9 1999by Humana Press Inc.

All rights of any nature, whatsoever, reserved.

0163-4984/99/6703-0205 $12.25

State of Pregnancy Modifies Lead

Toxicity in Mice
J. SINGH, P. PARKASH,1 AND G. S. GUPTA*
Department of Biophysics, Panjab University,
Chandigarh, India; and 'Department of Physiology,
Government Medical College, Chandigarh, India
Received April 22, 1998; Revised June 22, 1998; Accepted July 25, 1998

ABSTRACT
Toxicity of lead acetate after administration through the oral
route at 0-50 m g / k g body weight of animal has been assessed in the
liver of pregnant mice and compared with the effect in the liver of
nonpregnant dams. Analysis showed that the basal level of hepatic
lead is considerably reduced during pregnancy as compared to that
in nonpregnant state. After administration of Pb-acetate, deposited
lead in liver of nonpregnant mice was 3- to 4-fold while in pregnant
mice was, it was 1.8- to 3.0-fold over their respective control values.
Although hepatic Fe, Cu, and Zn levels had a tendency to be lowered
during pregnancy, it appeared that the added trace quantity of lead
prior to and during pregnancy helped in the retention of these met-
als, which either remained unaffected (as Fe) or declined (Cu and Zn)
after lead administration during the nonpregnant state. The effect
of lead on Mn diminution, however, was visible at the dose of
50 m g / k g body wt of lead-acetate. Alkaline phosphatase, which
increased during pregnancy along with Mn, was reversed between
the pregnant and nonpregnant states after oral administration of
lead. On the other hand, the level of 5-aminolevolunic acid dehy-
dratase, which declined during normal pregnancy, continued to fall
further after lead exposure. It is concluded that the distribution of
basal or administered lead and its effect on enzyme activities and
trace metal composition in liver depends on the pregnant and non-
pregnant states of female hosts.
Index Entries: Lead toxicity; trace metals; hepatic enzymes;
hepatic lead; pregnancy.

*Author to whom all correspondence and reprint requests should be addressed.

Biological Trace Element Research 205 Vol. 67, 1999

206 Singh, Parkash, and Gupta

INTRODUCTION

It is known that stored lead in the body during pregnancy mobilizes

and distributes uniformally in embryos (1-4). The maternal blood, umbil-
ical cord blood, amniotic fluid, and placenta under fetal death seem to
accumulate much more lead as compared to those seen in normal gestating
mothers (5,6). Because Pb modifies the endocrine status during pregnancy
(7) and also influences the absorption of trace metals in the gastrointesti-
nal (GI) tract (8), it is not known how far pregnancy can modify the Pb
toxicity in liver in terms of trace metal composition and metalloenzymes
therein. Therefore, the present study was undertaken to address the ques-
tion of whether pregnancy has any effect on lead distribution and its con-
sequences on marker enzymes and trace metal composition of liver after
oral administration of Pb-acetate prior to and during gestation.

MATERIALS AND METHODS

Experimental Design and Lead Treatment

Sexually mature LACA-strain female mice 2 mo age (20-25 g weight)
were acclimatized and divided into various groups (3). Animals in group
I were given 0.5 mL saline through gastric lavage on alternate days. Ani-
mals in other groups were given Pb-acetate (10-50 m g / k g body wt) in
0.5 mL saline through gastric lavage for a period of 4 wk. After 4 wk
of Pb intubation, ovulation was induced in each mouse with two injec-
tions of 5 IU hCG in 0.1 mL saline (ip) within a week. After hCG injection,
dams were caged to mate with their male counterparts up to a ratio of
three to one male. Every other morning, darns were examined for the
presence of positive vaginal plugs to confirm successful mating. Mating
was discontinued after 7 d. Various doses of Pb-acetate in normal saline
continued during ovulation and gestation as scheduled before mating.
The blood was drawn by occular puncture just before laporatomy was
performed. Mice were sacrificed on day 16-18 after observation of vagi-
nal plugs. A weighed quantity of frozen liver was either lyophilized or
homogenized in distilled water, depending on the assay. Biochemical
parameters were expressed for dry weights obtained after freeze-drying
to a constant weight. In another set, similar experiments at different
doses of Pb-acetate in nonpregnant female mice were performed to com-
pare and ascertain if Pb toxicity is modified by the state of pregnancy.
For 8 wk, these mice were not allowed to come in contact with males at
any stage of Pb administration, nor they were given hCG (6).

