of changes in PRA. PRA
:|| " measured at the end of the 8-wk study (Figure 6C) also failed to demonstrate a differ-
^ ence between the control and the lead-exposed rats.
jj Webb and associates (1981) studied the effect of lead exposure on the vascular reactiv-
iity of the tail artery in the rat. They observed an increase in BP in adult male Wistar rats
given drinking water containing 100 ppm lead, as lead acetate, for 7 months (147±4
: versus 133±4 mmHg in age-matched controls, p<0.05). In a 1981 study, Webb et al.
obtained findings that were similar to ours; they noted no difference between the treated
and control rats in contractile response to high concentrations of extracellular KC1 or to
electrical-field stimulation of the adrenergic nerve endings. They did demonstrate that at
||, a given concentration of either methoxamine or norepinephrine the contractile force was
If greater in arteries form lead-treated rats. The authors suggested that the lead-induced
£ hypertension was associated with an increased vascular responsiveness to a-adrenergic
J| agonists. However, neither their study nor the present data demonstrated a difference
if in sensitivity to adrenergic stimuli (as reflected by ED$Q values). In addition to differ-
j| ence in rat strain and blood vessel preparation, Webb's group extended lead treatment
'"* to 7 months, which increased the likelihood of vascular structural adaptations to hyper-
tension. Our study thus suggests that lead-induced hypertension can occur prior to the
development of adrenergic hyperresponsiveness.
Blaustein (1977) and Haddy and Overbeck (1976) have reviewed the importance of the
Na electrochemical gradient across the sarcolemma in the regulation of vascular smooth
muscle tension and contractility. For example, an inhibition of Na+ -K+ -ATPase activ-
ity by a humoral agent such as the natriuretic hormone can lead to a reduction in the
Na-gradient directed into the cell, the driving force for extruding intracellular Ca
through the Na-Ca exchanger. The consequence is an increase in intracellular Ca activ-
ity and thereby an augmentation in vascular smooth muscle tension and contractility.
Kramer et al. (1986) reported a 50% inhibition (150) of Na+ K+ -ATPase activity in rat
renal cortical homogenate incubated in 7 x 10-5M lead chloride. A subsequent study
from the same laboratory using purified Na+ -K+ -ATPase from hog cerebral cortex
also demonstrated an ISQ of 8 x 10-5M for lead (Weiler et al., 1988). In our in vivo
study the blood lead level attained ranged between 2 to 4 x 10-7M (4 to 8 ug/1), a con-
centration two orders of magnitude lower than those used in in vitro studies. This may
explain our failure to demonstrate a change in Na+ -K+ -ATPase activity .and a and 6
-------
100 Nakhouletal.
subunit abundance in either the cortical or the medullary homogenates of kidneys from
lead-treated rats. Fox et al. (1991) also noted that renal Na+ -K+ -ATPase activity in
adult (90 days) rats with blood lead levels of 5-7 ug/dl was not reduced compared to
that found in control rats not previously exposed to lead. Our finding that potassium-
dependent" vascular relaxation was not different between the control and the lead-
exposed rats was also consistent with a lack of an effect of lead treatment on the Na+
K+ -ATPase system in the vascular smooth muscle. Previous vascular contractility
studies have shown that potassium-induced relaxation of precontracted vascular
smooth muscle is ouabain-sensitive and correlates with Na+ -K> -ATPase activity
(Webb and Bohr, 1978). The data taken together would suggest that the effect of lead
on blood pressure is not a direct consequence of changes in the Na-Pump activity or its
pool size.
In conclusion, we found that modest lead exposure used to induce hypertension in
chronic animal studies can cause rapid BP elevation in rats genetically susceptible to the
development of hypertension. In such early-phase lead exposure the initiation of the
BP elevation effect appears to be independent of the conventional mechanisms
discussed. This acute lead-induced hypertension model provides an additional
approach to the study of this important disorder.
ACKNOWLEDGMENTS
This study was supported in part by funds provided by the Department of Veterans
Affairs and the Center for Internal, Occupational and Toxicological Medicine, Los
Angeles, California. Drs. Farid and Kayne were supported by fellowship awards from
the American Heart Association, Greater Los Angeles Affiliate, and Dr. Hu was
supported by a research fellowship from the National Kidney Foundation of Southern
California.
REFERENCES
AVIV, A., JOHN, E., BERNSTEIN, J., GOLDSMITH, I., and SPITZER, A. (1980). "Lead intoxication
during development: Its late effect on kidney function and blood pressure." Kidney Int. 17:
430-437.
BEVAN, J.A. and OSHER, J.V. (1972). "A direct method for recording tension changes in the wall
of small blood vessels in vitro." Agent actions 2: 247-260.
