Masatake Fujimura

Methylmercury induces neuropathological changes with tau hyperphosphorylation mainly through the activation of the c-jun-N-terminal kinase pathway in the cerebral cortex, but not in the hippocampus of the mouse brain.

Methylmercury (MeHg) is a well-known neurotoxicant inducing neuronal degeneration in the central nervous system. This in vivo study investigated the involvement of tau hyperphosphorylation in MeHg-induced neuropathological changes in the mouse brain, because abnormal tau hyperphosphorylation causes significant pathological changes associated with some neurodegenerative diseases. Mice that were administrated to 30 ppm MeHg in drinking water for 8 weeks exhibited neuropathological changes, e.g. a decrease in the number of neuron; an increase in the number of migratory astrocytes and microglia/macrophages; necrosis and apoptosis in the cerebral cortex, particularly the deep layer of primary motor cortex and prelimbic cortex. Western blotting revealed that MeHg exposure increased tau phosphorylation at Thr-205, Ser-396 and Ser-422 in the cerebral cortex, consistent with the phosphorylation patterns noted in Alzheimers disease and frontotemporal dementia. Immunohistochemical analyses revealed that the distribution of tau-phosphorylated (Thr-205) neurons corresponded with the areas showing considerable neuropathological changes. Among the kinases and phosphatases related to tau hyperphosphorylation, the activation of mitogen-activated protein kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) was recognized. Neither neuropathological changes nor tau hyperphosphorylation was detected in the hippocampus in this study although the mercury concentration here was twice that in the cerebral cortex. These findings suggest that MeHg exposure induces tau hyperphosphorylation at specific sites of tau mainly through the activation of JNK pathways, leading to neuropathological changes in the cerebral cortex selectively, but not in the hippocampus of mouse brain.

Methylmercury exposure downregulates the expression of Racl and leads to neuritic degeneration and ultimately apoptosis in cerebrocortical neurons.

Methylmercury (MeHg) has been recognized as a neurotoxicant targeted on the central nervous system including cerebellum and cerebral cortex. Some molecular targets of MeHg have been identified using cerebellar neuronal cells, but little is known in the cerebrocortical neuronal cells. In this study, the molecular mechanism underlying MeHg-induced cell death in cerebrocortical neurons was investigated using a primary culture of embryonic rat cortical neuronal cells. The cultured cells exhibited apoptosis 3 days after exposure to 100 nM MeHg, suggesting the involvement of caspase-dependent apoptotic pathways. We demonstrated for the first time that neuritic degeneration precedes MeHg-induced apoptotic death in neurons exposed to 100 nM MeHg. Immunocytochemical and ELISA analyses for neurite-specific proteins namely, tau and MAP2, showed that injury to tau-positive axons was first induced followed by damage to the dendrites and cellular bodies. To further investigate the factors responsible for neuronal death, we investigated the expression levels of Rho-family proteins (Rac1, Cdc42, and RhoA), which regulate neuritic functions and apoptosis in neurons. Western blot analysis demonstrated that MeHg downregulated the expression levels of Rac1 and Cdc42 but did not affect RhoA. The exposure concentration and time course studies confirmed that Rac1 is targeted during an early stage of MeHg-induced cytotoxicity. The results indicate that neuritic degeneration, in particular axonal degeneration triggered by the downregulation of Rac1 expression, contributes to MeHg-induced apoptotic cell death in cultured cerebrocortical neurons.

Deleterious effects in mice of fish-associated methylmercury contained in a diet mimicking the Western populations' average fish consumption.