Elemental Analysis
One hundred milligrams of lyophilized tissue was digested in 5 mL
of digestion mixture in Corning tubes (AR nitric acid + AR perchloric

Biological Trace Element Research Vol. 67, 1999

Lead Burden During Pregnancy 207

acid in the ratio of 5 to 1) for 6 h at room temperature. When the diges-

tion was complete, the tubes were cooled. The white ash was dissolved
in 5 mL of 10 mM HNO3. The analysis was performed by atomic absorp-
tion spectrophotometry. The detection limit of Cu, Zn, and Mn was i ~tg/L,
whereas that of Fe and Pb was 3 ~tg/L.

Alkaline Phosphatase
Alkaline phosphatase (AP) activity was assayed according to the
method reported earlier (6). One milliliter of p-nitrophenyl phosphate (Na
salt) was incubated in glycine buffer (0.05M, pH 10.5) for 15 rain at 37~
having 0.1 mL of suitable diluted enzyme. After incubation, the reaction
was terminated by 5 mL 0.1N NaOH. The color of p-nitrophenol was mea-
sured at 420 nm. A unit of enzyme was defined as the amount of protein
that liberated 1 Mmol p-nitrophenol/g dry wt of tissue/rain at 37~

8-Aminolevulenic Acid Dehydratase

The assay has been described earlier (6). To 0.25 mL tissue
homogenate, 1 mL of Triton-X-100 (0.2% by volume), and 1 mL of 0.01M
8-amino-levulenic acid containing 0.2M dithiothreitol in phosphate buffer
(0.1M, pH 6.80) were incubated in the dark at 37~ for 1 h. The reaction
was stopped with 10% trichloric acid (TCA) containing 0.05M of satu-
rated copper sulfate. For the experiment in which the activation of the
enzyme was determined, copper sulfate was replaced with 0.05M mer-
curic chloride in 10% TCA. One milliliter of supernatant obtained at
27,000g was colored with 1 mL of modified Ehrlich's reagent and read at
555 nm after 15 min. A unit was defined as that amount of protein which
formed 1 ~tmol porphobilinogen/min/g dry wt of tissue at 37~

Statistics
The significance of changes between two groups was tested by the
Student's t-test, whereas the coefficient of correlation (r) between two
parameters among all the groups of pregnant or nonpregnant mice was
tested by a direct method without taking deviations of items from the
actual mean or the assumed mean. The direction of change of averages
was evaluated by the trend test across the dose of lead-acetate of various
groups by a least squares fit. The coefficients of correlation of these best-
fit lines are given where felt necessary.

RESULTS

Trace Element Lead Levels

The normal concentration of lead in the liver of nonpregnant mice
was 5.0 + 0.9 ~tg/g dry wt, whereas the corresponding value in pregnant

