Methylmercury is the causative organometallic compound of Minamata disease. Pathological changes in the cerebrum of patients are localized along the deep sulci and fissures of the cerebral cortex such as the calcarine fissure. It has been suggested that the occurrence of brain edema is important for the cerebral damage caused by methylmercury. Previously, we found that methylmercury induces vascular endothelial growth factor-A in cultured human brain microvascular pericytes, which may increase permeability of the brain microvasculature. In the present study, our findings suggest that this induction is mediated via the epidermal growth factor receptor-p38 mitogen-activated protein kinase-cyclooxygenase-2-protein kinase A pathway in cultured human brain microvascular pericytes. These results partly support our hypothesis that methylmercury causes neurotoxicity by activation of intracellular signaling pathways in various cell types, including neurons, macrophages, vascular endothelial cells, and pericytes.

We received Dr. Takaoka's responses. The nine major points described in these responses were reviewed from the basic perspectives of epidemiology, toxicology and neuroscience. The responses indicate important failures in our views: exposure and health hazards are misconsidered from the toxicological perspective, mass biases in epidemiological surveys, and ignorance from the pathological viewpoint. We are looking forward to scientific discussion.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

Sensory disturbances and central nervous system symptoms are important in patients with Minamata disease. In the peripheral nervous system of these patients, motor nerves are not strongly injured, whereas sensory nerves are predominantly affected. In this study, we investigated the mechanisms underlying the sensory-predominant impairment of the peripheral nervous system caused by methylmercury. We found that the types of cell death in rat dorsal root ganglion (DRG) neurons caused by methylmercury included apoptosis, necrosis, and necroptosis. Methylmercury induced apoptosis in cultured rat DRG neurons but not in anterior horn neurons or Schwann cells. Additionally, methylmercury activated both caspase 8 and caspase 3 in DRG neurons. It increased the expression of tumor necrosis factor (TNF) receptor-1 and the phosphorylation of receptor-interacting protein kinase 3 (RIP3) and mixed-lineage kinase domain-like pseudokinase (MLKL). The expression of TNF-α was increased in macrophage-like RAW264.7 cells by methylmercury. The increase was suggested to be mediated by the NF-κB pathway. Moreover, methylmercury induced neurological symptoms, evaluated by a hindlimb extension response, were significantly less severe in TNF-α knockout mice. Based on these results and our previous studies, we propose the following hypothesis regarding the pathogenesis of sensory nerve-predominant damage by methylmercury: First, methylmercury accumulates within sensory nerve neurons and initiates cell death mechanisms, such as apoptosis, on a small scale. Second, cell death triggers the infiltration of macrophages into the sensory fibers. Third, the macrophages are stimulated by methylmercury and secrete TNF-α through the NF-κB pathway. Fourth, TNF-α induces cell death mechanisms, including necrosis, apoptosis through the caspase 8/3 pathway, and necroptosis through the TNFR1-RIP1-RIP3-MLKL pathway, activated by methylmercury in sensory neurons. Consequently, methylmercury exhibits potent cytotoxicity specific to the DRG/sensory nerve cells in the peripheral nervous system. This chain of events caused by methylmercury may contribute to sensory disturbances in patients with Minamata disease.

Although pregnant women's fish consumption is beneficial for the brain development of the fetus due to the DHA in fish, seafood also contains methylmercury (MeHg), which adversely affects fetal brain development. Epidemiological studies suggest that high DHA levels in pregnant women's sera may protect the fetal brain from MeHg-induced neurotoxicity, but the underlying mechanism is unknown. Our earlier study revealed that DHA and its metabolite 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) produced by cytochrome P450s (P450s) and soluble epoxide hydrolase (sEH) can suppress MeHg-induced cytotoxicity in mouse primary neuronal cells. In the present study, DHA supplementation to pregnant mice suppressed MeHg-induced impairments of pups' body weight, grip strength, motor function, and short-term memory. DHA supplementation also suppressed MeHg-induced oxidative stress and the decrease in the number of subplate neurons in the cerebral cortex of the pups. DHA supplementation to dams significantly increased the DHA metabolites 19,20-epoxydocosapentaenoic acid (19,20-EDP) and 19,20-DHDP as well as DHA itself in the fetal and infant brains, although the expression levels of P450s and sEH were low in the fetal brain and liver. DHA metabolites were detected in the mouse breast milk and in human umbilical cord blood, indicating the active transfer of DHA metabolites from dams to pups. These results demonstrate that DHA supplementation increased DHA and its metabolites in the mouse pup brain and alleviated the effects of MeHg on fetal brain development. Pregnant women's intake of fish containing high levels of DHA (or DHA supplementation) may help prevent MeHg-induced neurotoxicity in the fetus.

