NIMD Forum 2022

Theme Aiming to Prevent Methylmercury Poisoning
Date November 29,30,2022
Venue Minamata Disease Archives Hall


Subject1 「Protective Effect of Oleanolic Acid-3-Glucoside on Methylmercury Toxicity」
Ryosuke Nakamura(School of Pharmacy, Kitasato University, Japan)

Methylmercury (MeHg) causes serious damage to various organs in both animals and humans. Bioaccumulation of MeHg occurs through the food chain, and consumption of contaminated fish and other aquatic seafood is the primary source of exposure to humans. Severe neurological disorders occur in victims of MeHg poisoning, which was the cause of Minamata disease. Several studies have evaluated the effects of toxic or subtoxic doses of MeHg in vivo and in vitro. However, the molecular responses and effects of low doses of MeHg to which humans are exposed through daily food intake are not well-understood, and there is a demand for anti-MeHg medicines especially during pregnant period. Therefore, in this study, we evaluated the effectiveness of various synthetic oleanane-type saponin derivatives in alleviating MeHg toxicity, using in vitro and in vivo models. We synthesized a saponin derivative in which one glucose molecule was bound to the Oleanolic acid (OA) skeleton, i.e., a simplified onjisaponin structure. The synthetic OA-3-glucoside (OA3Glu), in which glucose was bound to the C3 position of OA, suppressed MeHg-induced cell death in Caco-2 colon carcinoma cells and mercury accumulation in the liver, kidney, and brain of MeHg-exposed mice. These results suggested that OA3Glu would be a potential anti-MeHg toxicity compound1,2). Recently, we exposed mice to MeHg at a higher concentration than the previous reports and verified the effect of OA3Glu, as an anti-MeHg toxicity compound especially focusing on dynamic weight bearing test and electrophysiology in MeHg-exposed mice. We newly found that OA3Glu can alleviate MeHg-induced Purkinje cell death and synaptic damage. Therefore, we propose OA3Glu as a candidate agent against MeHg toxicity.

Subject2 「Bacteria as Dietary Supplements to Mitigate Damage Associated to Mercury Exposure」
Manuel Zúñiga(Department of Food Biotechnology, Institute of Agrochemistry and Food Technology (CSIC), Spain)

Food represents the major risk factor for exposure to mercury in most human populations. In particular, large predatory fishes are the major contributors to Hg dietary burden while at the same time they constitute the main source of essential nutrients such as polyunsaturated fatty acids. Therefore, devising food supplements that reduce Hg intestinal assimilation is a promising strategy to diminish the risk associated to the consumption of these fish products. The use of lactic acid bacteria (LAB) as probiotics for counteracting the detrimental effects of heavy metal exposure at the gastrointestinal tract has been proposed. In order to test the efficacy of LAB in this aspect we employed a bicameral model consisting of Caco-2 and HT29-MTX intestinal epithelial cells and THP-1-derived macrophages (1). Exposure to 1 mg/ml mercury [Hg(II) or methyl-Hg)] for seven days in this model resulted in an inflammatory and pro-oxidant response mainly driven by macrophages. However, the presence of heat-killed LAB cells (a strain of Lactobacillus intestinalis or a strain of Lactobacillus johnsonii) during Hg exposure reverted these effects, and most of the parameters recovered values similar to control cells. Both lactobacilli showed the capacity to bind Hg(II) and methyl-Hg under the cell culture conditions. This points to Hg sequestration as the main mechanism that counteracted Hg toxicity. However, differences in the Hg binding capacity and in the effects between both strains suggest that other mechanisms may play a role in the alleviation of the damage elicited by Hg. These results were confirmed in vivo with mice exposed to Hg(II) or MeHg for two months. Supplementation with either LAB strain (live cells) resulted in diminished inflammatory and pro-oxidant responses and intestinal mucosa damage. Again, differences between strains were observed, stressing that alleviation of mercury damage by LAB is a strain-dependent character.



