Heavy Metal Lead (Pb) and Cadmium (Cd) Cytotoxicity Assessment in Newborn Rats - Research Paper Example

Heavy Metal Lead (Pb) and Cadmium (Cd) Cytotoxi Assessment in Newborn Rats A cytotoxi assessment of the effects of the heavy metals lead (Pb) and Cadmium (Cd) on the activity of the anti-oxidant enzymes catalase and glutathione peroxidase, and the biomarker malondialdehyde, was carried out in rat pups at birthday (Day 0) and at weaning (Day 21). The data showed that prenatal and perinatal exposure to these toxic metals affected the anti-oxidant activity levels and the level of MDA, suggesting that the cytotoxicity of these metals is associated with the production of free radicals and membrane lipid peroxidation. Introduction The purpose of this study was to assess the cytotoxic effects of prenatal and perinatal exposure to the environmental metal toxins, cadmium and lead, in rat pups. Data obtained from this type of study may be useful in assessing the potential cytotoxic effects of metal exposure in humans at early stages of development. The specific aims of this research study were to evaluate the effects of metal exposure on (I) tissue catalase activity; (ii) the comparative levels of glutathione peroxidase activity at parturition and at weaning in pups exposed to cadmium postnatally; and (III) on the levels of malondialdehyde at weaning. These tests were performed on blood samples and brain tissues obtained from rat pups on the day of parturition (Day 0) and postnatally at weaning (Day 21). Toxic metals cadmium and lead constitute important environmental pollutants that are found in contaminated water, air soil, manufacturing products and certain foods. These redox inactive metals cause oxidative tissue damage by decreasing the levels of antioxidants in the body and also antioxidant enzymes, especially by means of interacting with their critical sulfhydryl SH groups (Ercal et al, 2001). These toxic metals induce the formation of hydroxyl radicals (OH-) and superoxide radicals (O2-) and peroxide (H2O2). Oxidative stress occurs in response to the elevated production of these highly reactive oxygen moieties, and may result in the depletion of critical anti-oxidant enzymes necessary for free radical inactivation. Lipids, proteins and DNA structure can be directly affected by ree radical s in the cell, resulting in significant physiological damage. The production of oxidative stress by these metals appears to be a significant cause of their cytotoxic effects in the body (Ercal et al, 2001). A great deal of experimental evidence indicates that exposure to the environmental toxins, lead and cadmium, may have serious consequences on neural development and multiple organ system function. Mahmodabdady et al (2006) have investigated the effects of lead acetate and cadmium chloride on the activity of these free radical scavenging enzymes in human fibroblasts. Cadmium is one of the most toxic environmental pollutants in terms of its widespread organ toxicity at very low concentrations. Nevertheless, it is an essential metal cofactor in the body. Cadmium exposure results in the depletion of selenium. Selenium is required for cadmium detoxification in the body. Selenium binds to cadmium and the complex is removed from the body in bile. This detoxification process results in bodily depletion of selenium, an essential cofactor in the formation of the antioxidant enzyme glutathione peroxidase (Mahmodabady, 2006). The depletion of reduced glutathione and the accumulation of oxygen free radicals in the body causes membrane peroxidation. Superoxide dismutase, catalase and glutathione peroxidase are critical enzymes involved in destruction of oxygen free radicals. Metallothionein is required for the detoxification of cadmium by the human body Research studies by Sandhir et al (1994) evaluated the effects of lead exposure on lipid peroxidation in the rat brain. In this study, exposure to 50 mg/kg body weight of lead over a period of eight weeks in adult rats was assessed. Lead exposure was found to result in the widespread accumulation of this toxic metal in the rat brain, with especially high levels in the hippocampus. The researchers observed that there was a linear relationship between the amount of lipid peroxidation and increasing levels of lead exposure. The activity of the enzyme antioxidants, superoxide dismutase, catalase and glutathione peroxidase were significantly reduced in neural tissue as a result of lead exposure. In addition, the levels of reduced glutathione and glutathione reductase were also significantly diminished by lead exposure. Additional research has shown that lead exposure is associated with iron-dependent membrane peroxidation. Lead also results in the oxidation of hemoglobin to metahemoglobin. Other effects of lead exposure were decreased acetylcholinesterase activity which showed a linear relationship to the level of membrane lipid peroxdation in response to lead exposure. The authors concluded that lead exposure in rats produced significant neurotoxicity resulting from a primary effect on membrane peroxidation damage (Sandhir, 1994). The purpose of