Granule cell-selective toxicity of methylmercury in the cerebellum is one of the main unresolved issues in the pathogenesis of Minamata disease. Rats were orally administered methylmercury chloride (10 mg/kg/day) for 5 consecutive days, and their brains were harvested on days 1, 7, 14, 21, or 28 after the last administration for histological examination of the cerebellum. It was found that methylmercury caused a marked degenerative change to the granule cell layers but not to the Purkinje cell layers. The generative change of the granule cell layer was due to cell death, including apoptosis, which occurred at day 21 and beyond after the methylmercury administration. Meanwhile, cytotoxic T-lymphocytes and macrophages had infiltrated the granule cell layer. Additionally, granule cells are shown to be a cell type susceptible to TNF-α. Taken together, these results suggest that methylmercury causes small-scale damage to granule cells, triggering the infiltration of cytotoxic T-lymphocytes and macrophages into the granule cell layer, which secrete tumor necrosis factor-α (TNF-α) to induce apoptosis in granule cells. This chain is established based on the susceptibility of granule cells to methylmercury, the ability of cytotoxic T lymphocytes and macrophages to synthesize and secrete TNF-α, and the sensitivity of granule cells to TNF-α and methylmercury. We propose to call the pathology of methylmercury-induced cerebellar damage the "inflammation hypothesis."

Even today, more than 60 years after the discovery of Minamata disease, many controversies continue to arise from various viewpoints. Recently, Dr. Shigeru Takaoka has published a book entitled "Minamata Disease and the Responsibility of Medical authorities" as a summary of his and colleagues' previous works in which he presented their objections to past academic theories. We, who were also engaged in this research at Kumamoto University, would like to address some substantial viewpoints. Drs. Nishimura and Okamoto clarified why a series of cases that were clearly Minamata disease were found only in the Minamata plant from late 1950 to 1975, even though many acetaldehyde plants have been operating in Japan for many years. Dr. Takaoka ignored this very important issue and we point out the lack of reliability of his data from their health examination of "10,000" people. From the pathology perspective, Dr. Takaoka mis-understood the location and plasticity of neurons. From a clinical viewpoint, he mentioned the poor evidence for the characteristics and courses of the patients' clinical symptoms.

Methylmercury (MeHg), the causal substrate in Minamata disease, can lead to severe and chronic neurological disorders. The main symptom of Minamata disease is sensory impairment in the four extremities; however, the sensitivity of individual sensory modalities to MeHg has not been investigated extensively. In the present study, we performed stimulus-response behavioral experiments in MeHg-exposed rats to compare the sensitivities to pain, heat, cold, and mechanical sensations. MeHg (6.7 mg/kg/day) was orally administered to 9-week-old Wistar rats for 5 days and discontinued for 2 days, then administered daily for another 5 days. The four behavioral experiments were performed daily on each rat from the beginning of MeHg treatment for 68 days. The pain sensation decreased significantly from day 11 onwards, but recovered to control levels on day 48. Other sensory modalities were not affected by MeHg exposure. These findings suggest that the pain sensation is the sensory modality most susceptive to MeHg toxicity and that this sensitivity is reversible following discontinuation of the exposure.

