Repair of wounded monolayers of cultured vascular endothelial cells after simultaneous exposure to lead and zinc

We investigated the interaction between lead and zinc on the repair of wounded monolayers of cultured bovine aortic endothelial cells. A half area of the monolayers was wounded and then incubated in the presence of lead (5.0 and 10 μM) and/or zinc (10 μM). It was morphologically observed that the appearance of the cells in the wounded area was strongly decreased by lead alone but considerably increased by zinc alone. The repair of wounded area after simultaneous exposure to lead and zinc showed that lead inhibits not only spontaneous but also zinc-promoted repair of endothelial cell layers without a change of the leakage of lactate dehydrogenase. Interaction between lead and zinc on the DNA synthesis of growing endothelial cells was similar to that on the repair, suggesting that the repair reflected the proliferation. In growing endothelial cells, the intracellular accumulation of lead was significantly increased by zinc; that of zinc was unaffected by lead; and that of metallothionein was slightly increased by zinc and lead but the effect of zinc was suppressed in the presence of lead. Although zinc significantly decreased the intracellular accumulation of radioactive calcium, lead increased it in the presence or absence of zinc. It was therefore concluded that lead inhibits not only spontaneous but also zinc-promoted repair of the damaged endothelial cell layers through an inhibition of the proliferation mediated by the calcium-mediated signalling pathways and/or a disturbance of intracellular calcium homeostasis.     

Responses of hepatocytes to DDT and methyl mercury exposure

The aim of the current work was to investigate the effects of dichlorodiphenyltrichloroethane (DDT) and monomethyl mercury (MeHg) on hepatocytes from tropical fish Hypostomus commersoni (cascudo). In order to verify DDT and MeHg impacts on the redox milieu, cells were exposed for 4 days to 50 nM of DDT, 0.25 and 2.5 μM of MeHg and to a combination of 50 nM of DDT and 0.25 μM of MeHg. These concentrations were compared with those previously published (Filipak Neto et al., 2008) for the predator fish Hoplias malabaricus (traíra). The effects were mostly noticeable on reduced glutathione concentration and δ-aminolevulinic acid dehydratase and glutathione S-transferase activity. Catalase activity increased in the group exposed to 2.5 μM of MeHg and hydrogen peroxide levels decreased in all exposed groups. Also, superoxide anion levels decreased in the groups exposed to 2.5 μM of MeHg and DDT1MeHg group. Cell viability decreased only in the DDT exposed group, demonstrating that the antioxidant defense mechanism of H. commersoni hepatocytes is more efficient than H. malabaricus. These results corroborate the resistance of H. commersoni to polluted areas and support the hypothesis that this species is more resistant to DDT and MeHg than H. malabaricus species.     

Selectivity of methyl mercury effects on cytoskeleton and mitotic progression in cultured cells

Methyl mercury (MeHg) disrupts microtubules, but effects on other cytoskeleton components are not well studied. Dose-effect relationships were used to determine the selectivity of MeHg effects on vimentin intermediate filaments, actin microfilaments, and microtubules. These were examined in PtK2 cells by immunofluorescence. At 0.5 microM MeHg the number of microtubules was reduced. After 1.0 or 2.0 microM MeHg, microtubules in most cells were disassembled except for a few "stable" microtubules. No effects on vimentin or actin filaments were observed except as secondary effects at concentrations of MeHg that caused extensive microtubule disassembly. Antimitotic effects of MeHg are well known. An assay based on fluid pinocytosis was used here to study kinetics of mitotic progression. HeLa cells were arrested at prometaphase with a lengthening of subsequent stages of mitosis. Progression from prophase to prometaphase was not affected. MeHg treatment also increased the number of micronucleated and multinucleated cells. Drugs specific for actin cause similar effects by blocking cytokinesis but the selective action of MeHg on microtubules argues against this mechanism. Data from both interphase and mitotic cultured cells indicate that MeHg acts selectively on microtubules. It further supports the hypothesis that the mechanism of damage of MeHg is related to its antimicrotubule activity.     

Blood and brain mercury levels after chronic gestational exposure to methylmercury in rats.