BLAUSTEIN, M.P. (1977). "Sodium ions, calcium ions, blood pressure regulation, and hyperten-
sion: A reassessment and a hypothesis." Am J. Physiology 232: C165-C173.
EGGENA, P., WILLSEY, P., TRUCKENBROD, L., JAMGOTCHIAN, N., HU, M.S., BARRETT, J.D.,
CLEGG, K., NAKHOUL, R, and LEE, D.B.N. (1991). "The influence of recombinant human ery-
thropoietin on blood pressure, and tissue renin angiotensin system of the rat." Am. J. Physi-
ology 261 (5) Part 1, pp. E642-646.
EVIS, MJ., DHALIWAL, K., KANE, K.A., MOORE, M.R., and PARRATT, J.R. (1987). "The effects
of chronic lead treatment and hypertension on the severity of cardiac arrhythmias induced by
-------
Toxicology and Industrial Health, Vol. 8, No. 1/2,1992 101
coronary artery occlusion or by noradrenaline in anaesthetized rats." Arch. Toxicol. 59: 336-
340.
FLEISHER, N., MOUN, D.R., and VANDER, AJ. (1980). "Chronic effects of lead on renin and renal
sodium excretion." J. Lab. Clin. Med. 95: 759.
FOX, D.A., RUBINSTEIN, S.D., and HSU, P. (1991). "Developmental lead exposure inhibits adult
rat retinal, but not kidney Na,K-ATPase." Toxicology and Applied Pharmacology 109: 482-
493.
FRIEDMAN, M. and FREED, S.C. (1949). "Microphonic manometer for indirect determination of
systolic blood pressure in the rat." Proc. Soc. Exp. Biol. Med. 70: 670-672.
GOLUB, M.S., LUSTIG, S., BERGER, M., and LEE, D.B.N. (1989). "Altered vascular responses in
cyclosporin-treated rats." Transplantation 48: 116-118.
HADDY, F.J. and OVERBECK, H.W. (1976). "The role of humoral agents in volume expanded
hypertension." Life Science 19: 935-948.
IANNACONE, A., CARMIGANI, M., and BOSCOLO, P. (1981). "Neurogenic and humoral mecha-
nisms in arterial hypertension of chronically lead-exposed rats." Med. Lav. 72: 13-21.
KEISER, J.A., VANDER, A.J., and GERMAIN, C.L. (1983). "Clearance of renin in unanaesthetized
rats: Effects of chronic lead exposure." Toxicology and Applied Pharmacology 69: 127-137.
KRAMER, H., GONICK, H., and LU, E. (1986). "In vitro inhibition of Na,K-ATPase by trace
metals: Relation to renal and cardiovascular damage." Nephron 44: 329-336.
LO, C.S., AUGUST, T.R., LIEBERMAN, U.A., and DELMAN, I.S. (1976). "Dependence of renal
Na,K-adenosine triphosphate activity on thyroid status." J. Biol. Chem. 251: 7826-7833.
LOWRY, O.H., ROSEBROUGH, N.J., FAW, A.L., and RANDALL, RJ. (1951). "Protein measure-
ment with the folin phenol reagent." J. Biol. Chem. 193: 256-275.
MOREAU, T., HANNAERT, P., ORSSAUD, G., HUEL, G., GARAY, R.P., CLAUDE, J.R., JUGUET,
B., FESTY, B., and LEIIOUCH, J. (1988). "Influence of membrane sodium transport upon the
relation between blood lead and blood pressure in a general male population." Environmental
•Health Perspective 78: 47-51.
POUNDS, J.G. (1984). "Effect of lead intoxication on calcium homeostasis and calcium-mediated
cell function. A review." Neurotoxicology 5: 295-332.
RAGHVAN, S.R.V., CULVER, B.D., and GONICK, H.C. (1980). "Erythrocyte lead-binding protein
after occupational exposure. 1. Relationship to lead toxicity." Environmental Research 22:
264-270.
RENART, J., REISER, J., and STARK, G.R. (1979). "Transfer of proteins from gels to diazobenzy-
loxymethyl-paper and detection with antisera: A method for studying antibody specificity and
antigen structure." Natl. Acad. Sci. 76: 3116-3120.
ROSEN, J.F. and POUNDS, J.G. (1989). "Quantitative interaction between Pb2+ and Ca2+ home-
ostasis in cultured osteoclastic bone-cells." Toxicology and Applied Pharmacology 98: 530-
. 543.
SCHMITT, C.A. and MCDONOUGH, A.A. (1986). "Developmental and thyroid hormone regulation
of two molecular forms of Na.K-ATPase in brain." J. Biol. Chem. 261: 10439-10444.
SHARP, D.S., BECKER, C.E., and SMITH, A.H. (1987). "Chronic low-level lead exposure. Its role
in the pathogenesis of hypertension." Medical Toxicology 2: 210-232.