Subject1 「Usefulness of the Functional Food Ingredients on Reducing Methylmercury Burden: Wheat Bran and Fructooligosaccharides」
Masaaki Nagano(Department of Basic Medical Sciences, National Institute for Minamata Disease, Japan)

Methylmercury (MeHg) is a ubiquitous environmental pollutant and well-known neurotoxicant. MeHg exposure through the consumption of fish and shellfish, especially during pregnancy, has been a public health concern in many places because of potential health risks to the fetus. Bacteroides, bifidobacteria, Escherichia coli and lactobacilli have the high metabolic (demethylation) activity of MeHg,1) and may contribute to the excretion of MeHg into the feces. In this study, we investigated the effect of fructooligosaccharides (FOS) on the accumulation and excretion of Hg after MeHg administration in female mice. FOS, typical nondigestible oligosaccharides, are prebiotics. Mice were fed a basal diet or the same diet supplemented with 5% FOS. Six weeks after feeding, mice were orally administered MeHg chloride (4 mg Hg/kg). FOS reduced tissue Hg accumulation in MeHg-exposed mice, and enhanced fecal Hg excretion, but not urinary Hg excretion.2) Tissue concentrations of inorganic mercury (Hg2+), the proportion of Hg2+ in feces, and analysis of fecal bacterial population suggested that FOS accelerate MeHg demethylation by intestinal microbiota. Long-term intake of wheat bran (bran) which is the outer layer of wheat kernel, before MeHg administration has been shown to decrease total Hg concentrations in the brain and blood of male mice,3) but the mechanism remains unclear. Therefore, we also examined whether intake of bran after MeHg administration reduces Hg accumulation in female mice. Immediately after single administration of MeHg (4 mg Hg/kg, p.o.), the mice were fed a basal diet supplemented with 0%, 5%, or 15% bran. Bran decreased total Hg concentrations in the blood, brain, liver, and kidney of MeHg-exposed mice, and increased both urinary and fecal Hg excretions.4) In conclusion, daily intake of bran or nondigestible oligosaccharides such as FOS, might be useful for reducing Hg burden and the risk of MeHg to health in humans.

Subject2 「Microbial Derived Factors in Methylmercury Demethylation and Reduction of Toxicity」
Matthew D. Rand(Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, USA)

Biotransformation (demethylation) of MeHg by microbes in the gut lumen is thought to enhance the elimination rate, (reduce the biological half-life (t1/2)) of MeHg in the body. The half-life dictates the MeHg body burden that an individual will experience from repeated exposures, such as regular consumption of fish. Our studies with human volunteers indicate that the MeHg elimination rate correlates with the degree of demethylation seen in feces, and is furthermore decreased in subjects taking antibiotics. The bacterial enzymes encoded by the merB (organomercurial lyase) and merA (mercuric reductase) genes are the only known enzymes capable of MeHg biotransformation. MerB and MerA act sequentially to demethylate MeHg to Hg2+ and reduce Hg2+ to volatile elemental mercury (Hg0) that can escape from the cell. Evidence for merB/A containing bacteria in the human or rodent gut is inconsistent. Furthermore, there is no evidence for enzymes in eukaryotic cells capable of MeHg demethylation or reduction. Thus, mechanisms for demethylation in animals and humans remains unknown. We examined the role of MeHg demethylation in moderating MeHg toxicity by expressing the MerB enzyme in both bacterial and animal (Drosophila) systems. We find that MeHg demethylation, without subsequent reduction, creates greater toxicity in bacterial cells. In contrast, systemic MeHg demethylation in transgenic Drosophila expressing the bacterial merB gene results in a rescue of MeHg toxicity during development. Relative to control flies, MerB expression results in a significant decrease in MeHg body burden which correlates with a significant increase in the MeHg elimination rate. With neuron-specific merB expression, we observe a rescue of MeHg-induced development failure without a decrease in Hg body burden, but with a redistribution of Hg away from the brain. These results demonstrate a previously unidentified potential for intracellular MeHg demethylation to promote transport and elimination of Hg, and reduce developmental MeHg toxicity. (Funding: NIH R01ES030940, P30 ES001247, T32 207026)



Subject1 「Roles of DHA and Its Metabolites in Protection against Methylmercury-Induced Neurotoxicity」
Ami Oguro(Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan)

