Comparative study of activities in reactive oxygen species production/defense system in mitochondria of rat brain and liver, and their susceptibility to methylmercury toxicity.

The involvement of oxidative stress has been suggested as a mechanism for neurotoxicity caused by methylmercury (MeHg), but the mechanism for MeHg selective toxicity in the central nervous system is still unclear. In this research, to clarify the mechanism of selective neurotoxicity caused by MeHg, the oxygen consumption levels, the reactive oxygen species (ROS) production rates and several antioxidant levels in mitochondria were compared among the cerebrum, cerebellum and liver of male Wistar rats. In addition, the alterations of these indexes were examined in MeHg-intoxicated rats (oral administration of 10 mg/kg day, for 5 days). Although the cerebrum and cerebellum in intact rats showed higher mitochondrial oxygen consumption levels and ROS production rates than the liver, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities were much lower in the cerebrum and cerebellum than in the liver. Especially, the cerebellum showed the highest oxygen consumption and ROS production rate and the lowest mitochondrial glutathione (GSH) levels among the tissues examined. In the MeHg-treated rats, decrease in the oxygen consumption and increase in the ROS generation were found only in the cerebellum mitochondria, despite a lower Hg accumulation in the mitochondrial fraction compared to the liver. Since MeHg treatment produced an enhancement of ROS generation in cerebellum mitochondria supplemented with succinate substrates, MeHg-induced oxidative stress might affect the complex II-III mediated pathway in the electron transfer chain in the cerebellum mitochondria. Our study suggested that inborn factors, high production system activity and low defense system activity of ROS in the brain, would relate to the high susceptibility of the central nervous system to MeHg toxicity.

Mercury accumulation and its distribution to metallothionein in mouse brain after sub-chronic pulse exposure to mercury vapor.

Previously we found that exposure to mercury vapor effectively induced metallothionein (MT) biosynthesis in rat brain. Although the induction of not only MT-I/II but also MT-III was evident, the induction rate of the latter was much lower than that of the former. The brain of an MT-null mouse lacks MT-I/II, but has MT-III. Here we examined the effects of sub-chronic pulse exposure to mercury vapor on the brain MT in MT-null mice and their wild type controls. MT-null and wild type mice were preliminarily exposed to mercury vapor for 2 weeks at 0.1 mg Hg/m(3) for 1 h/day for 3 days a week, and then exposed for 11 weeks at 4.1 mg Hg/m(3) for 30 min/day for 3 days a week. This exposure caused no toxic signs such as abnormal behavior or loss of body weight gain in the mice of either strain throughout the experimental period. Twenty-four hours after the termination of the exposure, mice were sacrificed and brain samples were subjected to mercury analysis, MT assay, and pathological examination. The MT-null mice showed lower accumulation of mercury in the brain than the wild type mice. Mercury exposure resulted in a 70% increase of brain MT in the wild type mice, which was mostly accounted for by the increase in MT-I/II. On the other hand, the brain MT in the MT-null mice increased by 19%, suggesting less reactivity of the MT-III gene to mercury vapor. Although histochemical examination revealed silver-mercury grains in the cytoplasm of nerve cells and glial cells throughout the brains of both strains, no significant difference was observed between the two strains.

Alterations of metallothionein isomers in Hg(0)-exposed rat brain.

Previously we found that exposure to mercury vapor effectively induced brain metallothionein (MT) in rats. Here, using FPLC-gel chromatography, we examined time-dependent alterations in the MT isomers, MT-I/II and MT-III, following 3 weeks of exposure. Rats were exposed to mercury vapor at 8.3 mg/m3 for 15 h in total over 5 consecutive days. Total MT levels in rat cerebrum and cerebellum increased by 65% and 155%, respectively, 24 h after the final exposure. The increased levels in both tissues remained unchanged for at least 2 weeks after termination of exposure. Interestingly, most MT in control rat cerebrum and cerebellum was accounted for by MT-III, with MT-I/II being less than 10%. Through mercury vapor exposure, MT-I/II was quickly induced to a significant extent in both tissues, reaching a level comparable to that of MT-III. The induction rate of MT-I/II in the cerebellum was somewhat higher than in the cerebrum. Chromatograms showed that the MT-I/II thus induced began to decline at an early stage in both tissues. In the cerebrum, the amount of MT-I/II on day 22 was about 30% of the maximum level on day 1. On the other hand, the induction of MT-III was not that dramatic, but it did become evident, at least in the latter stage, when MT-I/II had begun to decrease. Thus, though the induction rate of MT-III was not as high as MT-I/II, it was sustained throughout the experimental period.

Reappraisal of the historic 1959 cat experiment in Minamata by the Chisso Factory.