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. Only a few contradictory epidemiological studies are currently available examining the impact of fish consumption on human populations. In the present study, we wanted to address whether a diet mimicking the fish consumption of Western populations could result in observable adverse effects in mice, and whether beneficial nutriments from fish were able to counterbalance the deleterious effects of MeHg, if any. In Europe and the United States, fish consumption varies widely between countries, from 11 to 100 g fish/day. A mid-range value of 25 g fish/day corresponds to a fish contribution to the total diet of 1.25% on a dry weight basis. We decided to supplement a vegetarian-based mouse diet with 1.25% of lyophilized salmon flesh (SAL diet), or 1.25% of a blend of lyophilized cod, tuna, and swordfish (CTS diet). Total mercury contents were 1.15±0.15, 2.3±0.1 and 35.75±0.15 ng Hg/g of food pellets for the control, SAL and CTS diets, respectively. After two months feeding, the CTS diet resulted in significant observable effects as compared to the control and SAL diets, encompassing decreased body growth, altered behavioral performance and increased anxiety level, modification of mitochondrial respiratory protein subunit concentrations in kidney and brain structures, modified gene expression patterns in kidneys, liver and muscles, and a decrease of dopamine concentrations in the hypothalamus and striatum. Our findings have health implications, firstly because 1.25% of CTS flesh in the diet corresponds to an average exposure to MeHg below the WHO provisory tolerable weekly intake (PTWI) (1.6 μg MeHg/kg of body weight/week), and secondly because many people in Western populations, among them women of child-bearing age, are exceeding the PTWI value (for instance, 35% of the French population inhabiting the Atlantic and Mediterranean coasts).

Differing effects of toxicants (methylmercury, inorganic mercury, lead, amyloid β, and rotenone) on cultured rat cerebrocortical neurons: differential expression of rho proteins associated with neurotoxicity.

Methylmercury (MeHg), inorganic mercury (IHg), lead (Pb), amyloid-β peptide (Aβ), and rotenone (RTN) are well-known toxicants. Here, we demonstrate that these five toxicants exhibit differing effects on cerebrocortical neurons. The concentration responsible for 30% loss of viability (EC30) values 3 days after exposure was approximately 100nM for MeHg, IHg, and RTN and 10μM for Aβ. Neuritic degeneration and subsequent apoptotic cell death were observed in these toxicant-treated cells. In contrast, the EC30 value 3 days after exposure to Pb was > 10μM. We clarified the differential expression of Ras homolog gene (Rho) family proteins (Ras-related C3 botulinum toxin substrate 1 [Rac1], cell division cycle 42, and Ras homolog gene family, member A [RhoA]) upon exposure to these five toxicants. Exposure to 100nM MeHg, IHg, or RTN downregulated the expression of Rac1, related to neuritic extension, but did not affect RhoA, related to retraction. At a higher concentration (1μM), IHg and RTN also acted through the suppression of Rac1, whereas increased MeHg toxicity was not associated with the expression of Rho family proteins. On the other hand, Pb and Aβ showed no effects on the expression of Rho proteins. Modification of the balance of neuritic extension and retraction by the suppression of Rho A rescued the neurotoxicity of 100nM MeHg, IHg, and RTN. The results indicate that the imbalance of neuritic extension and retraction by the suppression of Rac1 by 100nM MeHg, IHg, and RTN causes cerebrocortical neuron axonal degeneration and cell death. By contrast, the neurotoxicities of Pb, Aβ, and MeHg (at higher concentrations) are conferred by other toxic mechanisms.

Methylmercury causes neuronal cell death through the suppression of the TrkA pathway: In vitro and in vivo effects of TrkA pathway activators

Methylmercury (MeHg) is an environmental toxin which induces cell death specific for the nervous systems. Here we show that MeHg causes neuronal cell death through the suppression of the tropomyosin receptor kinase A (TrkA) pathway, and that compounds activating the TrkA pathway prevent MeHg-induced nerve damage in vitro and in vivo. We first investigated the mechanism of MeHg-induced neurotoxicity in differentiating neurons using PC12 cells. Exposure to 100nM MeHg for 1day induced apoptosis in differentiating PC12 cells. Further, MeHg-induced apoptosis was preceded by inhibition of neurite extension, as determined by ELISA analyses of the neurite-specific protein neurofilament triplet H protein (NF-H). To determine the mechanism of MeHg-induced apoptosis, we evaluated the effects of MeHg on the TrkA pathway, which is known to regulate neuronal differentiation and viability. Western blot analysis demonstrated that, like the TrkA phosphorylation inhibitor K252a, MeHg inhibited phosphorylation of TrkA and its downstream effectors. Furthermore, GM1 ganglioside and its analog MCC-257, which enhance TrkA phosphorylation, overcame the effect of MeHg in neurons, supporting the involvement of the TrkA pathway in MeHg-induced nerve damage. Finally, we demonstrated that MCC-257 rescued the clinical sign and pathological changes in MeHg-exposed rats. These findings indicate that MeHg-induced apoptosis in neuron is triggered by inhibition of the TrkA pathway, and that GM1 ganglioside and MCC-257 effectively prevent MeHg-induced nerve damage.