this research study was to assess the following parameters: the comparative effects of lead versus cadmium on catalase activity at day 21; the depletion effects of cadmium on glutathione peroxidase activity levels at birth (parturition) and at weaning (postnatally); the effects of metal exposure on the level of malondialdehyde at the time of weaning (day 21).Groups of female rats were exposed to lead and cadmium prenataly and perinatally during lactation. Exposure to lead was at the level of 300 mg/Kg body weight. Exposure to cadmium was at a concentration of 10 mg/kG body weight. The toxic metals were administered via the drinking water. The control and treatment groups of animals were randomized on the first day of pregnancy. Metal exposure levels in the rat pups were assayed by means of blood levels, brain tissue testing and microscopy of the brain cortex. The test data were conducted on the day of birth (Day 0) and also on the day of weaning (Day 21). Objective 1: Analysis of the effects of cadmium and lead exposure on catalase activity The objective of this experiment was to determine the effects of cadmium and lead exposure on the level of an essential anti-oxidant enzyme, catalase. The rat pups were exposed to this toxic metal while the females were pregnant via contaminated drinking water. The levels of the anti-oxidant enzyme were measured at birth. The data assessment involved a measurement of enzyme levels in 100 rat pups at the two designated time intervals. The data were recorded and statistical measurements were performed. These consisted of the mean, the median, the data range, the standard deviation, the variance and the Anderson-Darling Statistical Test to evaluate the data distribution curve. 95% confidence levels were determined for the mean, the median and the standard deviation. The Anderson-Darling statistical test is a normality test that evaluates data that is significantly different from a normal distribution. It is considered an excellent tool for this type of data analysis. In this test, a hypothesis is rejected if the p-value (probability) is less than or equal to .05. If so, then one would conclude that with 95% confidence the data do not fit a normal distribution. At p-values > .05, one can conclude that the data do not constitute a significant deviation from a normal distribution. The test is limited by poor precision; if too many data points are coincident, the hypothesis may be rejected as the data will not constitute a good fit for a normal distribution. This must be kept in mind when one uses this statistical test. The confidence interval provides a data range that would be expected from additional untested members of a group. The range is estimated based on the data produced by the test group. If the confidence interval is large, this may indicate a lack of precision requiring more data sampling. Hypothesis The research hypothesis is that exposure to the toxic metals cadmium and lead will affect the activity of the anti-oxidant enzyme catalase. The hypothesis is derived from research indicating that toxic metal exposure causes oxidation and this induces the activation of enzymes that protect the cell from these destructive effects of oxidation. Assumptions The assumptions made in the experimental design are that the rat pups will respond to toxic metal exposure in ways that are similar to humans and other experimental subjects, to ensure study relevance. The physiological mechanisms of response to toxic metal exposure are assumed to operate by similar mechanisms in these different species. It is also assumed that the exposure levels are sufficient to cause the expected results on redox metabolism. Results Figure 1 shows the data for catalase activity measurements made in rat pups after exposure to the toxic metal cadmium. The data show a normal distribution range of catalse activity for 100 rat pups tested in this research study. The mean catalase activity was 2.2108 units. The minimum activity recorded was 1.6579 and the maximum activity was 2.6755. The 95% confidence interval for the mean was 2,1628-2.2587. The confidence ntern=val for the median was 2.1330-2.2679. The 95% confidence level for the standard deviation was .2121-.2807. The p-value was.605 indicating that the data are consistent with a normal distribution of values for enzyme activity. The data obtained indicate that cadmium exposure in this group of rat pups was associated with activation of catalase anti-oxidant activity. This observation activity is consistent with research data indicating that an important physiological response to cadmium is the production of H2O2, O2- and OH-. The presence of elevated levels of free radicals due to exposure to this toxic metal is associated with increased catalase activity as a cellular protective response against free radical damage. The catalase enzyme specifically targets peroxide and catalyzed its reduction to H20 an02. The data produced in this experiment are consistent with this model of catalase activation in response to cadmium exposure. Figure 1. Efect of Cadmium (Cd) on catalase activity. One hundred rats were tested