SHIONO, K. and SOKABE, H. (1976). "Renin-angiotensin system in spontaneously hypertensive
rats." Am. J. Physiol. 231: 1295-1299.
VANDER, A.J. (1988). "Chronic effects of lead on the renin-angiotensin system." En •ironmental
Health Perspective 78: 77-83.
VICTERY, W. (1988). "Evidence for effects of chronic lead exposure on blood pressure in experi-
mental animals: An overview. Environmental Health Perspective 78: 71-76.
VICTERY, W., VANDER, A.J., SCHOEPS, P., and GERMAIN, C. (1983). "Plasma renin is increased
in young rats exposed to lead in utero and during nursing." Proc. Soc. Biol. Med. 172: 1-7.
VICTERY, W., VANDER, A.J., SHULAK, J.M., SCHOEPS, P., and JULIUS, P. (1982a). "Lead,
hypertension, and the renin-angiotensin system in rats." J. Lab. Clin. Med. 99: 354.
VICTERY, W., VANDER, A.J., MARKEL, A.J., KATZMAN. L., SHULAK, J.M., and GERMAIN, C.
(1982b). "Lead exposure begun in utero decreases renin and angiotensin II in adult rats." Proc.
Soc. Exp. Biol. Med. 170: 63-67.
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102 Nakhouletal.
WEBB, R.C, WINQWIST, RJ, VICTERY, W.. and VANDER "-W^™* and
effects of lead on vascular reactivity in rats." Am. J. PhysioL 241. H21 1-H216.
WEBB, R.C. and BOHR, D.F. (1978). "Potassium-induced relaxation as an indicator of Na -K
ATPase activity in vascular smooth muscle." Blood vessels 15: 1V5-2U/.
WffiCEK A MANN J.F E., NOWACK, R., and EBERHARD, R. (1986). "Effects of lead mtoxica-
Sn 'on blood ^cnuM and vascular reactivity in spontaneously hypertensive rats." Journal of
-^ GONICK, H. (1988), "Effects of lead and na^uretic
horrnone on kinetics of sodium-potassium-activated, adenosme tnphosphates: Possible rele-
vance to hypertension." Environmental Health Perspective 78:^ 113-115.
WINER, B.J. (1962). Statistical Principles in Experimental Design. McGraw-Hill, New York, NY.
-------
A SURVEY STUDY OF LEAD IN
DRINKING WATER SUPPLIED BY
TRANSIENT WATER SYSTEMS
'Richard P. Maas
Steven C.'Patch
Diane M. Morgan
Geoffrey M. Brown
US EPA Project Manager: Jeff Cohen
Technical Report #5-019
DECEMBER 1995
ENVIRONMENTAL QUALITY INSTITUTE
- THE UNIVERSITY OF NORTH CAROLINA Af ASHEVIUE
-------
-------
I. BACKGROUND AND INTRODUCTION
The National Primary Drinking Water Regulation for Lead ("The Rule") requires many
- public water systems to monitor for lead in tap water, and to take various steps (including corrosion
control, public education, and lead service live line replacement) to reduce lead exposure for their
customers if the levels are found to be elevated. The Rule applies to "community" water svstems
'(those which serve at least 25 people year round), and to "non-transient, non-corrmiunity"' water
' systems (serving at least 25 of the same people for at least six months of the year). The Rule
excludes systems referred to as "transient non-community" water systems. These systems cater
to transitory customers in non-residential areas such as campgrounds, motels, and gas stations. The
US EPA excluded transient systems because adverse health effects' caused by lead hi drinking
water would generally be associated with long-term cumulative exposures rather than the shon-
•term exposures predicted from these types of systems. The purpose of this study was to collect
• actual data on lead in drinking water from transient systems-in order to better characterize potential
exposure'risks. '
II. METHODOLOGY
The names and addresses of 8000 transient water systems were supplied to the EQI by the
US EPA, Office of Groundwater and Drinking Water. A letter was sent to all 8000 systems
• (approximately 800 in each of the ten EPA regions) asking for'their participation in the study in
return for the EQI laboratory providing free lead testing of their drinking water. The letter
emphasized that no action, regulatory or otherwise, would be-taken contingent on sample results
and that all individual results would be kept confidential. Two-hundred-eleven request forms were
returned from the pool of systems. A test kit with laboratory cleaned bottles, sampling instructions
(Attachment 21), and a research questionnaire (Attachment £2) was sent to'each of these 211
respondents. Over a three month period, which included two sets of follow-up phone calls, a total
of 115 systems returned their samples, with these participants then forming the database for the
survey analysis described herein.