The chemokine CCL2 protects against methylmercury neurotoxicity.

Industrial pollution due to heavy metals such as mercury is a major concern for the environment and public health. Mercury, in particular methylmercury (MeHg), primarily affects brain development and neuronal activity, resulting in neurotoxic effects. Because chemokines can modulate brain functions and are involved in neuroinflammatory and neurodegenerative diseases, we tested the possibility that the neurotoxic effect of MeHg may interfere with the chemokine CCL2. We have used an original protocol in young mice using a MeHg-contaminated fish-based diet for 3 months relevant to human MeHg contamination. We observed that MeHg induced in the mice cortex a decrease in CCL2 concentrations, neuronal cell death, and microglial activation. Knock-out (KO) CCL2 mice fed with a vegetal control food already presented a decrease in cortical neuronal cell density in comparison with wild-type animals under similar diet conditions, suggesting that the presence of CCL2 is required for normal neuronal survival. Moreover, KO CCL2 mice showed a pronounced neuronal cell death in response to MeHg. Using in vitro experiments on pure rat cortical neurons in culture, we observed by blockade of the CCL2/CCR2 neurotransmission an increased neuronal cell death in response to MeHg neurotoxicity. Furthermore, we showed that sod genes are upregulated in brain of wild-type mice fed with MeHg in contrast to KO CCL2 mice and that CCL2 can blunt in vitro the decrease in glutathione levels induced by MeHg. These original findings demonstrate that CCL2 may act as a neuroprotective alarm system in brain deficits due to MeHg intoxication.

Feeding mice with diets containing mercury-contaminated fish flesh from French Guiana: a model for the mercurial intoxication of the Wayana Amerindians.

BackgroundIn 2005, 84% of Wayana Amerindians living in the upper marshes of the Maroni River in French Guiana presented a hair mercury concentration exceeding the limit set up by the World Health Organization (10 μg/g). To determine whether this mercurial contamination was harmful, mice have been fed diets prepared by incorporation of mercury-polluted fish from French Guiana.MethodsFour diets containing 0, 0.1, 1, and 7.5% fish flesh, representing 0, 5, 62, and 520 ng methylmercury per g, respectively, were given to four groups of mice for a month. The lowest fish regimen led to a mercurial contamination pressure of 1 ng mercury per day per g of body weight, which is precisely that affecting the Wayana Amerindians.ResultsThe expression of several genes was modified with mercury intoxication in liver, kidneys, and hippocampus, even at the lowest tested fish regimen. A net genetic response could be observed for mercury concentrations accumulated within tissues as weak as 0.15 ppm in the liver, 1.4 ppm in the kidneys, and 0.4 ppm in the hippocampus. This last value is in the range of the mercury concentrations found in the brains of chronically exposed patients in the Minamata region or in brains from heavy fish consumers. Mitochondrial respiratory rates showed a 35–40% decrease in respiration for the three contaminated mice groups. In the muscles of mice fed the lightest fish-containing diet, cytochrome c oxidase activity was decreased to 45% of that of the control muscles. When mice behavior was assessed in a cross maze, those fed the lowest and mid-level fish-containing diets developed higher anxiety state behaviors compared to mice fed with control diet.ConclusionWe conclude that a vegetarian diet containing as little as 0.1% of mercury-contaminated fish is able to trigger in mice, after only one month of exposure, disorders presenting all the hallmarks of mercurial contamination.