Background: Human exposure to mercury (Hg) is widespread and both organic and inorganic Hg are routinely found in the human brain. Millions of people are exposed to methyl Hg (MeHg) due to the consumption of fish and to inorganic Hg from dental amalgams, small scale gold mining operations, use of Hg containing products, or their occupations. Neuropathology information associated with exposures to different species of Hg is primarily based on case reports of single individuals or collections of case studies involving a single species of Hg at toxic exposure levels such as occurred in Japan and Iraq. Methods/Results: This study brings together information on the neuropathological findings and deposition of Hg in the central nervous system of people exposed to different species of Hg at varying concentrations. The low dose exposures were lifetime exposures while the high dose exposures were generally acute or short term by different exposure routes with survival lasting various lengths of time. Total and inorganic Hg deposits were identified in formalin-fixed, paraffin embedded tissues from both low and high exposure Hg cases. Low concentration exposures were studied in adult brains from Rochester, New York (n = 4) and the Republic of Seychelles (n = 17). Rochester specimens had mean total Hg concentrations of 16-18 ppb in the calcarine, rolandic, and cerebellar cortices. Inorganic Hg averaged between 5-6 ppb or 30-37% for the cerebral and cerebellar cortices of the Rochester subjects. Total Hg was approximately 10-fold higher in specimens from Seychelles, where consumption of ocean fish is high and consequently results in exposure to MeHg. The predominant Hg species was MeHg in both the Rochester and Seychelles brain specimens. Histologically, cerebral and cerebellar cortices from Rochester and Seychelles specimens were indistinguishable. High concentration exposures were studied in brains from four adults who were autopsied at variable time periods after exposure to organic Hg (methyl or dimethyl) or inorganic Hg (inhaled vapor or intravenous injection of metallic Hg). In contrast to the Seychellois adults, these individuals had acute or subacute exposures to lethal or significantly higher concentrations. The pattern of Hg deposition differed between subjects with high organic Hg exposure and high inorganic Hg exposure. In the organic Hg cases, glia (astrocytes and microglia) and endothelial cells accumulated more Hg than neurons and there were minimal Hg deposits in cerebellar granule and Purkinje cells, anterior horn motor neurons, and neocortical pyramidal neurons. In the inorganic Hg cases, Hg was seen predominantly in neurons, vascular walls, brainstem, and cerebellar and cerebral deep gray nuclei. The presence of inorganic Hg in neural and neural supporting cells in the four high exposure Hg cases was not closely correlated with cellular pathology; particularly in the inorganic Hg cases. Conclusions: Different Hg species are associated with differing neuropathological patterns. No neuropathological abnormalities were present in the brains of either Rochester or Seychelles residents despite substantial differences in dietary MeHg exposure. Increasing concentrations of inorganic Hg were present in the brain of relatively low exposure subjects with increasing age.

Methylmercury (MeHg) exposure is known to induce neurodegeneration in both the central nervous system (CNS) and peripheral nervous system (PNS). Molecular mechanisms of MeHg-induced neurotoxicity have been well investigated in the CNS, however, it remains unclear in the PNS. In the present study, comprehensive gene expression analysis was performed by analyzing MeHg-exposed adult rat dorsal root ganglion (DRG) by DNA microarray. Methylmercuric chloride (6.7 mg/kg/day) was administered to nine-week-old male Wistar rats for five days, followed by two days without administration; this cycle was repeated once. Rats were anesthetized at 7 or 14 days after commencement of MeHg exposure, and their DRGs were removed and homogenized to make total RNA samples. DNA microarray data from Day 7 samples identified 100 out of 18,513 detected genes as annotated genes with more than two-fold upregulated or downregulated expression compared with controls. Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested strong involvement of immune activation and inflammation pathways in rat DRG exposed to MeHg, and some genes overlapped with previously reported genes affected by MeHg exposure in the cerebellum. The present results suggest that MeHg-induced neurotoxicity is associated with immune activation and inflammatory responses in rat DRG.

Exposure to organic mercury, especially methylmercury (MeHg), causes Minamata disease, a severe chronic neurological disorder. Minamata disease predominantly affects the central nervous system, and therefore, studies on the mechanisms of MeHg neurotoxicity have focused primarily on the brain. Although the peripheral nervous system is also affected by the organometallic compound and shows signs of neural degeneration, the mechanisms of peripheral MeHg neurotoxicity remain unclear. In the present study, we performed quantitative immunohistochemical analyses of the dorsal root ganglion (DRG) and associated sensory and motor fibers to clarify the mechanisms of MeHg-induced peripheral neurotoxicity in Wistar rats. Methylmercury chloride (6.7 mg/kg/day) was orally administrated for 5 days, followed by 2 days without administration, and this cycle was repeated once again. Seven and 14 days after the beginning of MeHg exposure, rats were anesthetized, and their DRGs and sensory and motor nerve fibers were removed and processed for immunohistochemical analyses. The frozen sections were immunostained for neuronal, Schwann cell, microglial and macrophage markers. DRG sensory neuron somata and axons showed significant degeneration on day 14. At the same time, an accumulation of microglia and the infiltration of macrophages were observed in the DRGs and sensory nerve fibers. In addition, MeHg caused significant Schwann cell proliferation in the sensory nerve fibers. In comparison, there was no noticeable change in the motor fibers. Our findings suggest that in the peripheral nervous system, MeHg toxicity is associated with neurodegenerative changes to DRG sensory neurons and the induction of a neuroprotective and/or enhancement of neurodegenerative host response.

