Mechanism of methylmercury cytotoxicity

Although a large number of epidemiological, clinical, and pathological studies on methylmercury intoxication have been published, these investigations have not been able to elucidate the detailed mechanisms by which the metal alkyl causes a wide variety of biological dysfunctions. Thus, the cultured cells which are free from the influence of whole body complexities, such as absorption, distribution, metabolism, etc., which complicate the interpretation of in vivo experimental results, attract the attention of many scientists who are interested in clarifying the mode of toxic action of methylmercury. The aim of this article is to review the recent studies on the toxicity of methylmercury at the cellular level and to outline the mechanisms which have been proposed to be responsible for cell injuries.     

Behavioral teratology of methylmercury

Behavioral effects of experimental perinatal methylmercury exposure are reviewed. Studies were summarized by classification based on examined behaviors and functions as follows; Development of reflexive behaviors, Swimming ability, Spontaneous activity, Open-field behavior, Maze learning, Avoidance learning, Operant learning, Susceptibility to induced convulsion and seizure, Ultrasonic vocalization, Visual function. Findings suggest that perinatal methylmercury exposure caused changes in a wide spectrum of behaviors in offspring. It is suggested that further researches on neuro-behavioral teratogenicity of methylmercury will be awaited, especially in the area investigating interaction of the other environmental factors.     

Neurotoxic mechanisms of fish-borne methylmercury

Methylmercury (MeHg) epidemics and studies in fish-eating populations have provided invaluable insights into the neurotoxic sequelae of MeHg exposure. MeHg is a ubiquitous environmental contaminant, and its accumulation in the food chain will continue to pose health risks given anthropogenic and natural contamination. This review briefly addresses recent epidemiological data associated with exposure to MeHg in fish-eating populations, identifies mechanisms of MeHg transport into the central nervous system (CNS), and discusses various theories on cellular processes afflicted by MeHg, which most certainly include astrocytic failure to maintain the composition of the extracellular fluid.     

Glia and methylmercury neurotoxicity

Methylmercury (MeHg) is a global environmental pollutant with significant adverse effects on human health. As the major target of MeHg, the central nervous system (CNS) exhibits the most recognizable poisoning symptoms. The role of the two major nonneuronal cell types, astrocytes and microglia, in response to MeHg exposure was recently compared. These two cell types share several common features in MeHg toxicity, but interestingly, these cells types also exhibit distinct response kinetics, indicating a cell-specific role in mediating MeHg-induced neurotoxicity. The aim of this study was to review the most recent literature and summarize key features of glial responses to this organometal.     

Necrophagy by a benthic omnivore influences biomagnification of methylmercury in fish

Omnivory has an important role in the movement of energy, nutrients, and contaminants between benthic and pelagic food webs. While top-predator fish are known to supplement a mostly piscivorous diet with benthic organisms, a more obscure benthic-pelagic coupling occurs when benthic invertebrates forage on fish carcasses, referred to as necrophagy. The combination of these two benthic-pelagic links, top-predator fish feeding on benthic organisms that have fed on dead fish, can generate a trophic feedback cycle that conserves energy and nutrients and may have implications for biomagnification of methylmercury (MeHg) in fish. We investigated the role of necrophagy by crayfish (Procambarus clarkii), via a trophic feedback cycle, on the biomagnification of MeHg in largemouth bass (Micropterus salmoides), a cosmopolitan top predator fish known to feed on crayfish. Controlled laboratory tests quantified the uptake of MeHg by both organisms from artificial and natural food (whole crayfish or bass tissue). Assimilation efficiency (AE) of MeHg was greater for bass fed crayfish (79±0.5%) than those fed artificial food (60±3%). Furthermore, AE of MeHg was greatest for largemouth bass fed crayfish that fed on MeHg-dosed dead fish (i.e., trophic feedback cycle; 94±17%). A model, parameterized with results of the laboratory experiments, was used to make steady-state projections of MeHg biomagnification factors. Model projections also indicate that MeHg biomagnification would be greatest for largemouth bass from a trophic feedback cycle. These results suggest that food web ecology has an important role in determining MeHg levels in predatory fish and underscore the need for further investigation into the magnitude that necrophagy may affect MeHg biomagnification in aquatic systems.     