The consumption of fish now involves a risk of methylmercury (MeHg) exposure but also provides the benefit of ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA). Some epidemiological studies have suggested that the intake of DHA can alleviate the neurotoxicity of MeHg1-2), but the underlying mechanism is not known. Herein, we observed that pretreatment with DHA suppressed MeHg-induced cytotoxicity in neuronal cells via activation of RXR. DHA also suppressed the MeHg-induced production of reactive oxygen species (ROS) via an induction of antioxidant genes (catalase and SOD1). We showed previously that in the brain, the intake of DHA increased the level of 19,20-DHDP, which is the metabolite produced by cytochrome P450 and soluble epoxide hydrolase from DHA. We observed that 19,20-DHDP also suppressed neurotoxicity from MeHg, and promoted stabilization of antioxidant protein Nrf2. These results indicate that DHA and its metabolites have a protective role in MeHg-induced neurotoxicity. In addition, supplementation of DHA to the pregnant mouse suppressed MeHg-induced impairment of neuronal functions in pups. DHA supplementation also suppressed oxidative stress in the hippocampus and cortex of pups. The supplementation of DHA to the dam significantly increased DHA metabolites, 19,20-DHDP as well as DHA in the brain of fetus and infant, although the low expression levels of P450s and sEH in the brain and liver of fetus. DHA metabolites were detected in the breast milk and umbilical cord blood, indicating the active transfer of DHA metabolites from dams to pups. These results showed that supplementation of DHA is important to alleviate the effects of MeHg on fetal brain development by increased DHA and its metabolites in the pup brain. The intake of fish containing high levels of DHA or supplementation of DHA metabolites by pregnant women may be valuable to prevent neurotoxicity of MeHg for fetus.

Subject2 「The Human LRRK2 Modulates the Age-Dependent Effects of Developmental Methylmercury Exposure in Caenorhabditis elegans」
Michael Aschner, and Tao Ke(Department of Molecular Pharmacology, Albert Einstein College of Medicine, USA)

Methylmercury (MeHg) neurotoxicity exhibits age-dependent effects with a latent and/or persistent neurotoxic effect on aged animals. Individual susceptibility to MeHg neurotoxicity is governed by both exposure duration and genetic factors that can magnify or mitigate the pathologic processes associated with this exposure. We previously showed the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2) modulates the response of worms to high levels of MeHg, mitigating its effect on neuronal morphology in pre-vesicles in cephalic (CEP) dopaminergic neurons. Here we sought to better understand the long-term effects of MeHg exposure at low levels (100-fold lower than that in our previous report) and the modulatory role of the LRRK2 mutation. Worms exposed to MeHg (10 or 50 nM) at the larval stage (L1 stage) were compared at adult stages (young age: day 1 adult; middle age: day 5 adult; old age: day 10 adult) for the swimming speeds in M9 buffer, moving speeds during locomotion on an OP50-seeded plate, and the numbers of CEP dopaminergic pre-vesicles, vesicular structures originating from the dendrites of CEP for exportation of cellular content. In addition, the expression levels of Caenorhabditis elegans homologs of dopamine transporter (dat-1) and tyrosine hydroxylase (cat-2) were also analyzed at these adult stages. Our data showed that swimming speeds were reduced in wild-type worms at the day 10 adult stage at 50 nM MeHg level; yet, reduced swimming speeds were noted in the G2019S LRRK2 transgenic line upon MeHg exposures as low as 10 nM. Compared to locomotor speeds, swimming speeds appear to be more sensitive to the behavioral effects of developmental MeHg exposures, as the locomotor speeds were largely intact and indistinguishable from controls following MeHg exposures. Furthermore, we showed an age-dependent modulation of dat-1 and cat-2 expressions, which could also be modified by the LRRK2 mutation. Although MeHg exposures did not change the number of pre-vesicles, the LRRK2 mutation was associated with increased numbers of pre-vesicles in aged worms. Our data suggest that the latent behavioral effects of MeHg are sensitized by the G2019S LRRK2 mutation, and the underlying mechanism likely involves age-dependent changes in dopaminergic signaling.



Subject1 「Development of a Sensor for the methylmercury Toxicity」
Akio Sumioka(Department of Basic Medical Science, National Institute for Minamata Disease, Japan.)

