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In the present study, the role of vitamins C and E on blood parameters of lead exposed pregnant females during gestation and lactation were studied in Swiss mice. Pregnant mice were exposed to 266, 533 and 1066 mg/kg BW (8, 16 and 32 mg/animal/day) of lead acetate and blood parameters were monitored in pregnant females during gestation and after parturition. The effects of vitamins C (166 mg/kg BW) and E (133 mg/kg BW) separately and in combination with lead were also studied. The present study shows that exposure of lead from 10th day of gestation up to 21st day of lactation caused hematological changes in mothers during gestation and lactation. During the respective tenure of experiment, hemoglobin, RBC and WBC counts were recorded on 17th day of gestation and 1st, 7th, 14th and 21st days of lactation. The hemoglobin content and RBC counts were significantly decreased. These changes were not markedly improved with administration of vitamin C and E. Lead also adversely affects WBC count. Dose dependent significant increases in WBC counts were observed after administration of different dose levels of lead acetate. These effects were not reversed by the treatment of vitamins; higher doses of these vitamins generate negative effects on hematological parameters.

Exposure to lead results many toxicological problems. Vitamin C and E are the most comprehensively studied antioxidant with respect to lead intoxication. The purpose of the present study was to evaluate the ameliorative potential of vitamin C and vitamin E against lead induced toxicity in Swiss albino mice.

Heavy Metal Toxicity

Most heavy metals are not physiologically or biochemically essential to an organism. In many cases they are extremely dangerous, as they are easily absorbed and remain in tissues for a long time 40.

Heavy metals become toxic when they are not metabolized by the body and accumulate in the soft tissues. In recent years, a wide body of evidence involvement of oxidative stress in metal induced toxicity has been reported. The enhanced generation of highly reactive oxygrn species, such as hydroxyl radical (HO), superoxide radical (O -), hydrogen peroxide (H O ) and lipid peroxides (LPO), between heavy metals is known to cause lession in various cellular components including lipids, proteins, and DNA 41.

Lead is an indestructible heavy metal that can accumulate and linger in the body. Based on epidemiological and experimental data, the Working Group of the International Agency for Research on Cancer (IARC) concluded that inorganic lead compounds are probably carcinogenic to humans 12.

Lead toxicity can affect organ system. Environmental lead exposure remains a serious concern for the growth and development of children. Exposure of lead during pregnancy is one source from which a fetus can be exposed to lead. Mice exposed to lead continuously beginning at approximately 6 days prior to birth, showed significant decrease in their blood lead level 2 weeks after weaning, despite continued exposure to adult. Their result suggests maternal transfer of lead is more efficient than oral adult exposure and substantial lead transfer occurs both transplacentally and lactionally.

Effects of Lead

Lead adversely affects survival, growth, reproduction, development, and metabolism of most species under controlled conditions, but its effects are substantially modified by numerous physical, chemical, and biological variables.

Several reports have indicated that lead can cause neurological, hematological, gastrointestinal, reproductive, circulatory, immunological, histopathological and histochemical changes all of them related to the dose and time of exposure to Pb42.

Absorption

Lead is more readily absorbed in fasting individuals (up to 45% for adults) than when ingested with food. Inorganic lead absorbed into the mammalian body enters the bloodstream initially and attaches to the red blood cell. There is a further rapid distribution of the lead between blood extracellular fluid and other storage sites that is so rapid that only about half the freshly absorbed lead remains in the blood after a few minutes.

Lead Poisoning

Lead-induced oxidative stress has been identified as the primary contributory agent in the pathogenesis of lead poisoning 17. Lead poisoning is one of the commonest disease, although in recent years there has been a decline in both the number of reported cases and the severity of the symptoms presented, hence lead poisoning has shifted from an industrial hazard to an environmental. Lead poisoning has been recognized for at least 2,500 years. All credible evidence indicates that lead is neither essential nor beneficial to living organisms, and that all measured effects are adverse--including those on survival, growth, reproduction, development, behavior, learning, and metabolism. At present, there is no known dietary requirement for lead in domestic animals, nor has it been shown unequivocally that lead plays any beneficial role 43. On the contrary, lead demonstrably and adversely affects weight, survival, behavior, litter size, and skeletal development 44 and induces teratogenic and carcinogenic responses in some species of experimental animals 43 &45. Lead has been shown to alter RBC membrane flexibility and to increase RBC fragility, leading to increased risk for hemolysis46.