Behavior of methylmercury in mammalian erythrocytes

A low molecular weight substance bound to methylmercury in the human, rabbit, mouse, and rat erythrocytes was identified, and the relationship between the distribution pattern of methylmercury in erythrocytes and the behavior of methylmercury in the blood were studied using different species of animals. Results of gel filtration using Sephadex G-15 and thin-layer chromatography suggest that the low molecular weight substance which is bound to methylmercury in erythrocytes is glutathione (GSH). The in vitro releasing rate of methylmercury taken up in erythrocytes into bovine serum albumin solution was investigated, and a remarkably low value was obtained in the case of rat erythrocytes which have an extremely smaller percentage of methylmercury bound to the low molecular weight substance compared to the other animals used. Because the considerable amount of methylmercury are bound to the low molecular weight substance in men, rabbits, and mice, it is conceivable that methylmercury bound to the low molecular weight substance is more easily released out of erythrocytes. When time-dependent changes in the methylmercury concentration in the blood taken from rats, rabbits, and mice injected sc or iv with methylmercury were evaluated, the concentration in rat blood was maintained at a very high level compared to that in rabbit or mouse blood. This seems to reflect the results of the in vitro release experiment described above.     

Interaction of methylmercury compounds with albumin

The nature of interaction between bovine serum albumin (BSA) and methylmercurial compounds has been investigated by ultrafiltration analysis. Four types of BSA samples, mercaptalbumin, its mixed disulfides with glutathione (GSH) andl-cysteine (CySH), and the S-carbami-domethylated derivative, were used for binding assays with methylmercury (MM) chloride (MMC) and three kinds of MM mercaptides of low molecular weight thiols, GSH (GS-MM), CySH (CyS-MM) and cysteinylglycine (CG-MM). Among various ligands tested, MMC showed the highest affinity for all BSA species, and the BSA-bound fraction of the ligand did not change with ligand/protein ratio. MMC strongly and stoichiometrically bound to mercaptalbumin even at a molar ratio of 11. In contrast, the albumin bound fractions of three other MM ligands increased with concomitant decrease in ligand/protein ratio and with time except for the alkylated albumin, the highest binding being shown by mercaptalbumin. Binding of S-2-nitrophenyl-glutathione, a GSH analog with a hydrophobic S-substituent, to albumin species occurred similarly to that of GS-MM. However, GSH and oxidized glutathione (GSSG) interacted differently with albumin; mercaptalbumin showed the lowest affinity for GSH, and GSSG scarcely interacted with all BSA species. These results suggest that the sulfhydryl group at Cys-34 is not the only site of BSA that interacts with MM compounds and that albumin interacts preferentially with the hydrophobic domains of a mercurial ligand rather than its hydrophilic peptide moiety.     

Disposition of methylmercury over time in a 75% nephrectomized rat model

Chronic kidney disease (CKD) is a highly relevant clinical condition that is characterized by the permanent loss of functional nephrons. Individuals with CKD may exhibit impaired renal clearance, which may alter corporal handling of metabolites and xenobiotics. Methylmercury (MeHg) is an important environmental toxicant to which humans are exposed to on a regular basis. Given the prevalence of CKD and ubiquitous presence of MeHg in the environment, it is important to understand how mercuric ions are handled in patients with CKD. Therefore, the purpose of the current study was to characterize the disposition of MeHg over time in a rat model of CKD (i.e., 75% nephrectomized (NPX) rats). Control and NPX rats were exposed intravenously (iv) to a non-nephrotoxic dose of MeHg (5 mg/kg) once daily for1, 2, or 3 d and the amount of MeHg in organs, blood, urine, and feces determined. The accumulation of MeHg in kidneys and blood of controls was significantly greater than that of NPX animals. In contrast, MeHg levels in brain and liver of controls were not markedly different from corresponding NPX rats. In all organs examined, accumulation of MeHg increased over the course of exposure, suggesting that urinary and fecal elimination are not sufficient to fully eliminate all mercuric ions. The current findings are important in that the disposition of mercuric ions in rats with normal renal function versus renal insufficiency following exposure to MeHg for a prolonged period differ and need to be taken into account with respect to therapeutic management.     