Autopsy specimens from the historic cat experiment were recently discovered in a storage area at the Kumamoto University School of Medicine. The specimens were from an experiment prompted by physicians in the Chisso Minamata Plant following the announcement made by the Study Group for Minamata disease. On July 14, 1959 the Group announced that the disease was most likely caused by a kind of organic mercury. In order to prove or disprove that industrial waste from the Chisso Factory was the culprit in Minamata disease, a total of ten cats were fed food mixed with industrial waste produced in the acetaldehyde-producing plant. One of the ten cats, No. 717, was subsequently autopsied but the autopsy findings have never been published or recorded in the literature despite their historic significance. The rediscovered specimens were studied pathologically and biochemically, and were analyzed chemically with currently available techniques. Characteristic lesions of methylmercury poisoning were observed in the central nervous system, and the mercury levels in the cerebrum, cerebellum, liver and kidney were found to be markedly elevated in this animal.

Evaluation of methylmercury biotransformation using rat liver slices.

To examine the demethylation reaction of methylmercury (MeHg) in rat liver, slices prepared from MeHg-treated rats were incubated in L-15 medium under 95% O2/5% CO2 atmosphere. During the incubation, the amount of inorganic Hg in the slices markedly increased in a time-dependent manner, although the concentration of total Hg remained unchanged. Since the C-Hg bond in MeHg was demonstrated to be cleaved by the action of some reactive oxygen species, the effects on MeHg demethylation of several reagents that could modify reactive oxygen production were examined in the present system. Methylviologen was found to be an effective enhancer of the demethylation reaction with only a minor effect on lipid peroxidation. On the other hand, ferrous ion added to the medium showed no effect on demethylation in the presence or absence of methylviologen, although lipid peroxide levels were increased significantly by ferrous ion. Similarly, deferoxamine mesylate, which effectively suppressed the increase in lipid peroxide levels, also had no effect on demethylation. Furthermore, hydroxy radical scavengers, such as mannitol and dimethylsulfoxide, had no effect on inorganic Hg production. Rotenone, an inhibitor of complex I in the mitochondrial electron transport system, increased levels of both inorganic Hg and lipid peroxide. However, other inhibitors, such as antimycin A, myxothiazole and NaCN, significantly suppressed the demethylation reaction. Cell fractionation of the MeHg-treated rat liver revealed that the ratio of inorganic Hg to total Hg was highest in the mitochondrial fraction. Furthermore, superoxide anion could degrade MeHg in an organic solvent but not in water. These results suggested that the demethylation of MeHg by the liver slice would proceed with the aid of superoxide anion produced in the electron transfer system at the hydrophobic mitochondrial inner membrane. Furthermore, the involvement of hydroxy radicals, which have been demonstrated to be effective in cleaving the C-Hg bond in the aqueous media, might be minimal. Here, we also demonstrated that liver slices are a useful experimental model for mimicking the MeHg biotransformation reaction.

Methylmercury poisoning in common marmosets--a study of selective vulnerability within the cerebral cortex.

Neuropathological lesions found in chronic human Minamata disease tend to be localized in the calcarine cortex of occipital lobes, the pre- and postcentral lobuli, and the temporal gyri. The mechanism for the selective vulnerability is still not clear, though several hypotheses have been proposed. One hypothesis is vascular and postulates that the lesions are the result of ischemia secondary to compression of sulcal arteries from methylmercury-induced cerebral edema. To test this hypothesis, we studied common marmosets because the cerebrum of marmosets has 2 distinct deep sulci, the calcarine and Sylvian fissures. MRI analysis, mercury assays of tissue specimens, histologic and histochemical studies of the brain are reported and discussed. Brains sacrificed early after exposure to methylmercury showed high contents of methylmercury and edema of the cerebral white matter. These results may explain the selective cortical degeneration along the deep cerebral fissures or sulci.

In vivo protection of a water-soluble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat.

Methylmercury (MeHg) is a well-known neurotoxicant. MeHg-intoxication causes a disturbance in mitochondrial energy metabolism in skeletal muscle and apoptosis in cerebellum. We report the first in vivo effectiveness of antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carhoxylic acid), a water soluble vitamin E analog, against the MeHg-induced cellular responses. Treatment with Trolox (6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid) clearly protects MeHg-treated rat skeletal muscle against the decrease in mitochondrial electron transport system enzyme activities despite the retention of MeHg. Tdt-mediated dUTP nick-end-labeling method clarified that Trolox is effective for protecting cerebellum from MeHg-induced apoptosis. These data indicate that MeHg-mediated oxidative stress plays an important role in the in vivo pathological process of MeHg intoxication. Trolox may prevent some of clinical manifestations of MeHg-intoxication in humans.