Female rats were exposed to 0, 0.5, or 6 ppm Hg (as methylmercuric chloride, 10 rats/group) in drinking water. For half the rats, exposure began 4 weeks before mating and for the others, exposure began 7 weeks before mating. All mating was done with an unexposed male. Maternal exposure continued to post-natal day (PN) 16. Blood and whole-brain mercury concentrations were determined in pups on PN 0 (birth) and PN 21 (weaning). Maternal water consumption was monitored daily during gestation and lactation. Maternal water consumption increased 2- to 3-fold through gestation for all groups. Mercury levels in blood and brain were unrelated to the duration of exposure before mating, although reproductive success appeared to be so related. Mercury levels in both media were closely related to consumption during gestation, but apparently maternal exposure during lactation did not result in exposure to the nursing pups. Brain mercury in offspring decreased between birth and weaning from 0.49 to 0.045 ppm in the low-dose rats and from 9.8 to 0.53 ppm in the high-dose rats. The brain increased in weight only about 5.5-fold during this time, indicating that there was minimal mercury exposure and some net loss from brain during this period. Brain:blood ratios averaged about 0.14 at birth and 0.24 at weaning, suggesting differential loss from neural and non-neural tissue. These ratios are higher than those reported in studies using less chronic exposure conditions or with adult rats. Brain concentrations of mercury in females in the low-dose group were about 10-15% higher than those seen in their male siblings. At the higher dose, the males had slightly higher levels of mercury in the brain than did their female siblings at birth. The relationship between brain concentration (in ppm) and cumulative mercury consumption, also expressed on a ppm basis (cumulative mercury consumed divided by maternal body weight at parturition), was not linear but was well described by a power-function relationship: Hg = A*(cum exposure)b where the exponent, b, was 1.12 and 1.17 for blood and brain, respectively, at birth. This exponent was indistinguishable from 1.0 for both media at weaning, indicating that the relationship between exposure and blood and brain levels became linear.     

Kinetics of methyl mercury in blood and brain during chronic exposure in the monkey Macaca fascicularis

The disposition parameters derived from a compartmental model kinetic analysis of blood Hg levels in nonpregnant, adult female Macaca fascicularis given daily doses of MeHg did not vary with either dosage level (50, 70 or 90 micrograms MeHg/kg b.wt.day) or duration of exposure (up to 507 day). In contrast, blood clearance of Hg in pregnant females was dose-dependent; it being higher at the 90 micrograms MeHg/kg b.wt.day than at the lower dosage levels. Hg levels in the brain of adult fascicularis relative to blood Hg also increased at the highest level of exposure. Blood Hg half-life in neonate fascicularis was similar to half-life in their mothers (adults). Finally, the regional distribution of mercury in the brains of adult and neonate fascicularis exposed to low and intermediate levels of MeHg resembles the reported distribution of mercury in the brains of adult and neonate humans environmentally exposed to MeHg. Consequently, M. fascicularis may be an especially appropriate animal model for studying the neurotoxic mechanisms of chronic methyl mercury exposure.     

Mercury distribution in the rat brain after mercury vapor exposure.

Brown Norwegian rats were exposed to mercury vapor at a concentration of approximately 1 mg/m3 for 5 weeks 24 hr/day 7 days a week and 6 hr/day 3 days a week, respectively. The total mercury absorption was calculated to 264 and 35 micrograms per week and 100 g body weight. The mean blood mercury concentration was 0.25 +/- 0.03 and 0.09 +/- 0.01 microgram/g, and the total concentration in the brain was 5.03 +/- 0.73 and 0.71 +/- 0.10 microgram/g tissue, respectively. The mercury distribution in the brains was examined using a method based on chemographic principles. Mercury was found primarily in the neocortex, in the basal nuclei, and in the cerebellar Purkinje cells. This distribution pattern corresponded to the pattern of inorganic mercury described after exposure to methyl mercury. Distribution of mercury after administration of different mercury compounds is discussed.     

