Curcumin protects against methylmercury-induced cytotoxicity in primary rat astrocytes by activating the Nrf2/ARE pathway independently of PKCδ.

Methylmercury (MeHg) is a ubiquitous environmental toxicant that leads to long-lasting neurological deficits in animals and humans. Curcumin, a polyphenol obtained from the rhizome of turmeric, has well-known antioxidant functions. Here, we evaluated curcumin's efficacy in mitigating MeHg-induced cytotoxicity and further investigated the underlying mechanism of this neuroprotection in primary rat astrocytes. Pretreatment with curcumin (2, 5, 10 and 20 µM for 3, 6, 12 or 24 h) protected against MeHg-induced (5 µM for 6 h) cell death in a time and dose-dependent manner. Curcumin (2, 5, 10 or 20 µM) pretreatment for 12 h significantly ameliorated the MeHg-induced astrocyte injury and oxidative stress, as evidenced by morphological alterations, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, and glutathione (GSH) and catalase (CAT) levels. Moreover, curcumin pretreatment increased Nrf2 nuclear translocation and downstream enzyme expression, heme oxygenase-1 (HO-1) and NADPH quinone reductase-1 (NQO1). Knockdown of Nrf2 with siRNA attenuated the protective effect of curcumin against MeHg-induced cell death. However, both the pan-protein kinase C (PKC) inhibitor, Ro 31-8220, and the selective PKCδ inhibitor, rottlerin, failed to suppress the curcumin-activated Nrf2/Antioxidant Response Element(ARE) pathway and attenuate the protection exerted by curcumin. Taken together, these findings confirm that curcumin protects against MeHg-induced neurotoxicity by activating the Nrf2/ARE pathway and this protection is independent of PKCδ activation. More studies are needed to understand the mechanisms of curcumin cytoprotection.

Activation of interleukin-6 and -8 expressions by methylmercury in human U937 macrophages involves RelA and p50.

The accumulation of macrophages has been observed around lesions of the brain in patients with Minamata disease. In this condition, mercury has been detected histochemically in macrophages throughout the brain. However, the role of macrophages in the neurotoxicity of methylmercury (MeHg) and the molecular mechanisms of their response to MeHg exposure remain to be elucidated. Here, we investigated how MeHg affects the expression of proinflammatory cytokines such as interleukin (IL)-6 and IL-8 in cultured human U937 macrophages. Compared with controls, IL-6 and IL-8 mRNA expression was maximally induced in U937 macrophages after treatment with 10 µM MeHg for 6 h. The protein secretion of IL-6 and IL-8 was significantly stimulated by MeHg in U937 macrophages. Results from luciferase reporter assay indicated functional activation of nuclear factor kappa B and the involvement of subunit RelA and p50 in MeHg-induced IL-6 and IL-8 activation, which was confirmed by siRNA knockdown experiments. MeHg exposure at 4 µM also significantly induced IL-8 expression in U-87 MG cells at mRNA and protein level, indicating that IL-8 induction might be a general mode of action of MeHg treatment among different cell types. These results indicate a possible involvement of an early inflammatory response, including IL-6 and IL-8 expression in the pathogenesis of MeHg. N-acetyl-l-cysteine suppressed MeHg-induced activation of IL-6 and IL-8 mRNA expression in U937 macrophages, indicating the effectiveness of N-acetyl-l-cysteine as a therapeutic drug in MeHg-induced inflammation. Copyright © 2016 John Wiley & Sons, Ltd.

Arginine decarboxylase: A novel biological target of mercury compounds identified in PC12 cells.

Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl d-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg2+) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC50=7.96nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg>EtHg>PhHg>HgCl2), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity.

Methylmercury upregulates RE-1 silencing transcription factor (REST) in SH-SY5Y cells and mouse cerebellum.