Methylmercury (MeHg) is known to cause neurobehavioral impairment in human and experimental animals. We previously reported that MeHg (5 mg Hg/kg) induced severe neurobehavioral dysfunction in 4‐week‐old KK‐Ay mice, although it is difficult to evaluate quantitatively the neurobehavioral impairment in MeHg‐treated KK‐Ay mice because of their obesity. The aim of this study was to evaluate MeHg‐induced neurobehavioral dysfunction in KK‐Ay mice using the dynamic weight‐bearing test, which analyzes the animal's weight distribution between the four limbs. Male 12‐week‐old KK‐Ay mice were treated with MeHg (5 mg Hg/kg) three times per week for 5 weeks. Body weight loss began after approximately 2 weeks of MeHg treatment, and decreased significantly at 4 weeks. Seven of the nine MeHg‐treated mice exhibited overt neurological symptoms such as ataxia and gait disturbance. The weight‐bearing load was lower for the forelimb than for the hindlimb at baseline and until 1 week after MeHg treatment was initiated. In weeks 2‐4, the dynamic weight‐bearing loads on the forelimb and hindlimb were similar. The load on the forelimb exceeded the load on the hindlimb after 5 weeks of treatment. This finding indicates that the dynamic weight‐bearing test is useful for semi‐quantitative evaluation of neurobehavioral impairment in MeHg‐treated rodents, and is less stressful for the animals. Infiltration of CD204‐positive macrophages was observed in the sciatic nerve of MeHg‐treated mice, suggesting that CD204 can serve as a useful marker of tissue injury in peripheral nerves and a possible target in regenerating peripheral nerves and controlling neuropathies.

The main purpose of the present study was to compare the blood and brain mercury (Hg) accumulation and neurological alterations in adult male and pregnant female/fetal rats following stable and episodic/bolus patterns of methylmercury (MeHg) exposure. In addition, MeHg accumulation in the human body was estimated by a one-compartment model using three different patterns of MeHg exposure. In the adult male rat experiment, doses of 0.3 and 1.5mg MeHg/kg/day were orally administered to the stable groups for 5 weeks, while 7-fold higher doses of 2.1 and 10.5mg MeHg/kg/once a week were administered to the bolus groups. The blood Hg levels increased constantly in the stable groups, but increased with repeated waves in the bolus groups. At completion of the experiment, there were no significant differences in the brain Hg concentrations or neurological alterations between the stable and bolus groups, when the total doses of MeHg were the same. In the pregnant female rat experiment, a dose of 1mg MeHg/kg/day was administered orally to the stable group for 20 days (until 1day before expected parturition), while a 5-fold higher dose of 5mg MeHg/kg/once every 5 days was administered to the bolus group. In the brains of the maternal/fetal rats, there were no significant differences in the Hg concentrations and neurological alterations between the stable and bolus groups. The mean Hg concentrations in the fetal brains were approximately 2-fold higher than those in the maternal brains for both stable and bolus groups. Using the one-compartment model, the Hg accumulation curves in humans at doses of 7µg MeHg/day, 48µg MeHg/once a week, and 96µg MeHg/once every 2 weeks were estimated to be similar, while the bolus groups showed dose-dependent amplitudes of repeated waves. These results suggest that stable and episodic/bolus patterns of MeHg exposure do not cause differences in Hg accumulation in the blood and brain, or in neurological alterations, when the total doses are the same.

In a cerebrum damaged by methylmercury, where neuropathological lesions tend to localize along deep sulci and fissures, edematous changes in white matter have been proposed as the cause of such localization. Since hyaluronan has a high water-retention capability and can contribute to the progression of edematous changes, we hypothesize that methylmercury increases hyaluronan in brain microvascular cells. Our experimental results indicate that methylmercury induces the expression of hyaluronan in cultured human microvascular endothelial cells and pericytes through the induction of expressed UDP-glucose dehydrogenase and hyaluronan synthase 2, respectively. After exposure to methylmercury, hyaluronan largely accumulates in perivascular space, where it contributes to the progression of edematous changes.

Methylmercury is an environmental pollutant that exhibits neurotoxicity when ingested, primarily in the form of neuropathological lesions that localize along deep sulci and fissures, in addition to edematous and inflammatory changes in patient cerebrums. These conditions been known to give rise to a variety of ailments that have come to be collectively termed Minamata disease. Since prostaglandins I-2 and E-2 (PGI(2) and PGE(2)) increase vascular permeability and contribute to the progression of inflammatory changes, we hypothesize that methylmercury induces the synthesis of these prostaglandins in brain microvascular endothelial cells and pericytes. To test this theory, human brain microvascular endothelial cells and pericytes were cultured and treated with methylmercury, after which the PGI(2) and PGE(2) released from endothelial cells and/or pericytes were quantified by enzyme linked immunosorbent assay while protein and mRNA expressions in endothelial cells were analyzed by western blot analysis and real-time reverse transcription polymerase chain reaction, respectively. Experimental results indicate that methylmercury inhibits the activity of protein tyrosine phosphatase 1B, which in turn activates the epidermal growth factor receptor p38 mitogen-activated protein kinase pathway that induces cyclooxygenase-2 expression. It was also found that the cyclic adenosine 3',5'-monophosphate pathway, which can be activated by PGI(2) and PGE(2), is involved in methylmercury-induced cyclooxygenase-2 expression. Since it appears that protein tyrosine phosphatase 1 B serves as a sensor protein for methylmercury in these mechanisms, it is our belief that the results of the present study may provide additional insights into the molecular mechanisms responsible for edematous and inflammatory changes in the cerebrum of patients with Minamata disease.