III. RESULTS AND DISCUSSION
As noted above, 8000 geographically distributed transient water systems were invited to
participate in the survey study and thereby receive free confidential lead-testing. The actual survey
population is a self-selected group based on their commitment to follow through with testing and
returning samples'and, thus,'may be a biased sample in one respect or another. Table 1 below
• shows the number of sites separated by system classification and by their first draw lead levels.
Although no rigorous statistical analysis of the original potential study population of 8000 sites was
conducted, the actual 115 respondents appear to represent a wide range of the types of systems
envisioned for the study. There may be a slight over-representation of governmental buildings (15)
and park visitor centers (19),.which is probably to be expected because these personnel might be
• more apt to participate in a government-sponsored survey study.
-------
Lead Levels lnd
Type of System
Number
es
Gas Station
•' ' i.-
Campground Building
—— .—,
Campground Hookup
Motel/Hotel/Lodge
Highway Rest Area
Restaurant/Bar
Ski Resort
Store
Mobile Home
Visitor Center
•••'" -•• ._
Residence-
Government Building
Other
A Mantel.Haenszel Chi-Square test for differences between system types revealed no statistically
Significant differences in the risk of first draw lead contamination as a function of system type (p
Table 2 summarizes 'first draw lead concentration ranges as a function of the type of-water
dispensing device. Again, no statistically significant differences were observed between the
various types (p = 0.67).
-------
Some general descriptive statistics derived from the
Table 3.
survey data are presented below
in
TABLE 3. General Descriptive Statistics of Transient System Lead
Draw
(Max)'
Quantile Concentrations
One-Minute Purged Line Lead Quantile Concentrations
; • •
!0'0%(Max> -
99%
95%
90%
75%
=
"58.3
"30.4
10.4
3.40
1.80
50% (Median)
25%
10%
Arithmetic Mean
Percent > 15 '
=====
0.70
<0.3
<0.3
2.30
-2.60
-------
"' l
90th P€rcentile ^ draw concentration is ?0 3
l5u8fL ^— rage fot draw concenidon
nenn " *« Upper fa°md of *e ^lean exposure of a
person dnnkmg water from these, transient systems. The average one-minute pur^d-L
concentrate was 2.3, which probably represents a good estimate of the lower boL "
visitor exposure at facilities served by transient water systems. Also, it can be seen
minute purge reduces the mean lead exposure by approximately 75%.
It should be noted that -the statistics presented up to this point include plumbing systems
of all ages rather than just the 1983-I988.age range for Tier 1 residences under the Rule Table 4
shows the breakdown of first draw lead results by age-of-piumbing systems.
Description
Age Category (Yrs.)
0 --4.9
5.0 - 9.9
• 10-19.9
20+ •
.
Unknown
Number of
Samples
3
8
26
48
~30
• Lead Concentration Range (ug/1)
Percent
66.7
62.5
69.2
68.8 ...
• 67.6
Percent
5-15 '
• 33.3
37.5
15.4
14.6-
=======
22.4
Percent
0.0
0.0
15.4
• 16.7
=
10.0
As can be seen from Table 4, very few facilities had new plumbing systems, and the distribution
of elevated lead levels shows very little dependence on plumbing system age (p = .66). Previous
EQI research using much larger residential .databases have observed this same trend.
Table 5 shows first draw lead levels as a function of the age of the water dispensing device.
As can be seen from the data the percentage of samples having lead levels above 15 ug/1 was
virtually identical for all three age groups, and no statistically significant association between
dispenser age and.lead concentrations was observed (p = 0.83).
-------
Lead Levels by Reported
Lead Concentration Range (ug/1)
TTie statistical association between water standing time in the plumbic
°nm *™
system and
eh
the extent to which visitors to these transient systems might actually be exposed to water
with excessive standing times was beyond the scope of this study. In most cases it seems
likely that only small percentages of visitors would encounter such circumstances
on
TABLE 6. First Draw Lead Levels by Reported Water sendin
Descrip
v*
Standing Time
Category (Hrs)
<6
6 -.10
10 - 14
144-
Unknowh
tion
Number of
Samples
10
30
"7"1
34
18.
Concentration Range (ug/1)
Percent
<5
'90.0
-T*> 1
/J.J
• 73.9
55.9
72.2
Percent
5-15
10.0 _ '
13.3
17.4 '
23.5
16.7
Percent
>15
0.0
13.3
8.70
20.6
11.1
-------
sates M «£ onfstS? * "*? °f "« P'°^' Compassed 35 sta.es. Nine
Virginia f6), New Yo^fj T in?^ ""l^^^ -P-entation were U^h (U),
Nebraska (4). ( '' M'SSOUn (6)' North Carolina («>• Montana (5), aid
across tteUs ,te/QI "^ "*** °V" i0'°°° residences ^ «"™ercial
£^^TC4T^^^^^
^sient water systems included in this study were almost ail suppiied by welistd^.s
residences as a whole, especially those served by.private weils and springs
-------
APPENDICES
1. Attachments
. Lead test instructions
Questionnaire
2. Table A
Information on Transients Sites
-------
Attaint .