Methylmercury (MeHg) is a highly neurotoxic compound that, in adequate doses, can cause damage to the brain, including developmental defects and in severe cases cell death. The RE-1-silencing transcription factor (REST) has been found to be involved in the neurotoxic effects of environmental pollutants such as polychlorinated biphenyls (PCBs). In this study, we investigated the effects of MeHg treatment on REST expression and its role in MeHg-induced neurotoxicity in neuroblastoma SH-SY5Y cells. We found that MeHg exposure caused a dose- and time- dependent apoptotic cell death, as evidenced by the appearance of apoptotic hallmarks including caspase-3 processing and annexin V uptake. Moreover, MeHg increased REST gene and gene product expression. MeHg-induced apoptotic cell death was completely abolished by REST knockdown. Interestingly, MeHg (1µM/24h) increased the expression of REST Corepressor (Co-REST) and its binding with REST whereas the other REST cofactor mammalian SIN3 homolog A transcription regulator (mSin3A) was not modified. In addition, we demonstrated that the acetylation of histone protein H4 was reduced after MeHg treatment and was critical for MeHg-induced apoptosis. Accordingly, the pan-histone deacetylase inhibitor trichostatin-A (TSA) prevented MeHg-induced histone protein H4 deacetylation, thereby reverting MeHg-induced neurotoxic effect. Male mice subcutaneously injected with 10mg/kg of MeHg for 10 days showed an increase in REST expression in the granule cell layer of the cerebellum together with a decrease in histone H4 acetylation. Collectively, we demonstrated that methylmercury exposure can cause neurotoxicity by activating REST gene expression and H4 deacetylation.

Design of novel rho kinase inhibitors using energy based pharmacophore modeling, shape-based screening, in silico virtual screening, and biological evaluation.

Rho-associated protein kinase (ROCK) plays a key role in regulating a variety of cellular processes, and dysregulation of ROCK signaling or expression is implicated in numerous diseases and infections. ROCK proteins have therefore emerged as validated targets for therapeutic intervention in various pathophysiological conditions such as diabetes-related complications or hepatitis C-associated pathogenesis. In this study, we report on the design and identification of novel ROCK inhibitors utilizing energy based pharmacophores and shape-based approaches. The most potent compound 8 exhibited an IC50 value of 1.5 µM against ROCK kinase activity and inhibited methymercury-induced neurotoxicity of IMR-32 cells at GI50 value of 0.27 µM. Notably, differential scanning fluorometric analysis revealed that ROCK protein complexed with compound 8 with enhanced stability relative to Fasudil, a validated nanomolar range ROCK inhibitor. Furthermore, all compounds exhibited ≥96 µM CC50 (50% cytotoxicity) in Huh7 hepatoma cells, while 6 compounds displayed anti-HCV activity in HCV replicon cells. The identified lead thus constitutes a prototypical molecule for further optimization and development as anti-ROCK inhibitor.

Molecular mechanisms involved in the protective effect of selenocystine against methylmercury-induced cell death in human HepG2 cells.

Methylmercury (MeHg) has been recognized as a very toxic contaminant present in certain foodstuffs that adversely affects health and impairs the normal function of different organs. Experimental studies have shown that selenocompounds play an important role as cellular detoxificant and protective agents against the harmful effects of mercury. The present study examined the potential preventive activities of organic selenocompounds, focused on selenocystine (SeCys), against MeHg-induced toxicity in human HepG2 cells. Combined treatment of SeCys and MeHg protected HepG2 cells against MeHg-induced cell damage, showing this selenocompound a more relevant effect than those of selenium methylselenocysteine and selenium methionine. Co-treatment with SeCys exerted a protective effect against MeHg by restraining ROS generation and glutathione decrease, and through the modulation of antioxidant enzymes activities. In addition, SeCys delayed MeHg-induced apoptosis and prevented extracellular regulated kinases (ERKs) deactivation, as well as p38 and c-Jun N-terminal kinase (JNK) stimulations in comparison to MeHg-treated cells. ERK, JNK and p38 involvement on the protective effect of SeCys against MeHg-induced cell damage was confirmed by using selective inhibitors. All these results indicate that SeCys protects against MeHg-induced cell damage by modulating the redox status and key proteins related to cell stress and survival/proliferation pathways.

Comparative study on methyl- and ethylmercury-induced toxicity in C6 glioma cells and the potential role of LAT-1 in mediating mercurial-thiol complexes uptake.