Methylmercury (MeHg) is a pollutant in the environment. A complex of MeHg with cysteine (Cys) mimics Methionine and reaches the central nervous system through the blood-brain barrier. Because neuronal degeneration is irreversible, it is essential to prevent MeHg toxicity at an early stage of this process. To utilize to prevention study, we developed sensors for MeHg toxicity.
We focused on the impairment of selenoprotein translation mediated by MeHg exposure to develop sensor vectors. Cos-7 cells were transfected with cDNA of tagged-TrxR1 and treated with MeHg. Full-length TrxR1 signals were decreased by inhibiting selenocysteine (Sec) insertion in a MeHg-dependent manner. Luciferase substitutional mutant of Cys with Sec (Luc-Sec) was designed to utilize this event. As expected, culture cells transfected with Luc-Sec cDNA showed decreased Luciferase signal by MeHg treatment.
Next, it was attempted to develop sensors increasing signals MeHg-dependently. Luc-Sec fused with degradation signal (Luc-Sec-odc) was designed to monitor truncated form. Unpredictably Luc-Sec-odc showed signal mediated by truncated form whether absence or presence of MeHg. It means that Sec is inserted inefficiently in the cell-cultured system. Known factors for Sec incorporation, including flanking-sequence, SBP2 protein, etc., were examined, but it was only clear that unknown mechanisms mediate the efficiency. Then cDNAs of Krab-U, Krab transcriptional repressor fused with Sec and pCTre-Luc, regulated Luciferase (Krab-Sec/Luc) were designed. Cultured cells transfected with Krab-Sec/Luc increased signal by MeHg exposure. Furthermore, Krab-Sec/Luc cDNAs were validated for the MeHg toxicity sensor. As a result, Krab-Sec/Luc showed enough signals like known sensors, dose dependency, and specificity from other toxicants.
In this presentation, Krab-Sec/Luc as sensor vectors for MeHg toxicity were developed. Krab-Sec/Luc will monitor the early stage of MeHg-mediated impairment and help with the prevention study. Furthermore, by combination with other toxicity sensors, Krab-Sec/Luc will be available for understanding cell specificity and molecular mechanism of MeHg.

Subject2 「Demethylation of Methylmercury in Bird, Fish, and Earthworm」
Jean-Paul Bourdineaud(European Institute of Chemistry and Biology, University of Bordeaux & CNRS, France)

Clark’s grebes have been sampled from a metal-polluted site and the different mercurial species in animals’ tissues have been identified using high energy-resolution X-ray absorption near-edge structure spectroscopy, called HR-XANES. The feathers and brain contained 100 % of cysteinyl-methylmercury as total mercury. Conversely, the liver contained a tetrahedral species, corresponding to tetraselenocysteinate. The proportion of the tetraselenated species in Clark’s grebe organs was 86 % in the liver, 59 % in kidneys, 11 % in muscle and below the detection threshold in brain.
Earthworms have been sampled from an industrial site. The proportion of the methylated form of mercury in earthworms’ tissues was very high, ranging from 28 to 50 % whereas the soil contained less than 3 % of methylmercury. This was explained by the presence in the earthworms’ digestive tract of mercury-methylating bacteria from the Geobacteraceae genera. The methylated mercury in the intestine was further demethylated in the tissues through the formation of the tetraselenocysteinate species, which proportion varied between 25 to 72 %.
Demethylation of methylmercury was also observed in Amazonian fish liver and in this organ the tetraselenocysteinate species accounted for 9 to 62 % of the total mercury. The formation of this species only occurred in liver whereas the methylated species represented 100 % in muscle.
Methylmercury demethylation occurs through the intervening action of selenoprotein P for vertebrates and selenoneine or low molecular weight selenocompound in earthworm that catalyze the formation of mercury tetraselenocysteinate. This leads to the final deposition of nanoparticulate mercury selenide, or tiemannite.



Subject1 「The importance of Rabenstein's Reaction in the Toxicity of Methylmercury: What is missing?」
João B T Rocha(Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Santa Maria, Brazil)

Methylmercury (CH3Hg+) is a soft electrophile with strong affinities for soft nucleophiles moieties found in biomolecules. Basically, the thiol (-SH) and the selenol (-SeH) groups are the two types of soft nucleophile sites found in biomolecules. The -SH groups if found both in low- and high-molecular weight biomolecules (e.g., cysteine and reduced glutathione-GSH). In contrast, the -SeH group is rare and found in few types of selenoproteins and selenoenzymes with antioxidant properties. The constant rate for the reaction of CH3Hg+ with R-XH (where X=S or Se) is extremely high and diffusion-controlled. Despite of this, the CH3Hg-X-R complex can easily enter in exchange reaction with free R-XH-containing molecules (the Rabenstein’s Reaction). However, the Rabenstein’s reaction have not been systematically studied, possibly in view of the analytical requirements. The determination of the constant rates for the exchange of CH3Hg¬-XR with different relevant thiol groups (cysteine, N-acetylcysteine, human plasma albumin, hemoglobin, etc.) would be instrumental to better predict the fate of CH3Hg+ in the human body. The reaction with selenoproteins and other critical thiol-containing enzymes would be also important to understand which of the selenoproteins or thiol-proteins may be the preferential targets of CH3Hg+-S-R (R=cysteine or GSH). The identification of such proteins by omics approaches will be crucial to identify the molecular initiating events (MIEs) and the main adverse outcomes pathways (AOP) associated with CH3Hg+ toxicity. The potential interaction CH3Hg+-S-R with low-molecular organoselenium compounds can also be instrumental to develop new drugs to treat CH3Hg+ over exposure. (Supported by CNPq-INCT, CAPES, FAPERGS)