Noa-Community Water Systems
I. Name of system or business
2. Address
. Phone_
a. System number (as shown on address label on mailer)
4. Water source: well, supplied by a public water utility
other (please specify)
5. ,Do you treat the water in-any way besides chlorination? .
6. Water system type: ' ' •
Gas Station ' Motel/hotel/lodge Ski resort
Campground building ' - Highway rest area Store
Campground hookup "Restaurant Mobile home park
Other (please specify)__ • _^
7. Type of water service line from water source to building: copper, lead plastic,
galvanized steel, combination, don't know, • other
a. Age of this water line in years
S. Type of indoor plumbing within building to water outlet: copper, lead,. plastic,
galvanized steel. combination,- don't know, other
-a. Age of this water line in years
b. Age of building
9 . Water sample source: • - ' .
' self cpntained water cooler, water-fountain, kitchen faucet, bathroom faucet,
hookup, outdoor spigot
other (please describe)^ • '
a. Manufacturer of source noted in question 9
b. Age of source noted in question 9 •
c. Used primarily by: employees, transients (customers)
10. Do you have a water filter attached to the line from which this sample was taken?'
a. If there is a filter, is it a sediment filter only, carbon filter, cation exchange filter,
combination to" remove particles, chlorine and metals
b. Manufacturer
II. Do you have a water conditioner attached to the line-from which this sample was taken?
a. Manufacturer __-
12. Approximate standing time of water before first draw sample was taken (in hours) •
13.' Dateofsample__.
-------
Attachment #
. The Environmental Quality Institute
UNCA "One University Heights "Asheville NC 28804
(704)251-6800
Lead Test instructions for Non-Community Water Systems
Enclosed are two sample bottles for collecting your tap water and a questionnaire with labels.
First, you must decide which of your taps you want tested. Second, fill in your name and address on the
labds proved. These labels will identify your samples and will be used as address labels for
0^
bdow for
Procedure 1: First Draw Sample use the one liter container •' •
This sample should be taken from the cold water tap sometime when the water has been standing in the
plumbing lines for at east six hours and before the toilet is flushed or water is run in the building Fully
open the enclosed co apsible one-liter plastic container. Place it under the faucet and turn on the cold water
to a moderate flow Mien it is full, replace the cap. Dry the outside of the bottle. Immediately attach the
. t irst Draw label to the sample bottle. (Make sure the bottle is dry or the label won't stick well.)
Procedure 2: Purged Line use the small 30 ml bottle but you will need to supply avne quart pitcher
or container for measuring. rr j ^ y
This sample should be taken after water has' been purged through the plumbing system'. Run the cold water
tap at a high rate for exactly 60 seconds, then reduce the flow to a small trickle. Place a one-quart or one'- '
liter contamer (preferably a plastic one, but glass 'is acceptable) under the faucet and fill When the
container is full, stir the water in it briefly with a plastic (Not Metal) spoon or other stirrer. Pour water from
the container into the small sample bottle. Dry the outside of bottle. Immediately attach the "Pur-ed Line"
label co this bottle. , s
Be sure to fill out and enclose the questionnaire. Please leave the label with your address stapled to the
questionnaire, it will be the mailing label used by the lab to return your results to you. This will help ensure
proper identification of your samples. Place the label saying "Environmental Quality Institute UNCA, -
Asheville. NC 2S804" on the outside of the box and mail to the laboratory within 10 days of'sampling.
The laboratory results will be sent back to you within two to three weeks of receipt, along with information
on what the. results mean and whether any further action beyond line purging is recommended.
If you have any questions about how to take these samples or fill out the forms-please call Diane Morgan at
(704) ZD 1-6800. Your participation in this project will help determine the extent of the lead problem at your
facility and in transient, non-community water systems as a whole.
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1
I
I
I
I
I i
-------
March 20, 1996
TO: Judy Lebowich
Connie Bosma
FROM: Jeff Cohen
SUBJECT: Analysis of UNC-Ashville Survey of Lead at Transient Systems
This is to summarize results of the UNC-Asheville survey on lead levels in transient
water systems. The survey results are documented in the report, A Survey Study of Lead in
Drinking Water Supplied by Transient Water Systems, Technical Report #95-019, December
1995, prepared by the Environmental Quality Institute of the University of North Carolina at
Asheville.
UNC-Asheville Survey
Of 8,000 transient systems throughout the country invited to receive free lead testing, 115
participated. The relatively small number prevents meaningful analysis of the representativeness
of the respondents, although a wide range of system types across the country is included. First
draw-(1-liter) and one-minute purged (30 milliliters) samples were collected at each site.