Various forms of mercury possess different rates of absorption, metabolism and excretion, and consequently, toxicity. Methylmercury (MeHg) is a highly neurotoxic organic mercurial. Human exposure is mostly due to ingestion of contaminated fish. Ethylmercury (EtHg), another organic mercury compound, has received significant toxicological attention due to its presence in thimerosal-containing vaccines. This study was designed to compare the toxicities induced by MeHg and EtHg, as well as by their complexes with cysteine (MeHg-S-Cys and EtHg-S-Cys) in the C6 rat glioma cell line. MeHg and EtHg caused significant (p<0.0001) decreases in cellular viability when cells were treated during 30min with each mercurial following by a washing period of 24h (EC50 values of 4.83 and 5.05µM, respectively). Significant cytotoxicity (p<0.0001) was also observed when cells were treated under the same conditions with MeHg-S-Cys and EtHg-S-Cys, but the respective EC50 values were significantly increased (11.2 and 9.37µM). l-Methionine, a substrate for the l-type neutral amino acid carrier transport (LAT) system, significantly protected against the toxicities induced by both complexes (MeHg-S-Cys and EtHg-S-Cys). However, no protective effects of l-methionine were observed against MeHg and EtHg toxicities. Corroborating these findings, l-methionine significantly decreased mercurial uptake when cells were exposed to MeHg-S-Cys (p=0.028) and EtHg-S-Cys (p=0.023), but not to MeHg and EtHg. These results indicate that the uptake of MeHg-S-Cys and EtHg-S-Cys into C6 cells is mediated, at least in part, through the LAT system, but MeHg and EtHg enter C6 cells by mechanisms other than LAT system.

Vitamin K has the potential to protect neurons from methylmercury-induced cell death in vitro.

Vitamin K (VK) has a protective effect on neural cells. Methylmercury is a neurotoxicant that directly induces neuronal death in vivo and in vitro. Therefore, in the present study, we hypothesized that VK inhibits the neurotoxicity of methylmercury. To prove our hypothesis in vitro, we investigated the protective effects of VKs (phylloquinone, vitamin K(1); menaquinone-4, vitamin K(2) ) on methylmercury-induced death in primary cultured neurons from the cerebella of rat pups. As expected, VKs inhibited the death of the primary cultured neurons. It has been reported that the mechanisms underlying methylmercury toxicity involve a decrement of intracellular glutathione (GSH). Actually, treatment with GSH and a GSH inducer, N-acetyl cysteine, inhibited methylmercury-induced neuronal death in the present study. Thus, we investigated whether VKs also have protective effects against GSH-depletion-induced cell death by employing two GSH reducers, L-buthionine sulfoximine (BSO) and diethyl maleate (DEM), in primary cultured neurons and human neuroblastoma IMR-32 cells. Treatment with VKs affected BSO- and DEM-induced cell death in both cultures. On the other hand, the intracellular GSH assay showed that VK(2), menaquinone-4, did not restore the reduced GSH amount induced by methylmercury or BSO treatments. These results indicate that VKs have the potential to protect neurons against the cytotoxicity of methylmercury and agents that deplete GSH, without increasing intracellular GSH levels. The protective effect of VKs may lead to the development of treatments for neural diseases involving GSH depletion.

Antagonistic interaction of mercury and selenium in a marine fish is dependent on their chemical species.

