Prenatal methylmercury exposure hampers glutathione antioxidant system ontogenesis and causes long-lasting oxidative stress in the mouse brain.

During the perinatal period, the central nervous system (CNS) is extremely sensitive to metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-induced developmental neurotoxicity remains obscure, several studies point to the glutathione (GSH) antioxidant system as an important molecular target for this toxicant. To extend our recent findings of MeHg-induced GSH dyshomeostasis, the present study was designed to assess the developmental profile of the GSH antioxidant system in the mouse brain during the early postnatal period after in utero exposure to MeHg. Pregnant mice were exposed to different doses of MeHg (1, 3 and 10 mg/l, diluted in drinking water, ad libitum) during the gestational period. After delivery, pups were killed at different time points - postnatal days (PND) 1, 11 and 21 - and the whole brain was used for determining biochemical parameters related to the antioxidant GSH system, as well as mercury content and the levels of F(2)-isoprostane. In control animals, cerebral GSH levels significantly increased over time during the early postnatal period; gestational exposure to MeHg caused a dose-dependent inhibition of this developmental event. Cerebral glutathione peroxidase (GPx) and glutathione reductase (GR) activities significantly increased over time during the early postnatal period in control animals; gestational MeHg exposure induced a dose-dependent inhibitory effect on both developmental phenomena. These adverse effects of prenatal MeHg exposure were corroborated by marked increases in cerebral F(2)-isoprostanes levels at all time points. Significant negative correlations were found between F(2)-isoprostanes and GSH, as well as between F(2)-isoprostanes and GPx activity, suggesting that MeHg-induced disruption of the GSH system maturation is related to MeHg-induced increased lipid peroxidation in the pup brain. In utero MeHg exposure also caused a dose-dependent increase in the cerebral levels of mercury at birth. Even though the cerebral mercury concentration decreased to nearly basal levels at postnatal day 21, GSH levels, GPx and GR activities remained decreased in MeHg-exposed mice, indicating that prenatal exposure to MeHg affects the cerebral GSH antioxidant systems by inducing biochemical alterations that endure even when mercury tissue levels decrease and become indistinguishable from those noted in pups born to control dams. This study is the first to show that prenatal exposure to MeHg disrupts the postnatal development of the glutathione antioxidant system in the mouse brain, pointing to an additional molecular mechanism by which MeHg induces pro-oxidative damage in the developing CNS. Moreover, our experimental observation corroborates previous reports on the permanent functional deficits observed after prenatal MeHg exposure.

Mercurial-induced hydrogen peroxide generation in mouse brain mitochondria: protective effects of quercetin.

Plants of the genus Polygala have been shown to possess protective effects against neuronal death and cognitive impairments in neurodegenerative disorders related to excitotoxicity. Moreover, previous reports from our group have shown the neuroprotective effects of the plant Polygala paniculata against methylmercury (MeHg)-induced neurotoxicity. In this work, we have examined the potential protective effects of three compounds (7-prenyloxy-6-methoxycoumarin, quercetin, and 1,5-dihidroxi-2,3-dimethoxy xanthone) from Polygala species against MeHg- and mercuric chloride (HgCl2)-induced disruption of mitochondrial function under in vitro conditions using mitochondrial-enriched fractions from mouse brain. MeHg and HgCl2 (10-100 microM) significantly decreased mitochondrial viability; this phenomenon was positively correlated to mercurial-induced glutathione oxidation. Among the isolated compounds, only quercetin (100-300 microM) prevented mercurial-induced disruption of mitochondrial viability. Moreover, quercetin, which did not display any chelating effect on MeHg or HgCl2, prevented mercurial-induced glutathione oxidation. The present results suggest that the protective effects of quercetin against mercurial-induced mitochondrial dysfunction is related to the removal of oxidant species generated in the presence of either MeHg or HgCl2. Reinforcing this hypothesis, MeHg and HgCl2 increased the production of hydrogen peroxide in the brain mitochondria, as well as the levels of malondialdehyde. These oxidative phenomena were prevented by co-incubation with quercetin or catalase. These results are the first to show the involvement of hydrogen peroxide as a crucial molecule related to the toxic effects of both organic and inorganic mercurials in brain mitochondria. In addition, the study is the first to show the protective effect of quercetin against mercurial-induced toxicity, pointing to its capability to counteract mercurial-dependent hydrogen peroxide generation as a potential molecular mechanism of protection. Taken together, these data render quercetin a promising molecule for pharmacological studies with respects to mercurials' poisoning.

Lactational exposure to inorganic mercury: evidence of neurotoxic effects.