Ubiquitin-proteasome system as a factor that determine the sensitivity to methylmercury

To elucidate the mechanism of toxicity of methylmercury (MeHg), we searched for factors that determine the sensitivity of yeast cells to MeHg and found that overexpression of Cdc34 or Rad23, both proteins related to the ubiquitin-proteasome (UP) system, induces resistance to MeHg toxicity. The acquisition of resistance to MeHg in Cdc34-overexpressing yeast cells requires the ubiquitin-conjugating activity of Cdc34 and the proteolytic activity of proteasomes. Therefore, it seems likely that certain as-yet-unidentified proteins that increase MeHg toxicity might exist in cells and that the toxicity of MeHg might be reduced by the enhanced degradation of such proteins through the UP system when Cdc34 is overexpressed. Unlike Cdc34, Rad23 suppresses the degradation of ubiquitinated proteins by proteasomes. This activity of Rad23 might be involved in the acquisition of resistance to MeHg toxicity when Rad23 is overexpressed. Overexpression of Rad23 might induce resistance to MeHg by suppressing the degradation of proteins that reduce the MeHg toxicity. Moreover, when we overexpressed Cdc34 in normal and Rad23-defective yeasts, resistance to MeHg was enhanced to almost the same extent in both lines of yeast cells. Thus it is possible that the binding of Rad23 to ubiquitinated proteins might be regulated by a mechanism that involves the recognition of substrate proteins and that the functions of Rad23 might not affect the protein-degradation system in which Cdc34 is involved. Many proteins that reduce or enhance MeHg toxicity and are ubiquitinated might exist in cells. The UP system and related proteins might determine the extent of MeHg toxicity by regulating the cellular concentrations of these various proteins.     

Toxicity of ethylmercury (and Thimerosal): a comparison with methylmercury

Ethylmercury (etHg) is derived from the metabolism of thimerosal (o‐carboxyphenyl‐thio‐ethyl‐sodium salt), which is the most widely used form of organic mercury. Because of its application as a vaccine preservative, almost every human and animal (domestic and farmed) that has been immunized with thimerosal‐containing vaccines has been exposed to etHg. Although methylmercury (meHg) is considered a hazardous substance that is to be avoided even at small levels when consumed in foods such as seafood and rice (in Asia), the World Health Organization considers small doses of thimerosal safe regardless of multiple/repetitive exposures to vaccines that are predominantly taken during pregnancy or infancy. We have reviewed in vitro and in vivo studies that compare the toxicological parameters among etHg and other forms of mercury (predominantly meHg) to assess their relative toxicities and potential to cause cumulative insults. In vitro studies comparing etHg with meHg demonstrate equivalent measured outcomes for cardiovascular, neural and immune cells. However, under in vivo conditions, evidence indicates a distinct toxicokinetic profile between meHg and etHg, favoring a shorter blood half‐life, attendant compartment distribution and the elimination of etHg compared with meHg. EtHg's toxicity profile is different from that of meHg, leading to different exposure and toxicity risks. Therefore, in real‐life scenarios, a simultaneous exposure to both etHg and meHg might result in enhanced neurotoxic effects in developing mammals. However, our knowledge on this subject is still incomplete, and studies are required to address the predictability of the additive or synergic toxicological effects of etHg and meHg (or other neurotoxicants). Copyright © 2013 John Wiley & Sons, Ltd.     

Partial characterization of a low-molecular weight methylmercury complex in rat cerebrum.

Treatment of rats with methylmercury produced a stable low-molecular weight methylmercury-containing complex which could be isolated by gel chromatography from either a 10,000g supernatant or a 108,000g supernatant prepared from cerebrum. The complex appeared within 5 min after iv injection of methylmercury and persisted for at least 5 days. At all time points investigated, the low-molecular weight complex accounted for about 30% of the total cerebral soluble methylmercury burden. The complex had elution properties on gel and ion exchange chromatography similar to those found for methylmercury-glutathione. Its migration and staining in gel electrophoresis also suggested its identity as methylmercury glutathione.     