It is well-known that selenium (Se) shows protective effects against mercury (Hg) bioaccumulation and toxicity, but the underlying effects of Se chemical species, concentration, and administration method are poorly known. In this study, we conducted laboratory studies on a marine fish Terapon jurbua to explain why Hg accumulation is reduced in the presence of Se observed in field studies. When Se and Hg were administrated concurrently in the fish diets, different Se species including selenite, selenate, seleno-dl-cystine (SeCys), and seleno-dl-methionine (SeMet) affected Hg bioaccumulation differently. At high concentration in fish diet (20 µg g(-1) normally), selenate and SeCys significantly reduced the dietary Hg(II) assimilation efficiency (AE) from 38% to 26%. After the fish were pre-exposed to dietary selenite or SeMet (7 µg g(-1) normally) for 22 days with significantly elevated Se body concentrations, the Hg(II) AEs were pronouncedly reduced (from 41% to 15-26%), whereas the dissolved uptake rate constant and elimination rate constant were less affected. In contrast to Hg(II), all the MeHg biokinetic parameters remained relatively constant whether Se was administrated simultaneously with the fish diet or when the fish were pre-exposed to Se with elevated body concentrations. Basic biokinetic measurements thus revealed that Se had direct interaction with Hg(II) during dietary assimilation rather than with MeHg and that different Se species had variable effects on Hg assimilation.

Modulation of methylmercury uptake by methionine: prevention of mitochondrial dysfunction in rat liver slices by a mimicry mechanism.

Methylmercury (MeHg) is an ubiquitous environmental pollutant which is transported into the mammalian cells when present as the methylmercury-cysteine conjugate (MeHg-Cys). With special emphasis on hepatic cells, due to their particular propensity to accumulate an appreciable amount of Hg after exposure to MeHg, this study was performed to evaluate the effects of methionine (Met) on Hg uptake, reactive species (RS) formation, oxygen consumption and mitochondrial function/cellular viability in both liver slices and mitochondria isolated from these slices, after exposure to MeHg or the MeHg-Cys complex. The liver slices were pre-treated with Met (250 µM) 15 min before being exposed to MeHg (25 µM) or MeHg-Cys (25 µM each) for 30 min at 37 °C. The treatment with MeHg caused a significant increase in the Hg concentration in both liver slices and mitochondria isolated from liver slices. Moreover, the Hg uptake was higher in the group exposed to the MeHg-Cys complex. In the DCF (dichlorofluorescein) assay, the exposure to MeHg and MeHg-Cys produced a significant increase in DFC reactive species (DFC-RS) formation only in the mitochondria isolated from liver slices. As observed with Hg uptake, DFC-RS levels were significantly higher in the mitochondria treated with the MeHg-Cys complex compared to MeHg alone. MeHg exposure also caused a marked decrease in the oxygen consumption of liver slices when compared to the control group, and this effect was more pronounced in the liver slices treated with the MeHg-Cys complex. Similarly, the loss of mitochondrial activity/cell viability was greater in liver slices exposed to the MeHg-Cys complex when compared to slices treated only with MeHg. In all studied parameters, Met pre-treatment was effective in preventing the MeHg- and/or MeHg-Cys-induced toxicity in both liver slices and mitochondria. Part of the protection afforded by Met against MeHg may be related to a direct interaction with MeHg or to the competition of Met with the complex formed between MeHg and endogenous cysteine. In summary, our results show that Met pre-treatment produces pronounced protection against the toxic effects induced by MeHg and/or the MeHg-Cys complex on mitochondrial function and cell viability. Consequently, this amino acid offers considerable promise as a potential agent for treating acute MeHg exposure.

Effects of selenite and chelating agents on mammalian thioredoxin reductase inhibited by mercury: implications for treatment of mercury poisoning.

Mercury toxicity is a highly interesting topic in biomedicine due to the severe endpoints and treatment limitations. Selenite serves as an antagonist of mercury toxicity, but the molecular mechanism of detoxification is not clear. Inhibition of the selenoenzyme thioredoxin reductase (TrxR) is a suggested mechanism of toxicity. Here, we demonstrated enhanced inhibition of activity by inorganic and organic mercury compounds in NADPH-reduced TrxR, consistent with binding of mercury also to the active site selenolthiol. On treatment with 5 µM selenite and NADPH, TrxR inactivated by HgCl(2) displayed almost full recovery of activity. Structural analysis indicated that mercury was complexed with TrxR, but enzyme-generated selenide removed mercury as mercury selenide, regenerating the active site selenocysteine and cysteine residues required for activity. The antagonistic effects on TrxR inhibition were extended to endogenous antioxidants, such as GSH, and clinically used exogenous chelating agents BAL, DMPS, DMSA, and α-lipoic acid. Consistent with the in vitro results, recovery of TrxR activity and cell viability by selenite was observed in HgCl(2)-treated HEK 293 cells. These results stress the role of TrxR as a target of mercurials and provide the mechanism of selenite as a detoxification agent for mercury poisoning.