While the median and average concentration of the first draw samples were relatively low
(2.3 and 9.2 parts per billion (ppb), respectively), high levels were measured at some of the sites.
Ten percent of the sites had levels in excess of 20.3 ppb, with a maximum first draw sample of
229 ppb.
The average one-minute purged line sample was 2.3 ppb, with a 90th percentile of 3.4
ppb. The purged samples had much lower concentrations (75% lower on average), and less
variable readings, although two sites had levels above 30 ppb.
High lead readings were correlated with long standing times and with age of the
plumbing system. All of the sites with first flush levels above 50 ppb had standing times of six
hours or greater. Some standing times were as high as 96-120 hours, resulting in levels of 34 and
56 ppb, respectively.
Standing time is not the only explanatory variable for high lead levels, however. The
maximum reading of 228.8 ppb was measured in a mobile home park (self-classified as a
transient system), where the water had been standing for six hours. The fact that the purged
sample level was only 0.7 ppb raises doubt about the validity of the first flush sample. Assuming
no contamination occurred and it is a legitimate result, the use at the site of a water softener and a
sediment filter (which if not maintained, can build up excessive lead particle concentrations)
could explain the high reading.
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Implications of UNC Study Results
As is the case with most any survey of lead in drinking water, the study found elevated
lead levels at some locations that appear to be correlated with standing time and plumbing age.
The influence of water corrosivity and other variables was not measured, and the possibility of
sampling errors and contamination cannot be ruled out.
Given the body's ability to safely "filter" out most lead that is ingested, and the fact that
lead toxicity is typically associated with long-term exposure,1 isolated consumption of water with
even extremely high lead levels (e.g., >100 ppb) would not necessarily constitute a significant
health-risk.
Assuming the results are accurate and representative of nationwide exposure at transient
systems, the remaining question is whether sensitive groups (women of childbearing age,
pregnant women, and young children and infants) in transient systems may be regularly exposed
to the high lead levels found at,the relatively small percentage of sites in the study. This question
is outside he scope of the UNC-Ashville study and conducting a representative exposure
assessment for sensitive populations in transient systems would be difficult.
'U.S. Environmental Protection Agency. June 1986. Air Quality Criteria for Lead,
Volume IV. Environmental Criteria and Assessment Office, Research Triangle Park, NC.
EPA-600/8-83/0282F.
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HEALTH EFFECTS FROM SHORT-TERM LEAD EXPOSURE
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
1.0 BACKGROUND
The long-term effects of low level lead exposure are well documented and are the basis of
numerous regulatory programs aimed at reducing lead exposure of all segments of the population
but particularly among children and women of childbearing age. Less is know about the effects
of short-term exposures to low levels of lead, especially among the groups that are most sensitive
to the effects of long-term exposures.
The EPA drinking water lead and copper rule is one of the regulatory programs focused on
reducing environmental lead exposure. This rule was promulgated in 1991 (USEPA, 1991).
When promulgated, transient, noncornmunity water systems1 were excluded from the rule's
requirements based on the fact .that exposures to lead that would occur at such facilities would be
acute rather than chronic.
The decision to exclude the transient noncommunity systems was supported by lexicological
data from studies in adults which identified increased concentrations of erythrocyte
protoporphrine and depressed activity of aminolevulinic acid dehydratase as the critical effects
from short-term lead exposures (Cools et al., 1976; Schlegel and Kufner, 1979; Stuik, 1974 as
cited by Cohen, 1993). These effects are markers for inhibition of heme synthesis (ATDRS,
1992; Hendmarsh, 1986) but are reversible and do not persist after exposure has ceased.
Aminolevulinic acid dehydratase is the key enzyme regulating the rate of heme synthesis and
erythrocyte protoporphrine is a precursor to heme and, thus, a biomarker for heme production.
Heme is the oxygen-binding pigment in the red blood cell. Physiological replacement of red
cells is a continuous process. Therefore, the red cells in circulation cover the spectrum of
nascent cells to cells ready to be removed by the spleen for degradation. Thus, a short term
deficit in heme production is not immediately manifest in a decreased supply of red blood cells.
The gradual turnover of the red blood cells is one of the factors that protects against acute
hematological effects resulting from a short term lead exposure. A short term event influencing
heme synthesis will not be have a parallel impact on the number of functional red cells in
circulation.
A study by Stuik (1974) of the short-term effects of lead on heme synthesis was key in the
'•Transient noncommunity water systems are public water systems that serve fewer than 25
of the same people at least six months a year.