Review of the health effects of methylmercury

The study critically reviews recent data relating to the health effects of methylmercury in man and the attendant dose-response relationships. New data obtained from animal studies, including pre-and postnatal exposure, are also examined. The consumption of fish and fish produce represents the major source of methylmercury exposure in the general population. Reported mercury concentrations in fish throughout the world are examined, particularly in the Mediterranean Sea. Here there is limited knowledge of methylmercury intake in critically exposed populations such as fishermen, employees of the fish industries and their families. The measurement of mercury in hair is now regarded as the most useful indicator of exposure but more experimental data are still required to increase the value of this index. The threshold levels of methylmercury in blood, hair and for dietary intake, as estimated by the World Health Organization, have been largely endorsed. However, new information from Japan and Canada suggests the existence of a latency period for some effects, so that the frequency or probability of their occurrence is inversely related to the duration of exposure. Incorporation of such findings would therefore lead to the designation of lower threshold values than are presently recognized. Pregnant women and the fetus have been identified as groups that are at special risk. The fetal blood mercury level is up to twice that of the mother and the sensitivity of both mother and fetus may be higher than in non-pregnant adults. This should be taken into account when assigning protective threshold concentrations.     

The effect of lactation on methylmercury intoxication

Four days after parturition 17 weeks old rats of Porton Wistar strain were given 8 mg/kg mercury as methylmercury chloride for 5 days. Virgin females or mothers separated from their offspring immediately after delivery received the same treatment and served as controls. Compared with these controls, lactation delayed the onset of weight loss, shortened the time between the end of treatment and the onset of weight gain, accelerated the elimination of mercury from the whole body and prevented the development of severe co-ordination disorders. However, lactation had no detectable effect on the elimination of mercury from the brain. Moreover control and lactating females had the same degree of histological abnormalities both in the granular layer of the cerebellum and in the dorsal root ganglion cells.     

Species difference in biliary excretion of methylmercury

Species difference in the biliary excretion of methylmercury was studied in male rats, mice, rabbits and guinea pigs. The rates of mercury excretion (% dose/2 hr) into the bile of the rats, mice, rabbits and guinea pigs during the 2 hr from 2 to 4 hr after the administration of methylmercury were 0.61, 0.091, 0.036 and 0.019, respectively. These results suggest that biliary excretion and enterohepatic circulation of methylmercury in the latter three species may not influence the fate of this compound as significantly as in rats. Most of the methylmercury excreted into the bile of rats was bound to glutathione (GSH). In the mouse bile, 40% of the methylmercury was bound to GSH and the rest was found in a fraction eluted at the void volume of the Sephadex G-15 column. However, in the case of the rabbits and guinea pigs, methylmercury-GSH was scarcely detectable in the bile and almost all of the methylmercury was eluted at the void volume of the column.     

The comparative toxicology of ethyl- and methylmercury

Neurotoxicity and renotoxicity were compared in rats given by gastric gavage five daily doses of 8.0 mg Hg/kg methyl- or ethylmercuric chloride or 9.6 mg Hg/kg ethylmercuric chloride. Three or 10 days after the last treatment day rats treated with either 8.0 or 9.6 mg Hg/kg ethylmercury had higher total or organic mercury concentrations in blood and lower concentrations in kidneys and brain than methylmercury-treated rats. In each of these tissues the inorganic mercury concentration was higher after ethyl than after methylmercury.Weight loss relative to the expected body weight and renal damage was higher in ethylmercury-treated rats than in rats given equimolar doses of methylmercury. These effects became more severe when the dose of ethylmercury was increased by 20%. Thus in renotoxicity the renal concentration of inorganic mercury seems to be more important than the concentration of organic or total mercury. In methylmercury-treated rats damage and inorganic mercury deposits were restricted to the P₂ region of the proximal tubules, while in ethylmercury-treated rats the distribution of mercury and damage was more widespread.There was little difference in the neurotoxicities of methylmercury and ethylmercury when effects on the dorsal root ganglia or coordination disorders were compared. Based on both criteria, an equimolar dose of ethylmercury was less neurotoxic than methylmercury, but a 20% increase in the dose of ethylmercury was enough to raise the sum of coordination disorder scores slightly and ganglion damage significantly above those in methylmercury-treated rats.In spite of the higher inorganic mercury concentration in the brain of ethylmercurythan in the brain of methylmercury-treated rats, the granular layer damage in the cerebellum was widespread only in the methylmercury-treated rats. Thus inorganic mercury or dealkylation cannot be responsible for granular layer damage in alkylmercury intoxication. Moreover, histochemistry demonstrated no inorganic mercury deposits in the granular layer.     