In chronic, moderately severe lead poisoning, anemia is commonly found 47. Anemia in lead poisoning results from impairment of hemoglobin production and changes in the red blood cell membrane.

Lead Toxicity and Blood

All animals maintained fairly normal hemoglobin concentrations even though during excretion of intermediates in porphyrin and heme synthesis, it increased several fold. There is a substantial reserve capacity for formation of hemoglobin which is reflected in maintenance of effectively normal hemoglobin concentrations despitetwo to threefold increase in excretion of intermediate products. Lead has a destabilizing effect on cellular membranes, andin red blood cells (RBC) the effect decreases cell membrane fluidity and increases the rate of erythrocyte hemolysis. Hemolysis appears to be the end result of ROS-generated lipid peroxidation in the RBC membrane48.

A lead-related decrease in the duration of pregnancy, decrease in birth weight, and small-gestational- age deliveries have been detected at cord blood lead levels of 10 to 19 µg/dL 49. These findings have not been consistent through all studies. It has been found during the postnatal stage of the prospective studies that the growth rate of infants is slowed. This effect was noted among infants born to women with blood lead concentrations greater than 8 µg/dL during pregnancy. Where sudden changes in blood lead concentration occur, further investigation is necessary to confirm the change and find the reason for the change. A sudden increase in blood lead concentration may be due to a lead exposure.

Oxidative Stress

Lead affects mammalian systems by directly lowering antioxidant reserves and generating ROS, specifically  hydroperoxides and lipoperoxides. These ROS alter cellular membranes and tissue, resulting in vascular, neurological, and genetic damage. The pathogenesis of lead toxicity is multifactorial, as lead directly interrupts enzyme activation, competitively inhibits trace mineral absorption, binds to sulfhydryl proteins (interrupting structural protein synthesis), alters calcium homeostasis, and lowers the level of available sulfhydryl antioxidant reserves in the body 50.

Some toxic agents works by attacking functional macromolecules such as lipid, protein, and nucleic acids, either through the generation of free radicals, depletion of antioxidant molecules, inflammation, and apoptosis60.

Toxic metals increase production of free radicals and decrease availability of antioxidant reserves to respond to the resultant damage.

Antioxidants

The major mechanism of lead toxicity is oxidative stress; natural products rich in antioxidants can be a good antidote against lead poison and can be used along with common lead chelators. Several compounds from natural products with confirmed antioxidant activities have been used as a hepatoprotective agent against lead position61.

Antioxidants, such as ascorbic acid, α-tocopherol (vitamin E), endogenous glutathione peroxidase and the pineal hormone melatonin, have all been tested for efficacy in defending against free-radical-mediated tissue injuries. In gastroprotection, the first line of antioxidative enzyme is SOD which catalyses the dismutation of superoxide radical anion (O2) into less noxious hydrogen peroxide (H2O2). H2O2 is then inactivated by the degradation into water by catalase or glutathione peroxidase 51. Vitamin C is a known free-radical scavenger and has been shown to inhibit lipid peroxidation in liver and brain tissue of lead-exposed animals 27.

In other animal studies, the toxic effects of lead on heme production were reversed by a vitamin C dose of 100 mg/kg 52. Other studies indicate vitamin C might have significant chelation capacity for lead.

Individuals who consume more than 340 mg of vitamin C tend to have lower blood lead levels than those who consume less than 110 mg. Consumption of 1000 mg a day has been shown to significantly decrease lead levels in some, though not all, cases - apparently more through reduced absorption rather than increased excretion.

Vitamin E has a known protective action in membrane stability and prevents membrane lipoproteins from oxidative damage 53. Alpha-tocopherol was shown to prevent RBC membrane damage in lead toxicity by lowering lipid peroxide levels and increasing SOD and catalase activity 54. Animal studies have found vitamin E to effectively prevent lipoperoxide-related lead toxicity in sperm 55 and to be more effective than methionine or vitamin C at decreasing lipoperoxidation in the liver, brain, and kidney of lead-exposed rats when given in doses of 100 IU/kg body weight 52.