Biological Trace Element Research Vol. 67, 1999

208 Singh, Parkash, and Gupta
mice was 3.0 _+0.7 ~tg/g dry wt, suggesting that the stored lead declined
during pregnancy. After oral administration of Pb-acetate, the hepatic Pb
tended to increase in response to exposures of 10 and 50 mg lead-
acetate/kg body wt; the highest concentration of deposited lead in the
liver of pregnant was 1.8- to 3-fold, and in the liver of nonpregnant mice,
it was 3- to 4-fold over the concentration of basal value, thus lowering
the P / N P ratio (P/NP = pregnant [P]/Nonpregnant [NP] Pb ratio in nor-
mal [P/NP] or Pb administered P10/NP10 or P50/NP50 at 10 and 50
m g / k g body wt for mice) from a control value of 0.60 to 0.32 and 0.47 at
two different doses of Pb-acetate. Along with these changes NPe/NP0, or
PJP0 ratios (NPe/NP0 - ratio between experimental lead-administered
and sham-treated control groups as at 10 mg [NP10/NP0] and 50 m g / k g
body wt [NP50/NP0] in the nonpregnant state or as P10/P0 and P50/P0,
respectively, in the pregnant state) declined from 3.4 (NP10/NP0) in non-
pregnant mice to 1.83 (Pl0/P0) in pregnant dams at 10 m g / k g body wt of
Pb-acetate (Table 1), whereas NP50/NP0 and P50/P0 ratios at 50 m g / k g
body wt of lead acetate did not differ effectively.
Iron
The normal level of hepatic iron in nonpregnant mice was 934 _+ 200
~tg/g dry wt and the corresponding value during pregnancy was
678 + 89 ~tg/g dry wt, suggesting a tendency of Fe being lowered during
gestation. Although the administration of Pb did not change the hepatic Fe
of nonpregnant dams, the exposure of pregnant dams to a dose of 10 and
50 mg Pb-acetate increased the P / N P ratio of Fe from a control value of 0.72
to 0.92-1.11 (P10/NP10 and P50/NP50) and changing the NPe/NP0 ratio
from 1.01 to 1.30 (P10/P0 or P50/P0) at two different doses of Pb (Table 1).
Copper
In nonpregnant mice, the normal level of hepatic Cu was 42 _+ 9.0
~tg/g dry wt, which decreased to 24.0 _+ 2.0 ~tg/g dry wt during preg-
nancy (Table 1). Lead administration at 10 and 50 mg as lead-acetate
reduced the copper level in nonpregnant mice, raising a P / N P ratio from
a control value of 0.5 to 0.9-1.0 after lead treatment. The NP~/NP0 ratio
of 0.67-0.76 in nonpregnant mice increased to 1.04-1.25 in pregnant mice
after treatment with 10-50 mg Pb-acetate/kg body wt.
Zinc
Hepatic Zn during the pregnant state diminished from a normal
value of 155 _+ 12 to 106 _+ + 14 g g / g dry wt. Following the administra-
tion of 50 mg Pb-acetate/kg body wt, a significant fall of zinc occurred
in the liver of nonpregnant mice. On the other hand, the P / N P ratio was
found to be enhanced from 0.69 in the control group to 1.06-1.11 in
Pb-acetate-treated mice. In general, the level of zinc loss in nonpreg-
nant Pb-treated mice correlated with the endogenous level of Pb with a
coefficient of correlation of r = 0.945. However, the NPe/NP0 ratio

Biological Trace Element Research Vol. 67, 1999

Lead Burden During Pregnancy 209

Table 1
Effect of Pb-Acetate on Trace Elements of Liver and its Modification by
State of Pregnancy (Values are mean _+fiE)
Dose* Pb Fe Zn Cu Mn

NPo 0 5.0~0.9 934• 155~12 42.0• 5.0•

Po 0 3.0• 678~89 106Z14b 24.0• 6.7•
Po/NPo 0.60 0.72 0.69 0.51 1.34

NPe i0 17.0Z6.4 942• 124Z48 28Z2.0 6.9•

Pe i0 5.5• 869• 131~12 25Z2.0 8.0•
Pe/NPe 0.32 0.90 1.06 0.90 1.16

NPe/Po I0 3.40 1.01 0.80 0.67 1.38

Pe/Po i0 1.83 1.30 1.23 1.04 1.20

NPe 50 21.0Z5.0a 982• 104• 30Z3.0 4.0•

Pe 50 10.0Z3.3a,d 903~i09 I16Z20 30~6.0 5.0~I.0
Pe/NPe 0.47 0.92 i.ii 1.00 1.25

NPe/NPo 50 4.20 1.05 0.67 0.76 0.80

Pe/Po 50 3.33 1.33 1.09 1.25 0.75

*Lead acetate (mg/kg body wt).