Absorption of methylmercury compounds from rat intestine

Intestinal absorption of methylmercury complexed with non-protein sulfhydryl compounds (NPSHs) as occurs in bile was studied by means of direct injection of mercury compounds into ligated intestinal segments of rats. The extent of absorption of methylmercury-cysteinylglycine (MM-CysGly) was similar to that of methylmercury-cysteine (MM-Cys) and 1.5 times larger than that of methylmercury-glutathione (MM-GSH). This result suggested that MM-CysGly, which is recognized as a major component of methylmercury in rat bile, can be easily reabsorbed from the intestine. These results indicate that not only MM-GSH and MM-Cys but also MM-CysGly may play important roles in the intestinal reabsorption of methylmercury during its enterohepatic circulation.

When the ligated intestine was pretreated with probenecid and acivicin, the intestinal absorption of MM-GSH was depressed much more than in the case of treatment with acivicin alone. This indicates the possibility that there are at least two systems for intestinal transport of MM-GSH i.e. γ-glutamyltrans-peptidase (GGT)-dependent and -independent systems.     

Early morphological changes in rat cerebellum caused by a single dose of methylmercury

A single dose of 10 mg methylmercury chloride per kg body weight was given to 30 days old rats and to adult rats (180–200 g). This resulted in brain levels of 1.4–2.2 g Hg/g wet weight. In the young rats electron microscopic morphometry showed swelling of the granule cells. The extent of changes was more pronounced in the cerebellar hemispheres than in the vermais and flocculus. At 7 days after giving the methylmercury the granule cells appeared to have returned to normal. Methylmercury produced both light and electron microscopic changes in cerebellar neurons of adult (180–200 g) rats 3 days after dosing. 2.5–10% of the granule cells appeared dark and condensed in toluidine blue stained semithin sections of perfusion fixed and plastic embedded material. In control animals the comparable percentage never exceeded 1. By electron microscopic morphometry the dark cells proved to be shrunken to 70%, whereas the remaining light granule cells were swollen to 130% of the normal cell volume. The heterochromatin and mitochondrial volumes per cell remained constant in both dark and light cells from methylmercury treated animals.In the Purkinje cells from both young and adult rats, geometrical changes in the cisternae of the granulated endoplasmic reticulum were evident. The swelling and shrinkage of the granule cells is supposed to be due to impaired electrolyte control and the disorganized granulated endoplasmic reticulum of the Purkinje cells may be related to the deleterious effects on protein synthesis.     

Reappraisal of somatosensory disorders in methylmercury poisoning

The first well-documented methylmercury (MeHg) poisoning by consumption of fish arose in Minamata, Japan in 1953. MeHg had dispersed from Minamata to the Shiranui Sea. The temporal changes in MeHg in the umbilical cords indicate that residents living around that Sea had been exposed to low-dose MeHg through fish consumption for about 20 years (at least from 1950 to 1968). They have complained of paresthesia at the distal parts of the extremities and around the lip even 30 years after the cessation of exposure to anthropogenic MeHg. The thresholds of touch and two-point discrimination of those residents and Minamata disease (MD) patients were examined using the quantifiable instruments. They could perceive the stimulation of touch although their touch thresholds significantly increased in comparison to those of the control people. Their touch thresholds increased at the proximal extremities and the trunks as well as at the distal extremities. The evenly distributed increases at both distal and proximal parts revealed that the persistent somatosensory disturbances were not caused by the injuries to their peripheral nerves. The thresholds of two-point discrimination, which are associated with the function of the somatosensory cortex, increased at both forefingers and the lip in both groups. Taking into consideration that, the apraxia limb kinetics, astereognosis and disorder of active sensation, which are all associated with damage to the somatosensory cortex, were detected, it is proposed that the persisting somatosensory disorders after discontinuation of exposure to MeHg were induced by diffuse damage to the somatosensory cortex.