Mercury accumulation and its distribution to metallothionein in mouse brain after sub-chronic pulse exposure to mercury vapor

Previously we found that exposure to mercury vapor effectively induced metallothionein (MT) biosynthesis in rat brain. Although the induction of not only MT-I/II but also MT-III was evident, the induction rate of the latter was much lower than that of the former. The brain of an MT-null mouse lacks MT-I/II, but has MT-III. Here we examined the effects of sub-chronic pulse exposure to mercury vapor on the brain MT in MT-null mice and their wild type controls. MT-null and wild type mice were preliminarily exposed to mercury vapor for 2 weeks at 0.1 mg Hg/m³ for 1 h/day for 3 days a week, and then exposed for 11 weeks at 4.1 mg Hg/m³ for 30 min/day for 3 days a week. This exposure caused no toxic signs such as abnormal behavior or loss of body weight gain in the mice of either strain throughout the experimental period. Twenty-four hours after the termination of the exposure, mice were sacrificed and brain samples were subjected to mercury analysis, MT assay, and pathological examination. The MT-null mice showed lower accumulation of mercury in the brain than the wild type mice. Mercury exposure resulted in a 70% increase of brain MT in the wild type mice, which was mostly accounted for by the increase in MT-I/II. On the other hand, the brain MT in the MT-null mice increased by 19%, suggesting less reactivity of the MT-III gene to mercury vapor. Although histochemical examination revealed silver-mercury grains in the cytoplasm of nerve cells and glial cells throughout the brains of both strains, no significant difference was observed between the two strains.     

Distribution and retention of mercury in metallothionen-null mice after exposure to mercury vapor

We studied the role of metallothionein (MT) in the distribution and retention of mercury in the brain, lung, liver and kidney of MT-null and wild-type mice after exposure to mercury (Hg0) vapor. Mice were exposed to Hg0 vapor at 5.5-6.7 mg/m3 for 3 h and killed at 1, 24, 72 or 168 h after exposure. One hour after exposure to Hg0 vapor, there were no differences in mercury concentrations in these organs from MT-null and wild-type mice. However, the elimination rate of mercury from the organs, except the brain, were remarkably faster in MT-null mice than in wild-type mice. MT-I and -II levels in the lung and kidney were increased significantly in wild-type mice but not in MT-null mice at 24 h after exposure to Hg0 vapor. At this time point, over 65% of the mercury was retained in the MT fraction of the cytosol of organs from wild-type mice. In contrast, mercury appeared mainly in the high-molecular-weight protein fractions in the cytosol of organs from MT-null mice. In the brain, a large amount of mercury was bound to MT in both strains of mice immediately after exposure. No difference was observed in the elimination rate of mercury from the brain between both strains of mice. Brain MT levels were elevated slightly in wild-type mice at 168 h after exposure but could not be detected in MT-null mice. These data suggest that no detectable MT-I and -II levels were found in the brain of MT-null mice and that mercury was apparently bound to MT-III. Using MT-null mice, we showed also that MT-III may play an important role in the retention of mercury in the brain.     

Neurochemical changes in newborn rat's brain after gestational cadmium and lead exposure

Gestational administration of cadmium (10 mg/l) and lead (300 mg/l) produced a strong decrease in proteins at birth (-17%) and on day 5 (-31%) as well as in brain lipid amount on both days (-11 and -23%, respectively). At day 5 postnatal the exposure also produced a marked decrease in DNA and RNA concentrations with respect to the control group. On the other hand, we found a significant increase of indoleamine content in all brain areas studied in the cadmium-lead group and so the dopamine and its metabolite in mesencephalon, whereas dopamine levels in metencephalon decreased significantly. This data suggests that gestational and early lactational exposure to low dose of cadmium and lead could produce alterations in monoaminergic metabolism that can place the exposed animal to a significant risk in adulthood.     

Antioxidant defense in rat brain regions after developmental lead exposure.

Oxidative stress is considered a possible molecular mechanism involved in Pb neurotoxicity. Considering the vulnerability of the developing brain to Pb neurotoxicity, this study was carried out to investigate the effects of low-level developmental Pb exposure on brain regions antioxidant enzymes activities. Wister dams were exposed to 500 ppm of Pb, as Pb acetate, or to 660 ppm Na acetate in the drinking water during pregnancy and lactation. The activities of superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase were determined in the hypothalamus, hippocampus and striatum of male pups at 23 (weaned) or 70 days (adult) of age. In the Pb-exposed 23-day-old pups, the activity of SOD was decreased in the hypothalamus. Regarding adults, there was no significant treatment effect in any of the enzymes and regions evaluated. Based on the present results, it seems that oxidative stress due to decreased antioxidant function may occur in weaned rats but it is suggested that this should not be the main mechanism involved in the neurotoxicity of low-level Pb exposure.     