a,bSignificantlydifferent at p < 0.01 and p < 0.05, respectively,from the corre-
sponding control (NP0 or P0) groups.
c,dSignificantlydifferent at p < 0.01 and p < 0.05, respectively,from the cor-
responding nonpregnant experimental (NP~) group.
Note:
NP0: nonpregnant (NP) tmtreated control mice.
NPe: nonpregnant (NP) lead-treated experimental mice.
P0: pregnant (P) untreated control mice.
Pe: pregnant (P) lead-treated experimental mice.
P0/NP0: ratio of pregnant and nonpregnant untreated control mice.
PJNPe: ratio of pregnant and nonpregnant lead-treated experimental mice.
Pe/P0: ratio of lead-treated experimental and untreated pregnant mice.
NPJNP0: ratio of lead treated experimentaland untreated nonpregnant mice.

increased from 0.67-0.80 in the n o n p r e g n a n t state to a Pe/P0 value of

1.09-1.23 in the pregnant state after Pb-acetate exposure.
Manganese
It appears that Mn is enhanced during pregnancy to 6.7 _+ 0.6 ~tg/g
dry wt from a normal value of 5.0 _+ 0.4 g g / g dry wt, giving a ratio of
P / N P = 1.34. Following the exposure of mice at 10 m g / k g b o d y wt of Pb-
acetate, the hepatic Mn increased to 6.9 + 0.8, gg during the nonpreg-
nant state and to 8.0 + 0.7 jag during the pregnant state. On the other
hand, it t e n d e d to lower to 4.0 _ 0.2 gg in the n o n p r e g n a n t state and to
5.0 _+ 1.0 g g / g in the pregnant state after administration of 50 m g / k g
b o d y wt of Pb-acetate. However, P / N P as well as NPe/NP0 and Pe/P0
ratios of Mn in contrast to that of Zn and Cu, w h i c h e n h a n c e d after Pb
administration, remained u n c h a n g e d in the two states.

Biological Trace Element Research Vol. 67, 1999

210 Singh, Parkash, and Gupta

1"0,
A C
~2.o J

0.8 A-~ x,,

x

C 1.6
0.6'
._>

q 0-4.
o~ , oy
o Io 2o 40 so ; 6 20 3; 4? 50
D
~ 0.4 6.0.

0.2 ,~ ~.o. -4
0~ ,3"
o lo 20 ;o 0 10 20 30 40 50
Pb-ocetote (mg/kg b.wt.) Pb-ocetote ( m g / k g b.wt.)

Fig. 1. Effect of Pb-acetate at different doses on the activity of 6-ALAD-

activated form of 6-ALADa (a), nonactivated form of 3-ALAD (b), 3-ALADa/
3-ALAD ratio (c), and alkaline phosphatase (d) in the livers of pregnant (~1,-~)
and nonpregnant ( H ) mice.

Effect on G-Aminolevulinic Acid Dehydratase

and Alkaline Phosphatase
The normal levels of activated and nonactivated forms of hepatic 6-
aminolevulinic acid dehydratase (6-ALAD) in nonpregnant mice was
0.93 _+0.05 and 0.55 + 0.04 units/g dry wt, respectively; the correspond-
ing values of 6-ALAD during pregnancy were 0.73 + 0.06 and 0.39 _+0.05
units (Fig. la). In lead-treated mice, the level of the dithiothreitol-
activated form of 6-ALAD showed a significant decline in the liver of
both pregnant and nonpregnant dams, whereas the nonactivated form
of 6-ALAD decreased significantly in nonpregnant females only (Fig. lb).
It may be noted that the loss of 6-ALAD, which continued to increase up
to a 50-rag dose of Pb-acetate in nonpregnant mice, correlated with
hepatic Pb (r = -0.51). The ratio of activated to nonactivated 6-ALAD
seemed to depend on the endogenous Pb composition, which reduced
from 1.8 to 1.2 in the nonpregnant state and from 1.6 to 1.0 in the preg-
nant state after treatment with 50 m g / k g body wt of Pb-acetate (Fig. lc).
The AP of the liver increased from 4.0 + 0.79 units during the nonpreg-
nant state to 7.0 + 0.5 units during pregnancy, whereas the increase of
AP during pregnancy did not persist after Pb administration. On the
contrary, the enzyme activity of AP reversed between the two states,