for catalase activity levels after exposure to cadmium. The effect of lead exposure on catalase activity in 100 rat pups was also assessed in this experiment. The data obtained (see Figure 2) showed that the mean level of catalase activity in these animals was 2.2714 with a standard deviation of 0.3181. The median activity was 2.2714. The Anderson –Darling Normality Test showed a normal distribution of the data with a p-value 0f 0.517, indicating that the data are consistent with a normal distribution. The 95% confidence interval for the mean was 2.2083-2.3345. The 95% confidence level for the median was 2.1731-2.3708. The 95% confidence interval for the standard deviation was 0.2793-0.3695. Analysis of the data shows that both cadmium and lead exposure are associated with elevated levels of catalase activity. The degree of activation of this enzyme is similar in response to these two toxic metals; however, lead exposure produced an average activity of 2.2714, whereas cadmium’s effect on catalase activity was slightly less, with a mean activity of 2.2108, representing an average 3% difference in activity level. The observed similarity of the effect of cadmium and lead on catalase activity is consistent with the similar mechanisms of cytotocity of these toxic metals. Both are non-redox active metals that induce the formation of peroxide and free radicals. The free radical formation is associated with catalase activation. The similar effects of these two toxic metals on catalase activity suggest a similar mode of action for these metals. Figure 2. Effect of lead (Pb) exposure on catalase activity. One hundred rats were tested for levels of catalase activity after exposure to lead. Objective 1 outcome summary The data obtained indicate that cadmium exposure in this group of rat pups was associated with activation of catalase anti-oxidant activity. This observed activity is consistent with research data indicating that an important physiological response to cadmium is the production of H2O2, O2- and OH-. The presence of elevated levels of free radicals due to exposure to this toxic metal is associated with increased catalase activity as a cellular protective response against free radical damage. The catalase enzyme specifically targets peroxide and catalyzed its reduction to H20 an02. The data produced in this experiment are consistent with this model of catalase activation in response to cadmium exposure. The observed similarity of the effect of cadmium and lead on catalase activity is consistent with the similar mechanisms of cytotocity of these toxic metals. Both are non-redox active metals that induce the formation of peroxide and free radicals. The free radical formation is associated with catalase activation. The similar effects of these two toxic metals on catalase activity suggest a similar mode of action for these metals. Objective 2: Analysis of glutathione peroxidase (GSHPx) levels in rat pups on day of birth (Day 0) and at weaning (Day 21) following exposure to cadmium. The objective of this experiment was to determine the effects of prenatal cadmium exposure versus postnatal exposure on the levels of an essential anti-oxidant enzyme, glutathione peroxidase (GSHPx). The rat pups were exposed to this toxic metal while the females were pregnant via contaminated drinking water. The levels of the anti-oxidant enzyme were measured at birth. The exposure to cadmium was continued postnatally and the level of GSHPx was recorded at the time of weaning (Day 22).The data assessment involved a measurement of enzyme levels in 100 rat pups at the two designated time intervals. The data were recorded and statisticalmeasurements were performed. These consisted of the mean, the median, the data range, the standard deviation, the variance and the Anderson-Darling Statistical Test to evaluate the data distribution curve. 95% confidence levels were determined for the mean, the median and the standard deviation. Hypothesis The hypothesis of this experiment is that there is a difference between prenatal and postnatal exposure to cadmium on the activity of the anti-oxidant enzyme, glutathione peroxidase. The rationale for this hypothesis is that the mode of delivery of this toxic metal is different during gestation which involves placental delivery to the fetal bloodstream directly versus postnatal exposure by water consumption which travels first through the GI tract. In addition, the physiological effects of prenatal exposure may be different from postnatal exposure as research has shown that toxic metal exposure during embryonic and fetal development may have greater physiological consequences than later exposure. These differences may be reflected in differences in the enzyme activity of glutathione peroxidase. Assumptions The research assumptions are that underscore the experimental design are that prenatal exposure to environmental toxins affects the body differently than exposure postnally or later. The differences in mode of delivery, via the placenta versus by ingestion or breathing, are assumed to constitute difference with potentially important physiological