Effects of dietary sulfur-containing amino acids on oxidative damage in rat liver caused by N-nitrosodimethylamine administration.

Effects of dietary protein and S-containing amino acids on oxidative damage were investigated in rat liver. After feeding rats for 3 weeks from weaning, lower GSH levels and higher metallothionein (MT) levels were found in the liver of rats fed on a 10% soyabean-protein-isolate (SPI)-based diet than those fed on a 10% casein-based diet. After injection of N-nitrosodimethylamine (NDMA) at 20 mg/kg body weight, increases in lipid peroxide, determined as thiobarbituric-acid reactive substances (TBARS), and gamma-glutamyltransferase (GGT) activity in the liver were observed in the 10% SPI diet group. By supplementing the 10% SPI diet with 0.3% cystine or methionine, GSH levels were increased, while MT levels were decreased, and elevation in TBARS levels after NDMA injection was diminished. On the other hand, increase in GGT activity could be prevented only by methionine supplement. On a 20% SPI or casein diet, TBARS concentration and GGT activity were not altered after NDMA injection with concomitant increase in GSH levels and decrease in MT levels. These results indicate that sufficient amounts of methionine and cystine in a diet are important to protect the liver from oxidative damage after NDMA administration, and GSH plays a primary role in the cellular protective function when compared with MT.

Distribution and retention of mercury in metallothionen-null mice after exposure to mercury vapor.

We studied the role of metallothionein (MT) in the distribution and retention of mercury in the brain, lung, liver and kidney of MT-null and wild-type mice after exposure to mercury (Hg0) vapor. Mice were exposed to Hg0 vapor at 5.5-6.7 mg/m3 for 3 h and killed at 1, 24, 72 or 168 h after exposure. One hour after exposure to Hg0 vapor, there were no differences in mercury concentrations in these organs from MT-null and wild-type mice. However, the elimination rate of mercury from the organs, except the brain, were remarkably faster in MT-null mice than in wild-type mice. MT-I and -II levels in the lung and kidney were increased significantly in wild-type mice but not in MT-null mice at 24 h after exposure to Hg0 vapor. At this time point, over 65% of the mercury was retained in the MT fraction of the cytosol of organs from wild-type mice. In contrast, mercury appeared mainly in the high-molecular-weight protein fractions in the cytosol of organs from MT-null mice. In the brain, a large amount of mercury was bound to MT in both strains of mice immediately after exposure. No difference was observed in the elimination rate of mercury from the brain between both strains of mice. Brain MT levels were elevated slightly in wild-type mice at 168 h after exposure but could not be detected in MT-null mice. These data suggest that no detectable MT-I and -II levels were found in the brain of MT-null mice and that mercury was apparently bound to MT-III. Using MT-null mice, we showed also that MT-III may play an important role in the retention of mercury in the brain.

Effects of N-nitrosodimethylamine (NDMA) on the oxidative status of rat liver.

To investigate oxidative effects of N-nitrosodimethylamine (NDMA) on the liver, rats were challenged by the reagent with a dose range of 10 to 40 mg/kg. With lower dose levels, protective responses were prominent, such as elevation of the hepatic glutathione and metallothionein (MT) levels. Increased activities were also evident of gamma-glutamylcysteine synthetase, glucose-6-phosphate dehydrogenase (G6PD), and malic enzyme. In the high dose range, however, toxic responses, such as increases in lipid peroxide levels in liver and serum, and glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and ketone bodies in serum became marked. Some of the protective responses became less marked at the highest dose. Catalase and glutathione peroxidase activities in the liver were also inhibited by NDMA treatment. On the other hand, when NDMA was injected as a series of doses (10 mg/kg on four separate occasions), the effects were less marked, and the hepatic levels of MT and lipid peroxide remained unchanged even after the 4th injection. Only the increase in G6PD activity was more marked after four times repeated injection than after a single injection. These results suggest that oxidative and hepatotoxic effects of NDMA are more moderate when given in repeated doses than in a single dose. In contrast to the liver, elevation of MT levels was the only detectable change in the kidney.

Pulmonary toxicity caused by acute exposure to mercury vapor is enhanced in metallothionein-null mice.

This study examined the protective role of metallothionein (MT) against pulmonary damage caused by acute exposure to metallic mercury (Hg0) vapor using MT-null and wild-type mice. Both strains of mice were exposed to Hg0 at 6.6 to 7.5 mg/m3 for 4 hr each day for 3 consecutive days. This dosing protocol was lethal to over 60% of MT-null mice but did not kill any wild-type mice. More severe pulmonary damage was found by histopathological observation in MT-null mice than in wild-type mice. MT levels in the lung were elevated in wild-type mice after Hg0 vapor exposure, and gel filtration of the lung cytosol revealed that most of the mercury was associated with MT. In MT-null mice, MT levels were below the limit of detection (0.2 microg/g tissue) for the MT assay even after exposure. After exposure to Hg0 vapor for 3 consecutive days, the pulmonary mercury levels in wild-type mice were significantly higher than in MT-null mice. These findings suggest that MT plays a protective role against the acute pulmonary toxicity of Hg0 vapor.