Lead-induced alterations in the hemopoitic system are among the toxic effects of lead. Ascorbic acid has an important role in restoring lead- induced alterations in the hemopoitic system and drug metabolizing enzyme 52.   

Recent study has documented the beneficial effects of vitamin C, vitamin E, either alone or in combination with DMSA or MIDMSA, on the oxidative stress indices in the liver, kidney, brain and blood of lead exposed rats 56. 46 determined the filterability of RBC, as well as RBC lipid peroxidation, in vitamin E deficient and vitamin E supplemented lead-exposed rats. Lead exposed vitamin E deficient RBCs were mechanically fragile and were susceptible to  oxidative  stress. Decreased filterability of RBCs and increased lipid peroxidation  were observed  in the vitamin E deficient rats. With vitamin E supplementation, these effects were reversed. It is well documented that vitamin e reacts with lipid peroxyl radicals to form vitamin E radicals that are incapable of abstracting H- from the membrane lipids. the vitamin E radical then acts as a chain terminator by interrupting chain reactions during lipid peroxidation 57.

Vitamin C reduces the vitamin E radical by recovering the chain-breaking antioxidant capacity of vitamin E 58.

Combination treatment with vitamin E and chelators wes able to preveny lead-induced lipid peroxidative damage 27 & 59. Vitamin E is the natural most effective lipid soluble antioxidant, which protects biological membranes and lipoproteins from oxidative stress 53. The role of the stomach in vitamin E digestion is suspected to be limited as this vitamin is naturally present in its free form, but no study has assessed whether there is any degradation of this essential antioxidant in the stomach. 

Adult female Swiss mice weighing 28-30 gm were used in the present study. Sexually mature males and females were kept in cages for breeding. These cages were checked every day in the morning and females showing vaginal plug were isolated and duration of their gestation period were recorded. The experimental study was approved by the Institutional Animal Ethics Committee of the M. L. S. University no. CS/Res/07/759 and the guidelines were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) The pregnant Swiss mice were separated into 12 groups of 6 animals each. Group 1: Control group were administered with distilled water Group 2: Exposure of 266 mg/kg BW (8 mg/animal/day) of lead acetate from 10th day of gestation up to 21st day of lactation Group 3: Exposure of 533 mg/kg (16 mg/animal/day) BW of lead acetate from 10th day of gestation up to 21st day of lactation Group 4: Exposure of 1066 (32 mg/animal/day) mg/kg BW of lead acetate from 10th day of gestation up to 21st day of lactation Group 5: Exposure of 166 mg/kg BW of vitamin C from 10th day of gestation up to 21st day of lactation Group 6: Exposure of 133 mg/kg BW of vitamin E from 10th day of gestation up to 21st day of lactation Group 7: Exposure of 266 mg/kg BW of lead acetate + 166 mg/kg BW of vitamin C from 10thday of gestation up to 21st day of lactation Group 8: Exposure of 533 mg/kg BW of lead acetate + 166 mg/kg BW of vitamin C from 10th day of gestation up to 21st day of lactation Group 9: Exposure of 1066 mg/kg BW of lead acetate + 166 mg/kg BW of vitamin C from 10th day of gestation up to 21st day of lactation Group 10: Exposure of 266 mg/kg BW of lead acetate + 133 mg/kg BW of vitamin E from 10th day of gestation up to 21st day of lactation Group 11: Exposure of 533 mg/kg BW of lead acetate + 133 mg/kg BW of vitamin E from 10th day of gestation up to 21st day of lactation Group 12: Exposure of 1066 mg/kg BW of lead acetate + 133 mg/kg BW of vitamin E from 10th day of gestation up to 21st day of lactation. Numerical changes in red blood cells (RBC) and white blood cells (WBC) of the mothers were observed for hematological investigation. During the respective tenure of experiments hemoglobin, RBC and WBC counts of female Swiss mice were recorded on 17th day of gestation and on the 1st, 7th, 14th and 21st days of lactation. Blood samples for hemoglobin and blood cell counts were obtained from the tail of each mouse. The tip of the tail was cleaned with spirit before being cut with a sharp blade and was not squeezed to avoid dilution of blood by tissue fluid. The first few drops of blood were discarded and the blood was diluted, for cell counting with the help of haemocytometer. The hemoglobin was estimated by hemoglobinometer. Number of (RBCs) and (WBCs) were estimated with haemocytometer adopting the method described by 11.