1
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original EPA decision to exclude transient systems from lead monitoring requirements (Cohen,
1993). As reported hi ATSDR (1992) two groups of 5 women and one group of 5 men were
orally exposed to 0.02 mg/kg/day lead hi the form of lead acetate for 21 days during this study.
This dose is equivalent to 1 mg lead per day for a 50 Kg reference woman and 1.4 mg lead per
day for a 70 kg reference man. Under conditions where water consumption is 2 L/day, this dose
is equivalent to a water concentration of 500 Mg/L for the women and 700 Mg/L for the men.
0.02 ms/kg/dav x 50 kg = 0.5 mg/L = 500 Mg/L
2L/day
0.02 mg/kg/dav x 70 kg = 0.7 mg/L = 700 Mg/L
2 L/day
In this study, suppression of the activity of erythrocyte aminolevulinic acid dehydratase became
apparent by day 3 of exposure. The degree of suppression increased until day 14 and then
remained constant for the remainder of the study. Effects on erythrocyte protoporphrme were
noted hi the women but not the men after 2 weeks of exposure. Blood lead levels had increased
to 40 Mg/dL or higher before effects on erythrocyte protoporphrine were noted.
Unfortunately, no studies were identified that examined the effects of short-term exposure to lead
on heme synthesis in children. Lead absorption in children is greater than that in adults.
Chamberlain et al (1978) as cited in ATSDR (1992) found that children absorbed about 3.3 times
more lead than adults. Using this relationship one might infer that short- term lead exposures
that were about one third that hi adults (0.006 mg/kg/day) or greater could have an effect on
heme synthesis hi children equivalent to that seen in adults.
0.02mg/kg/dav = 0.006 mg/kg/day
For a 10 kg child consuming 1 L of water per day this would be equivalent to a concentration of
60 Mg/L.
0.006 mg/kg/dav x 10 kg = 0.06 mg/L = 60 Mg/L
1 L/day
2.0 RECENT DATA ON SHORT TERM LEAD EXPOSURE
2.1 Human Data
In order to determine if additional data have become available regarding the effects of short-term
exposure to lead since EPA's original evaluation, EPA conducted a search of the Medline
databases for acute studies published hi the last ten years. Only one article which included
primary data on the effects of short-term exposure to lead in humans was identified.
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This article presented a case study of a 26 month old black female who was hospitalized because
of anorexia, vomiting and lethargy (Khan et aL, 1983). The child had been seen three weeks
earlier at another hospital and found to have a blood lead level of 97 ,wg/dL. At that time she was
treated with calcium disodium ethylene diamine tetraacetic acid (EDTA) and dimercaptol every 4
hours for seven days. She was then discharged to her parents. When she returned to the hospital
2 weeks later her blood lead levels had declined to 32 /zg/dL. Three weeks after the original
hospital admission, the child was brought to the hospital because of the vomiting/anorexia
problem. Blood urea nitrogen (BUN), proteinuria, and serum creatinine values indicated acute
renal failure. The child also was found to suffer from a glucose-6-phosphate dehydrogenase
(G6PD) deficiency. With treatment the renal problems were resolved and kidney function
normalized allowing the patient to be discharged after 17 days.
The treating physicians attributed the renal problems to the amount of EDTA administered
during chelation therapy (Khan et aL, 1983). They felt that renal effects from the EDTA may
have been complicated by the patient's G6PD deficiency and the dimercaptol therapy. Potential .
contribution of lead to the renal failure could not be eliminated because of slight proteinuria and
minimal elevation of BUN present at the time of the initial hospital visit. However, the authors
felt that contribution of lead to the renal problems, if any, was minimal. However the potential
for an adverse effect of lead on renal function should have been considered when initiating the
EDTA treatment.
2.2 Animal Data
Several articles regarding short terms effects of lead on animals were also identified in the
literature search. Studies were classified as acute only if the exposures lasted 14 days or less;
this is the Agency for Toxic Substances and Disease Registry (ATSDR, 1992) classification for
an acute animal study. In evaluating the data from these studies it is important to remember that
they are not directly applicable to humans because of differences in physiology and life-span. A
one week exposure in a rat with a two-year life-span is not equivalent to a one week exposure in
a human with an average 70 year life-span.
Two of the animal studies looked at the effects of lead on renal, function and/ or blood pressure or
related end points. The shortest duration evaluated was 7 days. Giridhar and Isom (1990)
examined the response of the atrial natriuretic factor (ANF) to intraperitoneal injections of 0.01,
0.1, 0.5 or 1 mg/kg lead acetate solution in groups of 4 Sprague-Dawley rats. ATF is a peptide
hormone that acts as a neuromodulator to control blood pressure and fluid volume. At the end of
7 days, ANF levels decreased in the plasma and hypothalamus. The response was roughly
related to dose but the dose-response trends were not consistent across the doses. The ANF
levels in the atria were not dose-related, but were increased as compared to the controls for three
of four doses.