Low concentrations of methylmercury inhibit neural progenitor cell proliferation associated with up-regulation of glycogen synthase kinase 3β and subsequent degradation of cyclin E in rats

Methylmercury (MeHg) is an environmental neurotoxicant. The developing nervous system is susceptible to low concentrations of MeHg; however, the effect of MeHg on neural progenitor cell (NPC) proliferation, a key stage of neurogenesis during development, remains to be clarified. In this study, we investigated the effect of low concentrations of MeHg on NPCs by using a primary culture system developed using the embryonic rat cerebral cortex. NPC proliferation was suppressed 48h after exposure to 10nM MeHg, but cell death was not observed. Western blot analyses for cyclins A, B, D1, and E demonstrated that MeHg down-regulated cyclin E, a promoter of the G1/S cell cycle transition. Cyclin E has been shown to be degraded following the phosphorylation by glycogen synthase kinase 3β (GSK-3β). The time course study showed that GSK-3β was up-regulated 3h after exposure to 10nM MeHg, and cyclin E degradation 48h after MeHg exposure. We further demonstrated that GSK-3β inhibitors, lithium and SB-415286, suppressed MeHg-induced inhibition of NPC proliferation by preventing cyclin E degradation. These results suggest that the inhibition of NPC proliferation induced by low concentration of MeHg was associated with up-regulation of GSK-3β at the early stage and subsequent degeneration of cyclin E.

Dietary Mercury Exposure Resulted in Behavioral Differences in Mice Contaminated with Fish-Associated Methylmercury Compared to Methylmercury Chloride Added to Diet

Methylmercury (MeHg) is a potent neurotoxin, and humans are mainly exposed to this pollutant through fish consumption. However, in classical toxicological studies, pure methylmercury chloride (MeHgCl) is injected, given to drink or incorporated within feed assuming that its effects are identical to those of MeHg naturally associated to fish. In the present study, we wanted to address the question whether a diet containing MeHg associated to fish could result in observable adverse effects in mice as compared to a diet containing the same concentration of MeHg added pure to the diet and whether beneficial nutriments from fish were able to counterbalance the deleterious effects of fish-associated mercury, if any. After two months of feeding, the fish-containing diet resulted in significant observable effects as compared to the control and MeHg-containing diets, encompassing altered behavioral performances as monitored in a Y-shaped maze and an open field, and an increased dopamine metabolic turnover in hippocampus, despite the fact that the fish-containing diet was enriched in polyunsaturated fatty acids and selenium compared to the fish-devoid diets.

Effects of Methylmercury Contained in a Diet Mimicking the Wayana Amerindians Contamination through Fish Consumption: Mercury Accumulation, Metallothionein Induction, Gene Expression Variations, and Role of the Chemokine CCL2

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. We addressed the question of whether a diet mimicking the fish consumption of Wayanas Amerindians from French Guiana could result in observable adverse effects in mice. Wayanas adult men are subjected to a mean mercurial dose of 7 g Hg/week/kg of body weight. We decided to supplement a vegetarian-based mice diet with 0.1% of lyophilized Hoplias aimara fish, which Wayanas are fond of and equivalent to the same dose as that afflicting the Wayanas Amerindians. Total mercury contents were 1.4 ± 0.2 and 5.4 ± 0.5 ng Hg/g of food pellets for the control and aimara diets, respectively. After 14 months of exposure, the body parts and tissues displaying the highest mercury concentration on a dry weight (dw) basis were hair (733 ng/g) and kidney (511 ng/g), followed by the liver (77 ng/g). Surprisingly, despite the fact that MeHg is a neurotoxic compound, the brain accumulated low levels of mercury (35 ng/g in the cortex). The metallothionein (MT) protein concentration only increased in those tissues (kidney, muscles) in which MeHg demethylation had occurred. This can be taken as a molecular sign of divalent mercurial contamination since only Hg2+ has been reported yet to induce MT accumulation in contaminated tissues. The suppression of the synthesis of the chemokine CCL2 in the corresponding knockout (KO) mice resulted in important changes in gene expression patterns in the liver and brain. After three months of exposure to an aimara-containing diet, eight of 10 genes selected (Sdhb, Cytb, Cox1, Sod1, Sod2, Mt2, Mdr1a and Bax) were repressed in wild-type mice liver whereas none presented a differential expression in KO Ccl2−/− mice. In the wild-type mice brain, six of 12 genes selected (Cytb, Cox1, Sod1, Sod2, Mdr1a and Bax) presented a stimulated expression, whereas all remained at the basal level of expression in KO Ccl2−/− mice. In the liver of aimara-fed mice, histological alterations were observed for an accumulated mercury concentration as low as 32 ng/g, dw, and metal deposits were observed within the cytoplasm of hepatic cells.