Biological Trace Element Research Vol. 67, 1999

Lead Burden During Pregnancy 211

showing a significant decline in the liver of nonpregnant mice after lead

administration (Fig. ld).

DISCUSSION
In the present study, hepatic toxicity resulting from lead during the
pregnant and nonpregnant states has been assessed and compared. Dur-
ing pregnancy, the burden of hepatic Pb is considerably reduced and this
continued to be so even after gastric intubation of Pb-acetate. Although
the placenta has a tendency to retard Pb (3), the lead content of fetuses
increases throughout pregnancy. It is stated that the stored lead is mobi-
lized during pregnancy and transferred to the fetus (2,6,9). Part of the
reduced level of lead in body tissues during pregnancy in comparison to
nonpregnant state could, however, be due to the greater fluid intake and,
hence, greater excretion. Nevertheless, it appears that an added trace
quantity of lead in liver during pregnancy helps in the retention of
hepatic Fe, Zn, and Cu, which was evident from their P/NP, NPe/NP0,
and Pe/P0 ratios.
The present study revealed that oral administration of Pb disturbs
liver functions, depending on the accumulated Pb in pregnant and non-
pregnant conditions. Although limited information is available on the
influence of pregnancy-associated proteins and hormonal changes (10) in
the disposition and toxicity of lead, nutritional and endocrine factors
seem to play an important role during Pb exposure, influencing its
absorption and toxic manifestations (7,8). In fact, pregnant mice per se did
not suggest any effect of lead on the levels of Cu and Zn over their
untreated controls, whereas nonpregnant mice showed a decrease of
these metals, as suggested in early investigations (11,12) without a
change in Fe composition during pregnancy or otherwise. Very often,
deficiencies of Cu and Mn have been related to the lipid peroxidation of
membranes and formation of free radicals in proteins (13,14), where glu-
tathione is known to play a role in the defense of cell injury (3,6,14). In
contrast to observations in the kidney and placenta (3,6), the influence of
a low dose of Pb on the enhancement of hepatic Mn was followed by a
decline at the higher dose of the pollutant. Probably the most studied
effect of lead toxicity is related to the inhibition of heme synthesis due to
inhibition of 8-ALAD (6,15,16) and accumulation of 8-aminolevulinic
acid (8-ALA) (17,18). The autooxidation of 8-ALA and other mechanisms
within hepatocytes may form oxygen radicals and other oxidative radi-
cal species, which induces cellular damage (13,19,20).
However, in the absence of Zn loss during the pregnant state, the
question to be addressed is how 8-ALAD, a zinc metalloenzyme, is lost
after Pb toxicity. Possibly, the low sensitivity of hepatic 8-ALAD in com-
parison to kidney and blood (6) after lead exposure depends on the endo-
geneous Pb level and detoxifying mechanisms as well as the replacement