consequences. Another assumption is that the effects of cytotoxicity during early stage development are different from effects on a fully developed body. Yet another important assumption is that the effects on glutathione peroxidase constitute a meaningful experimental assessment of cadmium cytotoxicity, an assumption corroborated by extensive research indicating that the activity of this enzyme comprises a critical physiological response to toxic metal exposure. Results At Day 0 (see Figure 3a), the mean GSHPx value was 28.461, with a standard deviation of 2.075. The median was 28.559. The minimum was 22.923 and the maximum activity was 32.530. The 95% confidence interval for the mean was 28.049-28.873. The 95% confidence interval for the median was 27.826-29.235. The 95% confidence interval for the standard deviation was 1.822-2.411. The p-value of the Anderson-Darling Normality Test was 0.223, indicating a normal distribution of the study data. Figure 3. Comparison of enzymatic levels of glutathione peroxidase (GSHPx) at (a.) birth (Day 0) and at (b.) weaning (Day 21). a. b. At Day 22 (see Figure 3b), the mean GSHPx value was 28.461/26.788, with a standard deviation of 2.666. The median was 28.559/26.250. The minimum was 21.354 and the maximum activity was 35.172. The 95% confidence interval for the mean was 26.259-27.317. The 95% confidence interval for the median was 25.757-27.251. The 95% confidence interval for the standard deviation was 2.341-3.097.The p-value for the Anderson-Darling Statistical Test was greater than 0.05, indicating a normal distribution of the data. The data show that the average GSHPx level was 8.1% lower in the rat pups at day 21 than at day 0. This result suggests that the effect of this toxic metal in prenatal exposure on GSHPx activity is greater than its postnatal effect, since the level of this criticalanti-oxidant enzyme decrased by almost 10% during this postnatal period when the animals were continuously exposed to cadmium. The data indicate that prenatal exposure to cadmium is more likely to affect tissue levels of GSHPx than early postnatal exposure via the breast milk. Moreover, research data indicate that cadmium is removed from the body very slowly following exposure; therefore, the continued exposure to cadmium postnatally may be expected to result in ever –increasing levels of this toxic metal in bodily tissues. Recent research suggests that very high levels of cadmium may result in decreased GSHPx levels; therefore, it is possible that the decreased enzyme levels observed at day 21 may actually reflect increased cadmium accumulation due to cumulative effects of exposure. It would be important to test also the levels of catalase which may more directly reflect levels of free radical formation in response to high level cadmium exposure than GSHPx. Objective 2 outcome summary The data indicate that prenatal exposure to cadmium is more likely to affect tissue levels of GSHPx than early postnatal exposure via the breast milk. Moreover, research data indicate that cadmium is removed from the body very slowly following exposure; therefore, the continued exposure to cadmium postnatally may be expected to result in ever –increasing levels of this toxic metal in bodily tissues. Recent research suggests that very high levels of cadmium may result in decreased GSHPx levels; therefore, it is possible that the decreased enzyme levels observed at day 21 may actually reflect increased cadmium accumulation due to cumulative effects of exposure. It would be important to test also the levels of catalase which may more directly reflect levels of free radical formation in response to high level cadmium exposure than GSHPx. Objective 3: Analysis of the effect of lead exposure on the level of malondialdehyde (MDA) The objective of this experiment was to determine the effects of prenatal lead and cadmium exposure on the tissue level of malondialdehyde (MDA), a biomarker for membrane peroxidation associated with the oxidizing effects of these toxic metals. The rat pups were exposed to these toxic metals while the females were pregnant via contaminated drinking water. The levels of MDA were measured at birth in 100 rat pups and compared to the levels of MDA in 100 rat pups that were not exposed prenatally to lead or cadmium, which served as a control group. The data assessment involved a measurement of MDA levels in 100 rat pups for the control and experimental groups. The data were recorded and statistical measurements were performed. These consisted of the mean, the median, the data range, the standard deviation, the variance and the Anderson-Darling Statistical Test to evaluate the data distribution curve. 