The statistical analysis was performed by using completely randomized design analysis of variance (CRD-ANOVA) for the comparison of data between different experimental groups.

In the present study, the toxic effects of lead on hemoglobin content and blood cells (RBCs and WBCs) were measured in Swiss mice during 17^th day of gestation and the 1^st, 7^th, 14^th and 21^st day of lactation period. We also observed the effects of vitamin C and E alone and in combination with lead on same parameters.
Effects of lead exposure on mother during gestation and lactation
Administration of different doses of lead in Swiss mice during gestation and lactation period produces a significant decrease in hemoglobin content (Table 1) and RBC counts (Table 2) as compared to the control but there is non-significant decrease in 8 mg lead treated groups at the time of birth. However, WBC counts (Table 3) cause significant increase during gestation and lactation.
Effects of vitamin C and E exposure on mother during gestation and lactation
In vitamin C and Etreated groups; there is no significant change in hemoglobincontent and RBC counts during gestation an lactation period as compared to the control group. However hemoglobin content show significant decrease at the 14^th and 21^st Day of lactation and RBC counts show significant decrease at the 21^st day of lactation (Table 1 and 2). WBC counts show significant increase in vitamin C group, but non-significant increase at the time of birth. In vitamin E group, WBC counts show non-significant increase in gestation and lactation period but non-significant decrease at 14^th and 21^st day of lactation as compared to the control group (Table 3).
Effects of lead and vitamins exposure on mother during gestation and lactation
Treated mother with vitamin C and E concomitantly with lead groups show significant decrease in hemoglobin content and RBC counts during gestation and lactation period as compared to the control group, but hemoglobin content show non-significant decrease at the time of birth at various dose level (8 + C, 16 + C, 8 + E and 16 + E) (Table 1). At lower doses (8 + C and 8 + E), RBC counts show non-significant decrease during gestation and all lead + vitamins treated groups show non-significant decrease at the time of birth (Table 2). WBC counts show non-significant decrease in all combined groups, but at lower doses (8 + C and 8 + E), and WBC count show significant decreases during lactation period as compared with control (Table 3).
In all groups, the combined treatment of lead + vitamin C and lead + vitamin E show non-significant decrease in hemoglobin content and RBC counts during gestation and lactation period compared with lead groups but in 8 + E treated group, hemoglobin content show significant decrease at 14^th and 21^st day of lactation (Table 1). RBC counts of the 32 + C, 8 + E and 32 + E groups show significant decrease at later stages of lactation (Table 2). WBC counts show significant decrease in all stages of vitamin C and E treated groups, but non-significant decrease in gestation at lower dose as compare to lead treated group (Table 3).
The administration of lead + vitamin C group shows non-significant decrease in hemoglobin content and RBC counts during gestation and 1^st day of lactation as compared to the vitamin C treated Group. These groups show significant decrease at 7^th, 14^th and 21^st day of lactation.Treatment with lead + vitamin E caused a significant decrease in hemoglobin content and RBC counts during gestation and lactation when compared to the vitamin E (Table 1 and 2). WBC counts show decrease in vitamin C treated group, but this is non-significant at gestation and significant during lactation period as compared with vitamin C group. In lead + vitamin E treated groups, WBC counts caused significant decrease during gestation and lactation period as compared to the vitamin E group. This decrease is non-significant in gestation period and becomes significant at 1^st, 7^th and 14^th day of lactation with lower doses (8 + E and 16 + E) (Table 3).
All blood parameters show decrease from gestation up to the time of birth in all groups.
Table 1. Effect of vitamins on lead exposed mother’s hemoglobin (g/dL), levels