Carbon monoxide derived from heme oxygenase-2 mediates reduction of methylmercury toxicity in SH-SY5Y cells.

We examined the contribution of carbon monoxide (CO), an enzymatic product of heme oxygenase (HO), to methylmercury (MeHg) cytotoxicity in SH-SY5Y cells, because this gas molecule is reported to activate Nrf2, which plays a protective role against MeHg-mediated cell damage. Exposure of SH-SY5Y cells to CO gas resulted in protection against MeHg cytotoxicity, with activation of Nrf2. Interestingly, pretreatment with tin-protoporphyrin IX, a specific inhibitor of HO, caused a reduction in basal Nrf2 activity and thus enhanced sensitivity to MeHg. No induction of isoform 1 of HO (HO-1) was seen during MeHg exposure, but constitutive expression of isoform 2 (HO-2) occurred, suggesting that CO produced by HO-2 is the main participant in the protection against MeHg toxicity. Studies of small interfering RNA-mediated knockdown of HO-2 in the cells supported this possibility. Our results suggest that CO gas and its producing enzyme HO-2 are key molecules in cellular protection against MeHg, presumably through basal activation of Nrf2.

Allium sativum L. extract prevents methyl mercury-induced cytotoxicity in peripheral blood leukocytes (LS).

Adenosine deaminase (ADA) is involved in purine metabolism and plays a significant role in the immune system. The focus of this investigation was to examine the effects of low concentrations of organic mercury on ADA activity in human leukocytes and to investigate the relationship between these effects and cell death. We have examined the protective potential effects of Allium sativum extract (GaE) against Methylmercury (MeHg)-induced cytotoxic effects on human leucocytes under in vitro conditions. MeHg (0.05-10 microM) significantly decreased leukocyte viability (58.97% for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and 51.67% for Alamar Blue (AB) and this decrease was positively correlated to the MeHg-induced inhibition of ADA activity. N-acetylcysteine (NAC) and GaE prevented both the MeHg-induced cytotoxic effects on leukocytes according to MTT and AB assays and the effects on the ADA activity. The present results suggest that the protective effects of GaE against MeHg-induced leukocyte damage is related to the removal of oxidant species generated in the presence of MeHg due to the antioxidant efficacy of garlic constituents. It is important to point out that the intense presence of ADA in Leukocyte suspension (LS) highlights the relevant effects in the immune system and in vitro cytotoxicity of MeHg exposure.

Effects of chelators on mercury, iron, and lead neurotoxicity in cortical culture.

Chelation therapy for the treatment of acute, high dose exposure to heavy metals is accepted medical practice. However, a much wider use of metal chelators is by alternative health practitioners for so called "chelation therapy". Given this widespread and largely unregulated use of metal chelators it is important to understand the actions of these compounds. We tested the effects of four commonly used metal chelators, calcium disodium ethylenediaminetetraacetate (CaNa2EDTA), D-penicillamine (DPA), 2,3 dimercaptopropane-1-sulfonate (DMPS), and dimercaptosuccinic acid (DMSA) for their effects on heavy metal neurotoxicity in primary cortical cultures. We studied the toxicity of three forms of mercury, inorganic mercury (HgCl2), methyl mercury (MeHg), and ethyl mercury (thimerosal), as well as lead (PbCl2) and iron (Fe-citrate). DPA had the worst profile of effects, providing no protection while potentiating HgCl2, thimerosal, and Fe-citrate toxicity. DMPS and DMSA both attenuated HgCl2 toxicity and potentiated thimerosal and Fe toxicity, while DMPS also potentiated PbCl2 toxicity. CaNa2EDTA attenuated HgCl2 toxicity, but caused a severe potentiation of Fe-citrate toxicity. The ability of these chelators to attenuate the toxicity of various metals is quite restricted, and potentiation of toxicity is a serious concern. Specifically, protection is provided only against inorganic mercury, while it is lacking against the common form of mercury found in food, MeHg, and the form found in vaccines, thimerosal. The potentiation of Fe-citrate toxicity is of concern because of iron's role in oxidative stress in the body. Potentiation of iron toxicity could have serious health consequences when using chelation therapy.