95% confidence levels were determined for the mean, the median and the standard deviation. Hypothesis The research hypothesis is that exposure to the toxic metals cadmium and lead will affect tissue levels of malondialdehyde (MDA). The rationale for this hypothesis is that MDA has been shown by many research studies to represent a biomarker for toxic metal exposure. A determination of the difference between MDA levels in rat pups exposed to lead and cadmium compared to pups who were not exposed to these toxic metals are expected to yield differences in MDA levels that can be used as a relevant parameter to assess the physiological effects of toxic metal exposure. Assumptions The assumptions used to design this experiment are that MDA is a relevant physiological barometer of the effects of cadmium and lead exposure and that MDA will serve as a relevant physiological biomarker in this experimental system involving the analysis of rat pups. There is ample research evidence to support each of these assumptions. Another assumption is that the level of exposure and the time-frame of toxic metal exposure in this experiment will be sufficient to reveal physiological responses related to levels of MDA. Results Figure 4a shows that the mean MDA level recorded in the control group was 3.0046 with a standard deviation of0.5552. The median was 3.0488. The data range for MDA was 1.7589-4.2742. The 95% confidence interval for the mean was 2.8944-3.1147. The 95% confidence interval for the median was 2.8814-3.1615. The 95% confidence interval for the standard deviation was 0.4874-0.6449. The Anderson-Darling Statistical test showed a p-value of 0.876, indicating a normal distribution of the data. Figure 4. Measurement of MDA levels in (a.) control rat not exposed to either lead or cadmium; (b.) rats exposed to lead (Pb); and (c.) rats exposed to cadmium (Cd). a. b. Figure 4bsows that the mean MDA level recorded in the group exposed to lead was 3.0046 with a standard deviation of 0.5552. The median was 3.0488. The data range for MDA was 1.7589-4.2742. The 95% confidence interval for the mean was 2.8944-3.1147. The 95% confidence interval for the median was 2.8814-3.1615. The 95% confidence interval for the standard deviation was 0.4874-0.6449. The Anderson-Darling Statistical test showed a p-value of 0.876, indicating a normal distribution of the data. The data show that the average MDA level was 13% higher in the rat pups exposed to lead than in the normal control animals. c. The mean MDA level recorded in the group exposed to cadmium (Figure 4c) was 3.4710 with a standard deviation of 0.1363. The median was 3.4726. The data range for MDA was 3.1414-3.8464. The 95% confidence interval for the mean was 3.4440-3.4981. The 95% confidence interval for the median was 3.4409-3.5003. The 95% confidence interval for the standard deviation was 0.1197-0.1583. The Anderson-Darling Statistical test showed a p-value of 0.961, indicating a normal distribution of the data. The data show that the average MDA level was 13% higher in the rat pups exposed to lead than in the normal control animals. Taken together, these data indicate that exposure to lead or cadmium affects the MDA levels in the issues to a similar extent. This is a significant finding, since MDA is a biomarker to plasma membrane peroxidation, an important pathological response to toxic metal exposure that results in free radical formation. One would conclude that the effect of these toxic metals on free radical production is a significant molecular component of their cytotoxic effects and that the pathophysiology can manifest as a consequence of prenatal exposure. Objective 3 outcome summary The data show that the average MDA level was 13% higher in the rat pups exposed to lead than in the normal control animals. Taken together, these data indicate that exposure to lead or cadmium affects the MDA levels in the issues to a similar extent. This is a significant finding, since MDA is a biomarker to plasma membrane peroxidation, an important pathological response to toxic metal exposure that results in free radical formation. One would conclude that the effect of these toxic metals on free radical production is a significant molecular component of their cytotoxic effects and that the pathophysiology can manifest as a consequ ence of prenatal exposure. Discussion The results presented in this research study provide definitive evidence that prenatal and perinatal exposure to the toxic heavy metals lead and cadmium in rat pups produces significant effects on the activities of two essential anti-oxidant enzymes, catalase and glutathione peroxidase. In addition, these studies show that the levels of malondialdehyde (MDA), a biomarker for free radical damage associated with membrane peroxidation and pathophysiology, were also elevated in these test animals. MDA elevation is observed in response to these membrane toxic activities. Membrane peroxidation is the result of free radical activities of OH- and O2- that interact with unsaturated fatty acid double bonds in lipid membranes. H2O2 is the source of free radical formation and is the substrate of the anti-oxidant enzymes catalase and glutathione peroxidase. When the activities of these important enzymes are in habited by toxic heavy metal exposure, the free radicals accumulation and cause membrane peroxidation. Cadmium is actually a micronutrient found in certain food groups such as grains and vegetables. It is also an environmental pollutant of lead and zinc mining. Exposure is in the form of inhalation or oral ingestion. Within