Biological Trace Element Research Vol. 67, 1999

212 Singh, Parkash, and Gupta
of Zn by Pb at the Zn binding sites beyond the postabsorptive level
(21-25). Above all, liver 8-ALAD appears to be modified by state of preg-
nancy following lead administration.
Alkaline phosphatase, which increased marginally in Pb-treated
pregnant dams, declined significantly in Pb treated nonpregnant dams.
Therefore, reports on AP during lead toxicity are contradictory, indicat-
ing its dependence on the endogeneous lead content and its influence on
other metals (26-28). Very often, changes in AP have been related to lead
toxicity (29,30). As AP requires metal ions like Zn and Mn for maximum
activity and for growth of rats (31), it is likely that the decline in AP dur-
ing the nonpregnant state after Pb treatment is related to the Zn/Mn
ratio; a lower Zn/Mn ratio gives a higher AP activity (3,6).
It is concluded that the distribution of lead in the liver of normal and
lead-treated mice is greatly influenced by state of pregnancy. Conse-
quently, the effect of lead toxicity on the enzyme activity of 5-ALAD and
AP and the trace elements composition in liver depends on the pregnant
and nonpregnant states of animals. These observations support our ear-
lier conclusion on lead toxicity in kidney (6). The present study in preg-
nant and nonpregnant animals following lead exposure warns that
further studies are needed on the defense mechanisms operative in
two different states of animals, in addition to endocrine hormones and
pregnancy-associated proteins (10).

REFERENCES
1. N. E Angell and J. P. Lavery, The relationship of lead levels to obstetric outcome,
Am. ]. Obstet. Gynecol. 14, 4 0 4 6 (1982).
2. J. P. Buchet, R. Lauwerys, H. Roels, and G. Hubermont, Mobilization of lead during
pregnancy in rats, Int. Arch. Occup. Environ. Health 40, 33-36 (1977).
3. G. S. Gupta, J. Singh, and A. Gupta, Trace metals and metalloenzymes in placenta
after oral administration of lead acetate, Biol. Trace Element Res. 60, 145-152 (1997).
4. R. M. McClain and B. A. Becker, Teratogenicity, fetal toxicity and the placental trans-
fer of lead nitrate in rats, Toxicol. Appl. Pharmacol. 31, 443451 (1975).
5. S. Kaur, Lead and cadmium in maternal blood, umbilical cord blood, amniotic fluid
and placenta of cows and buffaloes after fetal death (abortion) and after normal par-
turition, Sci. Total Environ. 19, 2987-2900 (1989).
6. G. S. Gupta, J. Singh, and P. Prakash, Renal toxoicity after oral administration of lead
acetate during pre- and post-implantation periods: effects on trace metal composi-
tion, metalloenzymes and glutathione. Pharmacol. Toxicol. 76, 206-211 (1995).
7. E. M. Sierra and E. Tiffnay-Castiglioni, Effects of low level lead exposure on hypo-
thalamic hormones and serum progesterone levels in pregnant guinea pigs, Toxicol-
ogy 72, 89-97 (1992).
8. G. Singh, D. K. Saxena, R. C. Murthy, and V. Chandra, Embryo fetal development
influenced by lead exposure in iron deficient rats, Human Exp. Toxicol. 12, 25-28 (1993).
9. C. B. Ernhart, A. W. Wolf, R. J. Sokol, G. M. Brittenham, and P. Erhard, Fetal lead
exposure, antenatal factors, Environ. Res. 38, 54-66 (1985).
10. S. W. Rosen, New placental proteins: chemistry, physiology and clinical use, Placenta
7, 575-594 (1986).
11. D. S. Klauder and H. G. Petering, Anemia of lead intoxication, a role of copper,
]. Nutr. 107, 1779-1785 (1977).