Methylmercury increases N-methyl-D-aspartate receptors on human SH-SY 5Y neuroblastoma cells leading to neurotoxicity.

Methylmercury (MeHg) is a known neurotoxin, yet the mechanism for low dose chronic toxicity is still not clear. While N-methyl-D-aspartate receptors (NMDARs) were found to be induced after exposure to MeHg in a mink model, its role on neurotoxicity is not known. The aims of this study were to investigate the expression and the functional roles of NMDARs on the induction of cell death in the human SH-SY 5Y neuroblastoma cell line after exposure to MeHg. NMDARs were measured using a radiolabeled phencyclidine receptor ligand [(3)H] (MK801) and cell death was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and lactate dehydrogenase (LDH) release. We found a significant increase in NMDARs followed by increased caspase-3 activity after 4 h of exposure to MeHg (0.25-1 microM). Necrotic cell death was found after 4 and 24 h of exposure to MeHg (0.25-5 microM). The NMDAR antagonists dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate [(+)-MK801]) and Memantine (1-amino-3,5-dimethyl-adamantane) (10 microM) completely attenuated MeHg-mediated cell death by blocking NMDARs, thus demonstrating the importance of NMDARs in mercury neurotoxicity. Intracellular calcium chelator BAPTA-AM (1 microM) partially attenuated the neurotoxicity effect of 1 microM MeHg. These results suggest that MeHg toxicity can be mediated through the binding and increase of NMDARs.

Biomarkers of exposure and effect as indicators of the interference of selenomethionine on methylmercury toxicity.

The present study was conducted to clarify the interference of selenomethionine (SeMet) on methylmercury (MeHg) toxicity through the evaluation of changes in biomarkers of exposure and effect in rats exposed to MeHg and co-exposed to MeHg and SeMet. Male Wistar rats received two intraperitoneally (i.p.) administrations, either MeHg (1.5mg/kg body weight), SeMet alone (1mg/kg body weight) or combined MeHg and SeMet, followed by 3 weeks of rat urine collection and neurobehavioural assays. The effects of different administrations were investigated by the quantification of total mercury in kidney and brain, analysis of urinary porphyrins, determination of hepatic GSH and evaluation of motor activity functions (rearing and ambulation). MeHg exposure resulted in a significant increase of urinary porphyrins during the 3 weeks of rat urine collection, where as it caused a significant decrease in motor activity only at the first day after cessation of rat exposure. Additionally, SeMet co-exposure was able to normalize the porphyrins excretion, and a tendency to restore rat motor activity was observed, on the first day after cessation of exposure. Brain and kidney mercury levels increased significantly in rats exposed to MeHg; however, in co-exposed rats to SeMet no significant changes in Hg levels were found as compared to rats exposed to MeHg alone. Hence, the present study shows that urinary porphyrins are sensitive and persistent indicators of MeHg toxicity and demonstrates for the first time that SeMet reduces its formation. Finally, these results confirm that the mechanism of interaction between SeMet and MeHg cannot be explained by the reduction of Hg levels in target organs and suggestions are made to clarify the interference of SeMet on MeHg toxicity.

Modulatory effect of glutathione status and antioxidants on methylmercury-induced free radical formation in primary cultures of cerebral astrocytes.

Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-dependent neurotoxicity remains far from clear, overwhelming data give credence to a mediatory role for astrocytes, a major cell type that preferentially accumulates MeHg. To extend our recent findings of MeHg-induced increase in ROS formation (G. Shanker, J.L. Aschner, T. Syversen et al., Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury, Mol. Brain Res. 128 (2004) 48-57), the present studies were designed to assess the effect of modulating intracellular glutathione (GSH) content, on ROS generation, in the absence and presence of MeHg. Intracellular GSH was reduced by treatment with 100 microM buthionine-L-sulfoxane (BSO) for 24 h, and increased by treatment with 1 mM l-2-oxothiazolidine-4-carboxylic acid (OTC) for 24 h. Additionally, the effects of the selective antioxidants, catalase (1000 U/ml for 1 h), an H2O2 scavenger, and n-propyl gallate (100 microM for 1 h), a superoxide radical (*O2-) and possibly hydroxyl radical (*OH) scavenger on MeHg-induced ROS formation were examined. After these treatments, astrocytes were exposed to +/-10 microM MeHg for 30 min, following which the fluorescent probes, CM-H2DCFA and CM-H2XRos were added; 20 min later, laser scanning confocal microscopy (LSCM) images were obtained. Exposure of astrocytes for 24 h to 100 microM BSO, a GSH synthesis inhibitor, led to a significant increase in mitochondrial ROS (i.e., *O2-, *NO, and ONOO-) formation, as assessed with CM-H2XRos mitotracker red dye. Similarly, BSO increased ROS formation in various intracellular organelles, as assessed with CM-H2DCFDA. BSO in combination with MeHg increased fluorescence levels in astrocytes to levels above those noted with BSO or MeHg alone, but this effect was statistically indistinguishable from either of these groups (BSO or MeHg). Pretreatment of astrocytes for 24 h with 1 mM OTC abolished the MeHg-induced increase in ROS. Results similar to those obtained with OTC were observed with the free radical scavenger, n-propyl gallate (n-PG). The latter had no significant effects on astrocytic fluorescence when administered alone. This *O2- and possibly *OH radical scavenger significantly attenuated MeHg-induced ROS formation. Catalase, an H2O2 scavenger, was less effective in reducing MeHg-induced ROS formation. Taken together, these studies point to the important protective effect of adequate intracellular GSH content as well as antioxidants against MeHg-triggered oxidative stress in primary astrocyte cultures.

Additive pro-oxidative effects of methylmercury and ebselen in liver from suckling rat pups.

Oxidative stress has been pointed as an important molecular mechanism for liver injury in methylmercury (MeHg) poisoning. Ebselen, a seleno-organic compound that possesses anti-oxidant properties, is a useful therapeutic agent used in clinical situations involving oxidative stress. Here, we examined the possible in vivo protective effect of ebselen against the pro-oxidative effects of MeHg in liver from suckling rat pups. The effects of MeHg exposure (subcutaneous injections of methylmercury chloride: 2 mg/kg) on the hepatic levels of thiobarbituric acid reactive substances (TBARS) and non-ptotein thiols (NPSH), and on liver glutathione peroxidase (GSHPx) activity, as well as the possible antagonist effect of ebselen (10 mg/kg; subcutaneously) against MeHg effects, were evaluated during the post-natal period. In addition, the possible in vitro interaction between ebselen, glutathione (GSH) and MeHg was investigated by light/UV spectroscopy, with particular attention to the formation of complexes involving ebselen selenol intermediate and MeHg. After in vivo exposure, MeHg and ebselen alone increased hepatic TBARS levels. Moreover, simultaneous treatment with both compounds caused a higher increase in hepatic TBARS levels when compared to the treatments with individual compounds. Liver NPSH decreased after treatments with MeHg and ebselen alone. A significant negative correlation between hepatic TBARS and NPSH was observed. MeHg alone decreased liver GSHPx activity and ebselen, which did not affect this variable per se, reverted this inhibitory effect of MeHg. Light/UV spectroscopy showed that ebselen and GSH form a chemical intermediate that regenerates ebselen after MeHg addition. The presented results show that ebselen abolished the MeHg-induced inhibition on liver GSHPx activity, but did not prevent the oxidative effects of MeHg on liver lipids and NPSH. MeHg affects the in vitro interaction between ebselen and GSH and this phenomenon seems to be responsible for its inhibitory effect toward thiol-peroxidase activity. Additionally, ebselen presents pro-oxidative effects on rat liver, pointing to thiol depletion as a molecular mechanism related to ebselen-induced hepatotoxicity.