Values are expressed as mean ± S.D. for six female Swiss mice/group, P value >0.05 = non-significant (n.s.), <0.05 = significant (*) and <0.01 = highly significant (**).
A = compare with control, B = compare with 8 mg lead, C = compare with 16 mg lead, D = compare with 32 mg lead, E = compare with vitamin C and F = compare with vitamin E.
Table 2. Effect of vitamins C and E on lead exposed mother’s RBC counts (´10⁶ cells/mm³)


Values are expressed as mean ± S.D. for six female Swiss mice/group, P value >0.05 = non-significant (n.s.), <0.05 = significant (*) and <0.01 = highly significant (**).
A = compare with control, B = compare with 8 mg lead, C = compare with 16 mg lead, D = compare with 32 mg lead, E = compare with vitamin C and F = compare with vitamin E.
Table 3. Effect of vitamins on lead exposed mother’s WBC counts (´10³ cells/mm³)


Values are expressed as mean ± S.D.for six female Swiss mice/group, P value >0.05 = non-significant (n.s.), <0.05 = significant (*) and <0.01 = highly significant (**).
A = compare with control, B = compare with 8 mg lead, C = compare with 16 mg lead, D = compare with 32 mg lead, E = compare with vitamin C and F = compare with vitamin E.
Discussion
The body consists of an elaborate antioxidant defense system that depends on dietary intake of natural vitamins and minerals. Antioxidants are vitamins that supply missing electrons for unstable molecules in order to prevent free radical damage from external and internal sources. Lead toxicity leads to free radical damage via two separate, although related, pathways: (1) the generation of reactive oxygen species (ROS), including hydroperoxides, singlet oxygen, and hydrogen peroxide, and (2) the direct depletion of antioxidant reserves.
Antioxidants, such as ascorbic acid, α-tocopherol (vitamin E), endogenous glutathione peroxides and the pineal hormone melatonin, have all been tested for efficacy in defending against free-radical-mediated tissue injuries. There is evidence that some nutrients especially vitamin C exhibit some protective effects against lead intoxication. Vitamin C is a known free-radical scavenger and has been shown to inhibit lipid peroxidation in liver and brain tissue of lead-exposed animals ³⁰.
It is well known that lead passes through the placenta of mother to fetus and accumulates in fetal tissues during gestation ¹⁴ and it can be passed through mother’s milk during lactation ⁶. Moderate lead levels of 100 micrograms/L can also inhibit fetal haeme and erythropoiesis. Besides the classical signs of lead poisoning, pregnant women face the risk of spontaneous abortion and increased blood pressure.
The alterations in hematological parameters serve as the earliest indicator of toxic effects on tissue ²⁹. Therefore in the present investigation, toxic effects of lead are evaluated by using hemoglobin and blood cell counts (WBCs and RBCs) as the hematological parameters and effects of vitamins C and E studied in lead intoxicated Swiss mice.
The amount of lead in blood and tissues, as well as the time course of exposure, determines the level of toxicity ³¹. Blood often shows pathological changes before the external signs of poisoning become apparent. The absorbed lead enters the blood stream, where over 90 percent of it is bound to the red cells with a biological half-life of 25-28 days ⁴. Toxicological effects of lead have their origin in perturbation in cell function of various organ systems. The major biochemical effect of lead is its interference with heme synthesis which leads to hematological damage ³.
Table 1, 2 and 3 represent the changes in the hemoglobin, red blood cell and white blood cell counts during pregnancy and lactation. The maximum decrease in hemoglobin and RBCs was observed during the first 7 days after parturition. It was also observed that number of WBCs showed dose dependent significant increase throughout the period of lactation. This finding therefore corroborates with similar findings suggested by ²¹, who reported that administration of lead acetate to the female lactating rats caused a significant decrease in hemoglobin (Hb) concentration and red blood cell count (RBC), whereas the white blood cells count (WBC) significantly increased. Additionally in a recent study ³⁹ suggestedthat the significance reduction in packed cell volume( PCV), haemoglobin concentration (HBC), red blood cell count (RBC) and significant elevations in total white blood cell count (TWBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)  and platelets in animals administered lead only (Pb C) in comparison to the normal control (NC) 
Lead acetate affects the hematological system even at concentrations below 10μg/dl ² by inhibiting the activities of several enzymes (ALAD) involved in heme biosynthesis and by shortening of erythrocyte life span ³⁴ as well as by inducing inappropriate production of erythropoietin leading to inadequate maturation of red cell progenitors and affecting the introduction of Fe²⁺ into protoporphyrin IX ³⁷.
The effects of lead on hematopoietic system in adult animal models were also studied by many investigators. ²⁷ observed a significant decrease in erythrocytes number and hemoglobin concentration in rats that was injected with lead as acetic acid. ²³ also reported similar finding in mice.
Development of anemia in lead toxicity may be attributed to the decreased red blood cell survival because of increased membrane fragility, reduced RBC count, decreased hemoglobin production, or summation of all these factors ³³.Similar finding were recorded by ³⁸ that Pb A caused a significant reduction in packed cell volume, hemoglobin while the total white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils and basophiles increased.  According to ⁴¹ lowered RBC counts were observed in male weanling rats that were exposed to lead acetate.
In adult male rats, lead decreased RBC count (anemia) and increased leukocyte count (leukocytosis) ²⁷. Lead acetate treated Swiss mice exhibited dose-dependent significant decrease in RBC counts and increase in WBC count ⁵.
In the present investigation it is also observed that WBCs show dose dependent, significant increase in their counts. Opposite to this, decreased leukocyte count were noted in lead exposed groups by ¹⁸, where total leukocyte count were significantly decreased in treated mothers administered 2-6 mg lead/kg/day as lead nitrate by gavages once. Opposite results has been reported by ³⁸, who suggested no changes in the white blood cell count in rats treated with lead. The variation in the total leucocytes count may be due to different doses given to the animals. If vitamin C is taken in mega doses during pregnancy, this can have a negative effect on pregnancy and developing fetus. Some studies show that taking too much vitamin C in the form of supplements during pregnancy may increase the risk of preterm birth.
The present study exhibited no major changes by vitamin C and E to lead induced hematological toxicity in pregnant Swiss mice during gestation and lactation. Our finding suggested that supplementation with vitamins (C and E) and in combination with lead (L+C and L+E) produced a decrease in hemoglobin content and RBC counts, whereas increased in WBC counts and non-significant when treated with vitamin C and E. The toxic damages which are caused by lead is ameliorated to a great extent by the treatment of antioxidants (vitamin C and E). High levels of vitamins given during pregnancy and lactation do not show any beneficial results and induces negative impact.
Opposite results has been reported by 21 who suggested that concomitant administration of vitamin E and lead acetate during lactation produced significant increase in hemoglobin concentration in lactating rats. Vitamin C supplementation in lead-exposed animals significantly reduces blood lead levels, and associated biochemical changes indicating a significant protective action of vitamin that these dose levels are safe in adult system but during the period of gestation and lactation they are not beneficial.