This study examined the effects of inorganic mercury (mercuric chloride - HgCl2) exposure exclusively through maternal milk on biochemical parameters related to oxidative stress (glutathione and thiobarbituric acid reactive substances levels, glutathione peroxidase and glutathione reductase activities) in the cerebellum of weanling mice. These parameters were also evaluated in the cerebellum of mothers, which were subjected to intraperitoneal injections of HgCl2 (0, 0.5 and 1.5 mg/kg, once a day) during the lactational period. Considering the relationship between cerebellar function and motor activity, the presence of motor impairment was also evaluated in the offspring exposed to HgCl2 during lactation. After treatments (at weaning), pups lactationally exposed to inorganic mercury showed high levels of mercury in the cerebellar tissue, as well as significant impairment in motor performance in the rotarod task and decreased locomotor activity in the open field. Offspring and dams did not show changes in cerebellar glutathione levels or glutathione peroxidase activity. In pups, lactational exposure to inorganic mercury significantly increased cerebellar lipoperoxidation, as well as the activity of cerebellar glutathione reductase. However, these phenomena were not observed in dams. These results indicate that inorganic mercury exposure through maternal milk is capable of inducing biochemical changes in the cerebellum of weanling mice, as well as motor deficit and these phenomena appear to be related to the pro-oxidative properties of inorganic mercury.

Postnatal methylmercury exposure induces hyperlocomotor activity and cerebellar oxidative stress in mice: dependence on the neurodevelopmental period.

During the early postnatal period the central nervous system (CNS) is extremely sensitive to external agents. The present study aims at the investigation of critical phases where methylmercury (MeHg) induces cerebellar toxicity during the suckling period in mice. Animals were treated with daily subcutaneous injections of MeHg (7 mg/kg of body weight) during four different periods (5 days each) at the early postnatal period: postnatal day (PND) 1-5, PND 6-10, PND 11-15, or PND 16-20. A control group was treated with daily subcutaneous injections of a 150 mM NaCl solution (10 ml/kg of body weight). Subjects exposed to MeHg at different postnatal periods were littermate. At PND 35, behavioral tests were performed to evaluate spontaneous locomotor activity in the open field and motor performance in the rotarod task. Biochemical parameters related to oxidative stress (levels of glutathione and thiobarbituric acid reactive substances, as well as glutathione peroxidase and glutathione reductase activity) were evaluated in cerebellum. Hyperlocomotor activity and high levels of cerebellar thiobarbituric acid reactive substances were observed in animals exposed to MeHg during the PND 11-15 or PND 16-20 periods. Cerebellar glutathione reductase activity decreased in MeHg-exposed animals. Cerebellar glutathione peroxidase activity was also decreased after MeHg exposure and the lowest enzymatic activity was found in animals exposed to MeHg during the later days of the suckling period. In addition, low levels of cerebellar glutathione were found in animals exposed to MeHg during the PND 16-20 period. The present results show that the postnatal exposure to MeHg during the second half of the suckling period causes hyperlocomotor activity in mice and point to this phase as a critical developmental stage where mouse cerebellum is a vulnerable target for the neurotoxic and pro-oxidative effects of MeHg.

Cerebellar thiol status and motor deficit after lactational exposure to methylmercury.

This study examined the exclusive contribution of methylmercury (MeHg) exposure through maternal milk on biochemical parameters related to the thiol status (glutathione (GSH) levels, glutathione peroxidase (GPx) and glutathione reductase (GR) activities) in the cerebellums of suckling mice. The same biochemical parameters were also evaluated in the cerebellums of mothers, which were submitted to a direct oral exposure to MeHg (10 mg/L in drinking water). With regard to the relationship between cerebellar function and motor activity, the presence of signs of motor impairment was also evaluated in the offspring exposed to MeHg during lactation. After the treatment (at weaning period), the pups lactationally exposed to MeHg showed increased levels of mercury in the cerebellum compared to pups in the control group and a significant impairment in the motor performance in the rotarod apparatus. In addition, these pups showed decreased levels of GSH in the cerebellum compared to pups in the control group. In dams, MeHg significantly increased the levels of cerebellar GSH and the activities of cerebellar GR. However, this was not observed in pups. This study indicates that (1) the exposure of lactating mice to MeHg causes significant impairments in motor performance in the offspring which may be related to a decrease in the cerebellar thiol status and (2) the increased GSH levels and GR activity, observed only in the cerebellums of MeHg-exposed dams, could represent compensatory pathophysiologic responses to the oxidative effects of MeHg toward endogenous GSH.