The effect of methylmercury on skeletal muscle in the rat: a histopathological study.

Methylmercury (MeHg)-induced neurotoxicity includes skeletal muscle symptoms (extremity weakness and wasting, muscle cramp) in addition to ataxia and disturbances of sensory and visual function. The underlying mechanisms responsible for the skeletal muscle symptoms are still poorly understood. In this study the effects of MeHg exposure on skeletal muscle were investigated in rats receiving orally administered MeHgCl at 5 mg/kg/day for 12 days. MeHg-treated rats gradually lost body weight and showed muscle weakness and wasting. Seven days after the last MeHg dose, MeHg levels in the skeletal muscle were as high as those in liver, kidney, or cerebrum. The obvious histopathological finding in skeletal muscle was a decrease in mitochondrial enzyme activity. These changes were more prominent in mitochondria-rich soleus muscle than in extensor digitorum longus muscle. Our findings confirm that MeHg exposure disturbs mitochondrial energy metabolism in skeletal muscle.

Induction by mercury compounds of brain metallothionein in rats: Hg0 exposure induces long-lived brain metallothionein.

Metallothionein (MT) is one of the stress proteins which can easily be induced by various kind of heavy metals. However, MT in the brain is difficult to induce because of blood-brain barrier impermeability to most heavy metals. In this paper, we have attempted to induce brain MT in rats by exposure to methylmercury (MeHg) or metallic mercury vapor, both of which are known to penetrate the blood-brain barrier and cause neurological damage. Rats treated with MeHg (40 micromol/kg per day x 5 days, p.o.) showed brain Hg levels as high as 18 microg/g with slight neurological signs 10 days after final administration, but brain MT levels remained unchanged. However, rats exposed to Hg vapor for 7 days showed 7-8 microg Hg/g brain tissue 24 h after cessation of exposure. At that time brain MT levels were about twice the control levels. Although brain Hg levels fell gradually with a half-life of 26 days, MT levels induced by Hg exposure remained unchanged for > 2 weeks. Gel fractionation revealed that most Hg was in the brain cytosol fraction and thus bound to MT. Hybridization analysis showed that, despite a significant increase in MT-I and -II mRNA in brain, MT-III mRNA was less affected. Although significant Hg accumulation and MT induction were observed also in kidney and liver of Hg vapor-exposed rats, these decreased more quickly than in brain. The long-lived MT in brain might at least partly be accounted for by longer half-life of Hg accumulated there. The present results showed that exposure to Hg vapor might be a suitable procedure to provide an in vivo model with enhanced brain MT.

Chronic effects of methylmercury in rats. I. Biochemical aspects.

To examine chronic effects of methylmercury (MeHg), male Wistar rats were fed on MeHg-contaminated diet, 0, 1 and 5 ppm Hg, under a restricted feeding schedule of 16 g/rat/day for 6 days a week. Rats were killed at 6-month intervals for examination of Hg accumulation, tissue levels of glutathione, metallothionein and lipid peroxide, as well as anti-oxidative enzyme activities. The survival of the 5 ppm Hg group, 50% of which died by the end of 32nd month of the exposure, was somewhat shorter than control and 1 ppm Hg groups, 50% of which survived for 34 months. Although the rats showed no neurological signs or decreased body weight gain even in 5 ppm Hg-exposed group until the end of the 2nd year, crossing of hind limb was evident after 2.5 years in all three groups. Accordingly, the neurological sign observed here possibly due to aging rather than MeHg toxicity. Tissue Hg levels showed a dose-dependent accumulation except for the kidney, where the highest Hg accumulation was observed among tissues examined. Renal Hg levels in the 1 ppm group showed about 40% of those in the 5 ppm group. Significant effects by MeHg were evident only in the kidney, where glutathione and metallothionein levels increased in both MeHg-exposed groups. However, lipid peroxide levels elevated only in 1 ppm group. Among the antioxidative enzymes examined, the renal glutathione peroxidase was found to be the most labile enzyme against MeHg exposure. Renal dysfunction suggested by increased plasma creatinine levels was also significant in 5 ppm Hg rats at 2 years. Furthermore, anemia which would be caused by reduced erythropoietin production in the kidney was also evident in this group. The present study suggested that the kidney was the most susceptible organ against MeHg toxicity under the present exposure schedule and that the renal dysfunction might at least partly account for the shortened survival in 5 ppm Hg rats.