This study demonstrated the hematological changes in exposed mothers during gestation and lactation. The outcome of the studied parameters indicates that higher doses of vitamin C and E are not protective against lead toxicity during gestation and lactation. However, mega doses of vitamins could be a very effective in adult organ system against lead toxicity. present study shows that exposure of lead from 10th day of gestation cause hematological changes in mothers during gestation and lactation. The changes are dose dependent. The results of present investigation showed that hemoglobin content and RBC counts were significantly decreased. These changes were not improved with administration of vitamin C and E, either given individually or in combination. Lead also adversely affects WBC count. Dose dependent significant increase in WBC counts were observed after administration of 266, 533 and 1066 mg/kg BW of lead acetate. Our finding suggests that supplementation with antioxidants vitamin C and E alone and in combination with lead produced a decrease in RBC counts. Low dose, long-term exposure and high-dose, short-term exposure of toxicants, both needs to be studied, and the impact of lead toxicity on mammalian development needs to be evaluated. Furthermore, although safe threshold levels have been derived from adult animal data, factors such as size, relative differences in consumption, and different maturity of metabolic pathways suggest that these threshold levels could be misleading. Little is known about the specific changes and risks factors that occur during developing stages. Our findings suggest that administration of higher concentrations of antioxidant vitamins may be beneficial for adult animals which were exposed to lead but supplementation of these vitamins during gestation and lactation could not prevent the negative effects generated by lead.

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