the body, cadmium is stored in the soft tissues and accumulates for very long periods of time. This toxic metal is associated with membrane peroxidation in the tissues of the liver, kidneys, brain, heart and lung. Responses to cadmium exposure in rats are associated with elevated catalase and GSHPx activity as a stress induced response; however, at high dose exposure levels, GSHPx activity in rats was depressed (Latinwo et al, 2006), Experiments in mice have shown that a single dose of cadmium chloride can activate the levels of both catalase and glutathione peroxidase in fibroblasts. Moreover, exposure of skin fibroblasts to cadmium chloride was found to produce elevated levels of MDA. This effect was observed at all concentrations of cadmium and for all exposure times (Latinwo et al, 2006). The results of this study showed that lead exposure resulted in elevation of MDA levels compared to controls. An important physiological response to lead is the elevation of malondialdehyde levels in the body. Lipid peroxidation of brain tissue is a dose dependent physiological response to lead exposure. This is associated with fatty acid saturation and an increase in MDA levels. Therefore, elevated MDA represents an important biomarker for lead toxicity. Lead exposure alters the phospholipid composition of the cell membrane, resulting in peroxidation and pathophysiology associated with changes in membrane fluidity function and permeability. Recommendations for future research In contrast to the findings presented in this research study, other researchers did not observe an increase in the level of malondialdehyde response to exposure to lead acetate at any concentration (Mahmodabady et al, 2006). This represents an area for future research to resolve the discrepancies between these very different research findings. The data presented in the Mahmodabady study indicate that, in contrast to cadmium, short term exposure to lead was not associated with lipid peroxidation. The significant decrease in glutathione peroxidase levels in response to cadmium exposure was most likely a secondary consequence of the effect of cadmium on selenium depletion. The inhibitory effect of toxic metals on glutathione peroxidase is a biomarker for the cytotoxic effects of these metals on antioxidant activity in the body (Mahmodabady et al, 2006). Glutathione peroxidase is required for the inactivation of lipid peroxides. The activity of this enzyme is dependent on the presence of free SH groups. It is thought that the depletion of free SH by lead results both in the decreased activity of glutathione peroxidase and the increased activity of malondialdehyde (Kulikowska-Karpinska & Moniuszko-Jakoniuk, 2001). Toxic metals may reduce the activities of antioxidant enzymes by binding covalently to their active sites and by replacing the essential co-factor, zinc, in reductase reactions. A compensatory physiological response may be to elevate the production of anti-oxidant enzymes to restore redox equilibrium. These authors found that the administration of zinc after lead exposure causes a more rapid restoration of normal enzymatic levels of glutathione peroxidase and lower levels of malondialdehyde. It is thought that zinc may act to prevent the oxidation of enzymatically important SH groups and to block the peroxidation reactions associated with toxic metal cell membrane damage (Kulikowska-Karpinska & Moniuszko-Jakoniuk, 2001). It will be important to assess the levels of other physiological markers for toxic metal exposure to develop a better understanding of the effects of these toxins. Research on the effects of zinc may show that this metal may reduce the cytotoxic effects of heavy metal contaminant exposure. A second important outcome of this and similar research studies is the observation that GSHPx measurements may not be as reliable as MDA and catalase activity mesadurements as indictors of the lead and cadmium physiological responses. Catalase and MDA levels parallel exposure levels to each of these toxic metals and may provide useful biomoarkers of pathophysiological responses to toxic metal exposure. References Ercal, N, Gurer-Orhan H & Aykan-Burns, N 2001, ‘Toxic metals and oxidative stress part 1: mechanisms involved in metal induced oxidative damage’ Current Topics in Medicinal Chemistry, vol. 1, pp. 529-539. Kulikowska-Karpinska,E & Moniuszko-Jakoniuk, J 2001, ‘Lead and zinc influence on anti-oxidant activity and malondialdehyde concentrations’, Polish Journal of Environmental Studies, vol. 10, no. 3, pp. 161-5. Latinwo, L et al 2006, ‘Effect of cadmium induced oxidative stress on anti-oxidative enzymes in mitochondria and cytoplasm of CRL-1439 rat liver cells’, International Journal of Molecular Medicine, vol. 18, pp. 477-481. Mahmodabady, Z et al 2006, ‘Cytotoxic and oxidative stress caused by cadmium and lead in human skin fibroblast cells’, Yakhteh Medical Journal, vol. 8, no.3, pp.172-177. Sandhir, R, Julka, D & Dip Gill, K 1994, ‘Lipoperoxidative Damage on Lead Exposure in Rat Brain and its Implications on Membrane Bound Enzymes’, Pharmacology & Toxicology, vol. 74, pp. 66–71. Read More