Biological Trace Element Research Vol. 67, 1999

Lead Burden During Pregnancy 213
12. W. J. Rogan, J. R. Reigert, and B. C. Gladen, Association of 8-amino levulinate dehy-
dratase levels and ferrochelatase inhibition in childhood lead exposure, J. Pediatr. 109,
60-64 (1986).
13. M. C. Domiguez, E. Sole, C. Goni, and A. Ballabriga, Effect of aluminium and lead
salts on lipid peroxidation and cell survival in human skin fibroblasts, Biol. Trace Ele-
ment Res. 47, 57-67 (1995).
14. F. H. Nielson, Oxidant stress effects on clinical and nutritional significance of trace
elements, in Trace and Toxic Elements in Nutrition and Health, M. Abdulla, S. B. Vohra,
and M. Athar, eds., Wiley Eastern Ltd., New Delhi, pp. 227-241 (1995).
15. K. H. Astrin, D. F. Bishop, J. W. Wetmur, B. Kaul, B. Davidow, and R. J. Desmick,
8-Aminolevulenic acid dehydratase isozymes and lead toxicity, Ann. N Y Acad. Sci.
514, 23-29 (1987).
16. M. Schuhmacher, J. L. Paternain, J. L. Domingo, and J. Corbella, An assesment of
some biomonitors indicative of occupational exposure to lead, Trace Elements Elec-
trolytes 14, 145-149 (1997).
17. E. J. H. Bechara, Free radical generation during delta amino levulinic acid auto-
oxidation: induction of hemoglobin and concentrations with porphyinopathies, Arch.
Biochem. Biophys. 271, 206-216 (1989).
18. H. P. Monteiro, D. S. P. Abdalla, A. Faljoni Alario, and E. J. H. Bechara, Generation
of active oxygen species during coupled auto-oxidation of oxyhemoglobin and delta
amino-levulinic acid, Biochim. Biophys. Acta 881, 100-106 (1986).
19. J. Jehan and D. B. Motlag, Effect of lead, zinc and copper on lipid peroxidation in rat
liver, in Trace and Toxic Elements in Nutrition and Health, M. Abdulla, S. B. Vohra, and
M. Ather eds., Wiley Eastern Ltd., New Delhi, pp. 210-214 (1995).
20. R. Sandhir and K. D. Gill, Effect of lipid peroxidation in liver of rats, Biol. Trace Ele-
ment Res. 48, 91-97 (1995).
21. D. L. Eaton, N. H. Stacey, K. L. Wong, and C. D. Klassen, Dose response effects of
various metal ions on rat liver metallothionein, glutathione, heme oxygenase and
cytochrome P-450, Toxicol. Appl. Pharmacol. 55, 393-402 (1989).
22. V. N. Finelli, D. S. Klauder, M. A. Karaffa, and H. G. Petering, Interaction of zinc and
lead on 8-amino levulinic acid dehydratase, Biochem. Biophy. Res. Commun. 65,
303-311 (1975).
23. S. J. S. Flora, D. Kumar, and S. D. Gupta, Interaction of zinc, methionine or their com-
bination with lead at gastrointestinal or post-absorptive level in rats, Pharmacol. Tox-
icol. 68, 3-7' (1991).
24. H. Ikebuchi, R. Teshima, K Suzuki, T. Terao, and Y. Yamane, Simultaneous induction
of Pb-metallothionein like protein and Zn-thionein in the liver of rats given lead
acetate, Biochem. J. 233, 541-546 (1986).
25. K. Nakagawa, Decreased glutathione-S-transferase activity in mice livers by acute
treatment with lead, independent of alteration in glutathione content, Toxicol. Lett. 56,
13-17 (1991).
26. B. Nehru and S. Kaushal, Biochemical and histological alterations following experi-
mental lead poisoning, J. Trace Element Exp. Med. 4, 203-209 (1991).
27. D. M. Nicholls, K. Teichert-Kuliszewska, and M. J. Kuliszewski, The activity of mem-
brane enzyme in homogenate fractions of rat kidney after administration of lead,
Toxicol. Appl. PharmacoI. 67, 193-199 (1983).
28. C. V. Vacca, J. D. Hines, and P. W. Hall, Protein urea of industrial lead intoxication,
Environ. Res. 41, 440-446 (1986).
29. B. Sarkar and A. Mas, The metabolism of metals in rat placenta, Biol. Trace Element
Res. 18, 191-99 (1988).
30. S. Kosmider, Lead and alkaline phosphatase levels in experimental animals and
humans, Pol. Arch. Med. 32, 1253-1257 (1963).
31. K. J. Freundt and H. A. Ibrahim, Growth of rats during a subchronic intake of heavy
metals, Pb, Cd, Zn, Mn, Cu, Hg and Be, Pol. ]. Occup. Med. 3, 227-232 (1990).

Biological Trace Element Research Vol. 67, 1999

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.