In vitro and in vivo effects of lead,methyl mercury and mercury on inositol 1,4,5-trisphosphate and 1,3,4,5-tetrakisphosphate receptor bindings in rat brain

In vitro and in vivo effects of mercury (Hg), methyl mercury (MM) and lead (Pb) on [³H]inositol 1,4,5-trisphosphate (IP₃) and [³H]linositol 1,3,4,5-tetrakisphosphate (IP₄) receptor binding in the Sprague-Dawley rat brain cerebellar membranes were studied. In vitro studies indicate that binding of [³H]IP₃ and [³H]IP₄ to cerebellar membranes was inhibited by Hg while they were stimulated by MM or Pb in a concentration-ependent manner. MM was more potent (EC₅₀ 3,4 μM) than Pb (EC₅₀ 18.2 μM) in stimulating the [³H]IP₃ receptor binding activity whereas Pb (IC₅₀ 30 μM) was more potent than MM (IC₅₀ 133 μM) in stimulating the [³H]IP₄, receptor binding. When the rats were treated (i.p) with Hg (5 mg/kg body wt.) or MM (5 mg/kg body wt.) or Pb (25 mg/kg body wt.) for 3 or 24 h, no significant alterations in[³H]IP₃ receptor binding were observed in cerebellum and cerebral cortex. But the above treatment of Pb or MM for 3 or 24 h to rats resulted in an increase of [³H]IP₄ receptor binding in the membranes of cerebral cortex. However, the rats treated with Hg (1 mg/kg body wt./day) or Pb (25 mg/kg body wt./day) for 7 days did not show any alteration in binding of [³H]IP₃ to its receptors in cerebellar membranes but an increase in this receptor binding was noticed with the treatment of MM (2.5 mg/kg body wt./day) for 7 days. The cerebellum and cerebral cortex of rats with the above treatment of MM or Pb for 7 days exhibited an increase in [³H]IP₄ receptor binding. These in vitro and in vivo data suggest that alterations in inositol polyphosphate receptor binding by metals could result in alterations in intracellular calcium levels which may influence neuronal activity.     

Selenium concentrations in brain after exposure to methylmercury: relations between the inorganic mercury fraction and selenium

 Three groups of female monkeys (Macaca fascicularis) were exposed to methylmercury (MeHg, p.o. 50 μg Hg/kg body wt per day) for 6, 12, or 18 months. One group was exposed to MeHg for 12 months and kept unexposed for 6 months before sacrifice. Another group of three monkeys was exposed to HgCl₂ i.v. for 3 months. Total and inorganic mercury concentrations in occipital pole and thalamus were determined by cold vapor atomic absorption spectroscopy. Selenium concentrations were analyzed by hydride generation atomic absorption spectroscopy. The results indicated an association between concentrations of inorganic mercury and selenium in both occipital pole and thalamus in the MeHg-exposed animals. A linear regression model using concentrations of inorganic mercury (nmol/g wet wt) as independent variable, and selenium concentrations (nmol/g wet wt) as the dependent variable showed significant correlations between the variables in both occipital pole and thalamus (r = 0.85 and r = 0.91, P <0.0001). The intercept of the regression line was slightly lower (about 2 nmol Se/g wet wt) than the selenium concentrations found in control monkeys (about 3 nmol Se/g wet wt). There was a tendency to a “hockey stick”-shaped relationship between concentrations of selenium and inorganic mercury in the thalamus of monkeys with ongoing exposure to MeHg. An important role for selenium in the retention of mercury in brain is indicated. Received: 6 April 1994 / Accepted: 5 October 1994     

Effects of triethyl lead on various cholinergic parameters in the rat brain in vitro

The effects of triethyl lead acetate (triethyl Pb) on the cholinergic system in the brain of the rats were investigated in vitro. Triethyl Pb, at concentrations below 10(-4) M, inhibited the depolarized release of acetylcholine (ACh) from slices of cortex and they synthesis of ACh in such slices, while it potentiated in a dose-dependent manner the non-depolarized release of ACh. In contrast, lead inhibited noncompetitively the high-affinity uptake of choline into synaptosomes with a Ki of 4.03 X 10(-6) M and the activity of choline acetyltransferase with a Ki of 4.07 X 10(-5) M. Triethyl Pb has an inhibitory effect (IC50 not equal to 5 X 10(-5) M) on the binding of [3H]quinuclidinyl benzilate to muscarinic ACh receptors. Triethyl Pb inhibited acetylcholinesterase activity slightly at 5 X 10(-5) and 10(-4) M. It is suggested that ACh transmission, in particular the synthesis of ACh and the release of ACh, is susceptible to organolead neurotoxicity.     