Subject2 「Capture of Methylmercury by Super Sulfide Species」
Yumi Abiko(Department of Hygienic Chemistry, Nagasaki University, Japan)

Methylmercury (MeHg) is an electrophile that can disrupt the function of cellular proteins by covalent binding to their thiol groups, resulting in toxicity. Glutathione (GSH), which is a tripeptide containing thiol, binds to MeHg to form a MeHg–SG adduct associated with detoxification of MeHg. We have demonstrated that nucleophilic sulfides such as hydrogen sulfide (H2S) and super sulfide species such as glutathione persulfide (GSSH), glutathione polysulfide (GSSSG), and protein-bound persulfides and polysulfides readily react with MeHg to yield bismethylmercury sulfide ((MeHg)2S)1,2. This sulfur adduct is less toxic than MeHg and the MeHg–SG adduct in both in vitro and in vivo1,2. Although Allium sativum L. (garlic) is known to contain numerous sulfur compounds, the presence of such super sulfide species in garlic is not well understood. We found that numerous garlic constituents interact with MeHg to form (MeHg)2S and hexane extract of garlic decreased MeHg-mediated cytotoxicity, suggesting the existence of super sulfide species in the plant3. In addition, the levels of cysteine persulfide, GSSH, hydrogen persulfide, GSH, and H2S in mice plasma were significantly increased after 2 h of administration of the garlic hexane extract (250 mg/kg) 3. Although a single oral dose of MeHg (50 mg/kg) decreased the body weight and 40% of the mice died within 10 days of exposure, simultaneous administration of the garlic hexane extract and MeHg caused alleviation of the MeHg-induced toxic effects3. These results suggest that ingestion of garlic may decrease MeHg toxicity, at least in part, by the formation of (MeHg)2S that is able to inhibit covalent modification of cellular proteins of MeHg. We therefore claim that capturing MeHg by super sulfide species in vivo and in food such as garlic could be a strategy to decrease the health risks of intake of MeHg from contaminated food or environment.

Subject3 「Protective Function of Supersulfides against Methylmercury Toxicity」
Takamitsu Unoki(Department of Basic Medical Sciences, National Institute for Minamata Disease, Japan)

Methylmercury (MeHg) is an electrophile with electron-deficient moiety that forms covalent bonds with electron-rich nucleophilic substituents such as protein cysteine residues, thereby modulating protein function resulting in toxicity. Transcription factor NF-E2-related factor 2 (Nrf2) plays a role in detoxification of MeHg via formation of glutathione adducts and subsequent excretion into extracellular spaces. Alternatively, supersulfides that are endogenously produced by cystathionine γ-lyase (CSE) and have high nucleophilicity, react with MeHg to form bismethylmercury sulfide, a less toxic sulfur adduct than MeHg1). Hence, we evaluated the contributions of Nrf2 and CSE in the protection against MeHg. Both Nrf2 and CSE single knockout (KO) mice were highly susceptible to MeHg, and such sensitivity was further exacerbated in Nrf2/CSE double KO (DKO) mice2). Primary hepatocytes from DKO mice were significantly more sensitive to the environmental electrophiles including MeHg than each single KO counterpart2). These results suggest that Nrf2 is a key transcription factor for detoxification of MeHg, CSE is also crucial factor to repress their toxicity in a parallel mode. Furthermore, we assessed the dynamics of supersulfide distribution and evaluated its relevance in sensitivity to MeHg in rat brain. Analyses of fetal/juvenile rat brains showed low supersulfide levels in early developmental stages3). Site-specific analysis of adult rat brains revealed that cerebellar supersulfide levels were lower than those of the hippocampus3). Microscopically, supersulfide levels of the granular cell layer were lower than those of the molecular layer in the cerebellum3). Thus, low supersulfide levels corresponded with age and site of the brain that is vulnerable to MeHg. Taken together with the finding that brain supersulfides were consumed during MeHg exposure, these results indicate that supersulfide is a factor that defines the specificity of MeHg vulnerability in the brain.




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