Identification of a mechanism by which the methylmercury antidotes N-acetylcysteine and dimercaptopropanesulfonate enhance urinary metal excretion: transport by the renal organic anion transporter-1.

N-Acetylcysteine (NAC) and dimercaptopropanesulfonate (DMPS) are sulfhydryl-containing compounds that produce a dramatic acceleration of urinary methylmercury (MeHg) excretion in poisoned animals, but the molecular mechanism for this effect is unknown. NAC and DMPS are themselves excreted in urine in high concentrations. The present study tested the hypothesis that the complexes formed between MeHg and these anionic chelating agents are transported from blood into proximal tubule cells by the basolateral membrane organic anion transporters (Oat) 1 and Oat3. Xenopus laevis oocytes expressing rat Oat1 showed increased uptake of [(14)C]MeHg when complexed with either NAC or DMPS but not when complexed with L-cysteine, glutathione, dimercaptosuccinate, penicillamine, or gamma-glutamylcysteine. In contrast, none of these MeHg complexes were transported by Oat3-expressing oocytes. The apparent K(m) values for Oat1-mediated transport were 31 +/- 2 microM for MeHg-NAC and 9 +/- 2 microM for MeHg-DMPS, indicating that these are relatively high-affinity substrates. Oat1-mediated uptake of [(14)C]MeHg-NAC and [(14)C]MeHg-DMPS was inhibited by prototypical substrates for Oat1, including p-aminohippurate (PAH), and was trans-stimulated when oocytes were preloaded with 2 mM glutarate but not glutamate. Conversely, efflux of [(3)H]PAH from Oat1-expressing oocytes was trans-stimulated by glutarate, PAH, NAC, DMPS, MeHg-NAC, MeHg-DMPS, and a mercapturic acid, indicating that these are transported solutes. [(3)H]PAH uptake was competitively inhibited by NAC (K(i) of 2.0 +/- 0.3 mM) and DMPS (K(i) of 0.10 +/- 0.02 mM), providing further evidence that these chelating agents are substrates for Oat1. These results indicate that the MeHg antidotes NAC and DMPS and their mercaptide complexes are transported by Oat1 but are comparatively poor substrates for Oat3. This is the first molecular identification of a transport mechanism by which these antidotes may enhance urinary excretion of toxic metals.

Inhibitory effect of selenium on biliary secretion of methyl mercury in rats.

The inhibitory effect of sodium selenite on biliary secretion of methyl mercury was examined in rats. The biliary secretion of methyl mercury in rat treated with 1 mumol/kg of methyl mercury was significantly decreased by administration of selenite at doses of 0.05 mumol/kg or higher. In rats given 10 mumol/kg of methyl mercury, marked depression of biliary secretion of mercury was observed when selenite was injected at a dose of 0.2 mumol/kg. On the other hand, secretion of substantial amounts of selenium was observed when biliary secretion of mercury was depressed. When the concentration of selenium in the bile was higher than 5 nmol/ml, biliary secretion of mercury was markedly depressed independently of the dose of methyl mercury administered (1 mumol/kg or 10 mumol/kg). These results suggest that the degree of inhibitory effect of selenite may be determined by the selenium concentration in the liver or the bile after treatment with selenite rather than the molar ratio of the dose of methyl mercury and selenite. We concluded that the decrease in biliary secretion of methyl mercury induced by selenite may result from inhibition of pathway for secretion of methyl mercury from liver to bile rather than the direct formation of a complex between methyl mercury and selenium. Methyl mercury has been considered to be secreted from liver to bile as a complex with glutathione (GSH). However, administration of selenite did not affect biliary secretion of GSH or hepatic glutathione S-transferase activity. Moreover, gel filtration of liver cytosol demonstrated that the distribution pattern of hepatic methyl mercury between macromolecules and GSH was not significantly changed by administration of selenite. These results suggest that selenite does not affect complex formation of methyl mercury with GSH at least in the liver. Selenite might specifically inhibit the activity of the canalicular transporter(s) which transport complexes of methyl mercury and GSH from the liver to bile.