Water ingestion did not change over the period of lead exposure but urine volume decreased in a
dose-related fashion (Giridhar and Isom, 1990). Increases in body weight suggested that the
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decreased urine output was the result of fluid retention and that the fluid retention rather than the
lead was responsible for the lower levels of ANF in plasma. Since evidence of lead-induced
nephrotoxicity was not evaluated hi this study, it was not possible to conclude whether or not the
effects observed were the impact of the lead on the kidney. The use of intravenously
administered lead also reduces the value of this study for evaluating risk in humans from lead
exposure through drinking water.
In a second study, spontaneously hypertensive Sprague-Dawley rats were given drinking water
containing 100 mg/L (100,000 /ug/L) lead as lead acetate for 3 weeks (Nakhoul et al., 1992).
There was no change in the systolic blood pressure of the animals for the first 8 days of exposure
even in these genetically susceptible rats. However, by day 12 and through day 20, the systolic
pressure of the rats was significantly higher than that for the controls. These results suggest that
short-term, low-level lead exposures would not have a hypertensive effect in humans even those
with a genetic predisposition to hypertension comparable to that in the rats unless they persisted
for a month or more.
Histological changes induced by lead from lead acetate in intestines, kidney and liver were
evaluated in Sprague Dawley rats by Karmaker et al. (1986). A dose of 44 mg/kg for durations
of 9, 15 or 30 days was evaluated in groups of 5 rats. This dose is equivalent to 3,080 mg/day in
a 70 kg human or a concentration of 1,540,000 //g/L with ingestion of 2 L of water per day.
After 9 days, mild shortening of the intestinal villi were seen in 2 of 5 rats and histological
changes in the liver were observed in all rats. No renal abnormalities were observed. After 15
days, intestinal and liver abnormalities had progressed and affected more animals than at 9 days;
3 of 5 rats showed histological kidney abnormalities. The changes observed were probable given
the magnitude of the dose, even though the exposures were relatively short-term. Comparable
exposures from drinking water are unlikely even from very corrosive water in systems with lead-
containing components.
3.0 CONCLUSIONS AND RECOMMENDATIONS
An examination of recent data on the effects of short-term lead exposure do not change the
conclusions of the original review by EPA (Cohen, 1993). Effects on heme synthesis still appear
to be the critical effect from lead exposure. These effects are first manifest by decreased activity
of aminolevuluiic acid dehydratase within as few as three days and occur at exposures below
those from other lead-induced effects. Concentrations of between 500 and 700 ^ug/L were
observed to change the enzyme activity in adult humans. Due to differences in lead absorption,
the response in children might occur at an normalized concentration as low as 50//g/L. The
normalized concentration is the average concentration for all water consumed during the day and
is not the same as the "first draw" concentration. Continued ingestion of water with a
normalized concentration of this value for a period beyond several weeks would not be advisable
for children from a health perspective.
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4.0 REFERENCES
ATSDR. Agency for Toxic Substances and Disease Registry. 1992. Toxicological Profile for
Lead. U.S. Department of Health And Human Services. Public Health Service. Agency for Toxic
Substances and Disease Registry. Atlanta, GA. TP-92/12.
Cohen, J. 1993. Memo to Jim Elder of the US Environmental Protection Agency Office of
Ground-water and Drinking Water and an Attachment entitled: Draft Response to NRDC .
Assertion that Transient Systems be Regulated under Lead NPDWR. July 21.
Giridhar, J. And G.E. Isom. 1990. Interaction of lead acetate with atrial natriuretic factor in rats.
Life Sci. 46(8):569-576.
Hindmarsh. J.T.. 1986. The porphyrias: Recent advances. Clinical Chemistry 32 (7): 1255-1263.
Karmakar, N., Saxena, R. and S. Anand. 1986. Histopathological changes induced in rat tissues
by oral intake of lead acetate. Environ. Res. 41(l):23-28.
Khan, A.J. Patel, U., Rafeeq, M, Myerson, A., Kumar, K. and H.E. Evans. 1983. Reversible
acute renal failure in lead poisoning. J. Pediatr. 102 (1):147-149.
Nakhoul, F., Kayne, L.E., Brautbar, N., Hu, M., McDonough, A., Eggena, P., Golub, M.S.,
Berger, M., Chang, C., Jamgotcbian, N., and D.B.N. Lee. 1992. Rapid hypertensinogenic effect
of lead: studies in spontaneously hypertensive rat. Toxicol Ind. Health 8(l-2):89-102.
USEPA. U. S. Environmental Protection Agency. 1991. Maximum contaminant level goals and
national primary drinking water regulations for lead and copper; Final rule. Federal Register.56
(110):26460-26564.
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