Maternal-to-fetus transfer of mercury in metallothionein-null pregnant mice after exposure to mercury vapor

This study examined the role of placenta metallothionein (MT) in maternal-to-fetal mercury transfer in MT-null and wild-type mice after exposure to elemental mercury (Hg(0)) vapor. Both strains were exposed to Hg(0) vapor at 5.5-6.7 mg/m(3) for 3 h during late gestation. Twenty-four hours after exposure to Hg(0) vapor, accumulation of mercury in the major organs, except the brain, of MT-null maternal mice was significantly lower than that in organs of wild-type mice. In contrast to mercury levels in maternal organs, fetal mercury levels were significantly higher in MT-null mice than in wild-type mice. In placenta, mercury concentrations were not significantly different between the two strains. Although MT levels in major organs, except the brain, of wild type mice were markedly elevated after the exposure to Hg(0) vapor, the placental MT levels were not elevated. However, endogenous MT level in the placenta is significantly higher than that in other organs, except the liver. Gel filtration profile of the placental cytosol in the wild-type mice revealed that a large amount of placental mercury was associated with MT. In MT-null mice, mercury in placental cytosol appeared mainly in the high-molecular-weight protein fractions. Mercury in the placenta was localized mainly in the yolk sac and decidual cells in the deep layer of the decidua in both mouse strains. The similar localization of MT was found in the placenta of wild type mice. These results suggest that MT in the placenta has a defensive role in preventing maternal-to-fetal mercury transfer.     

Environmental exposure to lead and mercury in Mexican children: a real health problem

Exposure to lead (Pb) and mercury (Hg) remains a world public health problem, particularly for young children in developing countries. In Mexico, the main sources of exposure to Pb and Hg are wastes from human activities that increase the natural sources of these metals. Pb and Hg are highly toxic during development and maturation periods of the central nervous system (CNS); these effects are associated with the risk for neurodegenerative diseases. Mexico has numerous exposure sources to Pb and Hg; nevertheless, information on exposure in children is limited, particularly for Hg. Therefore, we conducted a review of the studies performed in children exposed to Pb and Hg. Data presented support that an important proportion of Mexican children have Pb levels above values associated with dangerous effects. On the other hand, studies on Hg-exposure are scarce, so we need more studies to estimate the magnitude of the problem and to determine exposure levels in Mexican children. Available data support the urgent need for coordinated actions among researchers, and health and environmental government authorities to implement education and nutritional campaigns, as well as to decrease exposure and effects of Pb and Hg. In addition, there must be a priority for the implementation of educational campaigns directed to the general population, but with emphasis in parents, education staff and health care providers to decrease both the risk of exposure of children to Pb and Hg and the effects of the exposure to these metals.     

Environmental exposure to lead and mercury in Mexican children: a real health problem

Exposure to lead (Pb) and mercury (Hg) remains a world public health problem, particularly for young children in developing countries. In Mexico, the main sources of exposure to Pb and Hg are wastes from human activities that increase the natural sources of these metals. Pb and Hg are highly toxic during development and maturation periods of the central nervous system (CNS); these effects are associated with the risk for neurodegenerative diseases. Mexico has numerous exposure sources to Pb and Hg; nevertheless, information on exposure in children is limited, particularly for Hg. Therefore, we conducted a review of the studies performed in children exposed to Pb and Hg. Data presented support that an important proportion of Mexican children have Pb levels above values associated with dangerous effects. On the other hand, studies on Hg-exposure are scarce, so we need more studies to estimate the magnitude of the problem and to determine exposure levels in Mexican children. Available data support the urgent need for coordinated actions among researchers, and health and environmental government authorities to implement education and nutritional campaigns, as well as to decrease exposure and effects of Pb and Hg. In addition, there must be a priority for the implementation of educational campaigns directed to the general population, but with emphasis in parents, education staff and health care providers to decrease both the risk of exposure of children to Pb and Hg and the effects of the exposure to these metals.