Identification of the proteinaceous components of the pathological inclusions is an important step in understanding the associated disease mechanisms. We immunohistochemically examined two previously reported cases with eosinophilic neuronal cytoplasmic inclusions (NCIs) (case 1, Mori et al. Neuropathology 2010; 30: 648–53; case 2, Kojima et al. Acta Pathol Jpn 1990; 40: 785–91) using 67 antibodies against proteins related to cytoskeletal constituents, ubiquitin‐proteasome system, autophagy‐lysosome pathway and stress granule formation. Regional distribution pattern of eosinophilic NCIs in case 1 was substantially different from that in case 2. However, NCIs in both cases were immunonegative for ubiquitin and p62 and were immunopositive for stress granule markers as well as autophagy‐related proteins, including valosin‐containing protein. Considering that eukaryotic stress granules are cleared by autophagy and valosin‐containing protein function, our findings suggest that eosinophilic NCIs in the present two cases may represent the process of autophagic clearance of stress granules.

The localization of neuropathological lesions along deep sulci and fissures is one of the characteristics of a cerebrum damaged by methylmercury. Edematous changes in white matter have been proposed as the cause of the localization of lesions; however, the molecular mechanisms underlying methylmercury-induced edema remain unclear. Since the vascular endothelial growth factor (VEGF) system regulates vascular permeability and can be involved in the progression of edematous changes, we examined the effect of methylmercury on the expression of VEGF-related proteins in cultured human brain microvascular endothelial cells and pericytes. After methylmercury exposure, mRNA and protein levels of VEGF-A in pericytes and placenta growth factor (PlGF) and VEGF-receptor-1/-2 in endothelial cells were elevated. The induction of pericyte VEGF-A expression was independent of hypoxia-inducible factor-α and hypoxia-response element signaling. Taken together, these results suggest that methylmercury activates the VEGF system in brain microvessels in a paracrine fashion. When the activation occurs in narrow areas such as along the deep sulci in the cerebrum, hyperpermeability and subsequent edematous changes would cause a circulatory disturbance and result in neural cell damage. We propose this as a reason for the localization of the neuropathological lesions along the deep sulci and fissures in the cerebral cortex, such as the calcarine fissure, in patients with Minamata disease.

ABSTRACTWe examined the toxic effects of methylmercury (MeHg) in KK‐Ay type 2 diabetic mice to clarify how metabolic changes associated with type 2 diabetes mellitus affect MeHg toxicity. MeHg (5 mg Hg kg –1day–1 p.o.) was given to 4‐week‐old male KK‐Ay and C57BL/6J (BL/6) mice three times per week for 6 weeks. Average body weights (BW) of vehicle‐treated BL/6 and KK‐Ay mice were 16.3 and 16.4 g respectively on the first day, and 24.8 and 42.3 g respectively on the last day of the experiment. MeHg‐treated KK‐Ay mice began to lose weight about 5 weeks after MeHg administration. Six of seven MeHg‐treated KK‐Ay mice showed hind‐limb clasping in the final stage of the experiment. The mean blood mercury level of MeHg‐treated KK‐Ay mice reached a maximum of 9.8 µg ml–1, whereas that of the MeHg‐treated BL/6 mice was 2.8 µg ml–1 after 10 days of treatment. The average total mercury concentrations in the cerebrum and epididymal fat pad were 7.4 and 0.57 µg g–1, respectively, for BL/6 mice and 27 and 1.6 µg g–1, respectively, for KK‐Ay mice. In MeHg‐treated KK‐Ay mice with neurological symptoms, CD204‐positive macrophages were observed in the brain, kidney and spleen, indicating CD204 could be a marker for injured tissues. BW loss and significant pathological changes were not observed in other groups of mice. These results indicate that body fat gain in type 2 diabetes mellitus and low mercury accumulation in adipose tissue increased MeHg concentrations in organs and enhanced toxicity in KK‐Ay mice at the same dose of MeHg per BW. Copyright © 2013 John Wiley & Sons, Ltd.