Deleterious effects in mice of fish-associated methylmercury contained in a diet mimicking the Western populations' average fish consumption.

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. Only a few contradictory epidemiological studies are currently available examining the impact of fish consumption on human populations. In the present study, we wanted to address whether a diet mimicking the fish consumption of Western populations could result in observable adverse effects in mice, and whether beneficial nutriments from fish were able to counterbalance the deleterious effects of MeHg, if any. In Europe and the United States, fish consumption varies widely between countries, from 11 to 100 g fish/day. A mid-range value of 25 g fish/day corresponds to a fish contribution to the total diet of 1.25% on a dry weight basis. We decided to supplement a vegetarian-based mouse diet with 1.25% of lyophilized salmon flesh (SAL diet), or 1.25% of a blend of lyophilized cod, tuna, and swordfish (CTS diet). Total mercury contents were 1.15±0.15, 2.3±0.1 and 35.75±0.15 ng Hg/g of food pellets for the control, SAL and CTS diets, respectively. After two months feeding, the CTS diet resulted in significant observable effects as compared to the control and SAL diets, encompassing decreased body growth, altered behavioral performance and increased anxiety level, modification of mitochondrial respiratory protein subunit concentrations in kidney and brain structures, modified gene expression patterns in kidneys, liver and muscles, and a decrease of dopamine concentrations in the hypothalamus and striatum. Our findings have health implications, firstly because 1.25% of CTS flesh in the diet corresponds to an average exposure to MeHg below the WHO provisory tolerable weekly intake (PTWI) (1.6 µg MeHg/kg of body weight/week), and secondly because many people in Western populations, among them women of child-bearing age, are exceeding the PTWI value (for instance, 35% of the French population inhabiting the Atlantic and Mediterranean coasts).

Protective role of metallothionein in benzo[a]pyrene-induced DNA damage.

Metallothionein (MT) is known to reduce chemical carcinogenesis. Carcinogenesis induced by benzo[a]pyrene (B[a]P) which is an environmental chemical carcinogen is related to DNA adduct formation and oxidative damage through metabolic activation. Ten-week-old male MT-I/II null mice and wild-type mice were given a single injection of B[a]P (250 mg/kg, p.o.), and B[a]P-induced DNA damage was evaluated at 6-48 hr later. The frequencies of micronucleated reticulocytes (MNRET) in MT-I/II null mice were significantly increased compared with that of wild-type mice at 48 hr after B[a]P administration. At 48 hr after B[a]P administration, comet scores were significantly increased in MT-I/II null mice but not in wild-type mice. 8-Hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, was significantly increased in liver of MT-I/II null mice at 6 and 12 hr after B[a]P administration, although that of wild-type mice was only slightly changed. Because cytochrome P450 (CYP) plays a major role in the process of B[a]P metabolic activation, we attempted to reveal the effect of MT on metabolic activation of B[a]P. Although CYP1A activities were elevated in the livers of MT-I/II null mice and wild-type mice treated with B[a]P, it was not different between both strains of mice. In addition, MT levels in the livers of wild-type mice were significantly increased by the B[a]P treatment, whereas MT was not detected in livers of MT-I/II null mice with or without B[a]P treatment. These results demonstrate that MT acts as an endogenous defensive factor against B[a]P-induced DNA damage.

Methylmercury induces neuropathological changes with tau hyperphosphorylation mainly through the activation of the c-jun-N-terminal kinase pathway in the cerebral cortex, but not in the hippocampus of the mouse brain.

Methylmercury (MeHg) is a well-known neurotoxicant inducing neuronal degeneration in the central nervous system. This in vivo study investigated the involvement of tau hyperphosphorylation in MeHg-induced neuropathological changes in the mouse brain, because abnormal tau hyperphosphorylation causes significant pathological changes associated with some neurodegenerative diseases. Mice that were administrated to 30 ppm MeHg in drinking water for 8 weeks exhibited neuropathological changes, e.g. a decrease in the number of neuron; an increase in the number of migratory astrocytes and microglia/macrophages; necrosis and apoptosis in the cerebral cortex, particularly the deep layer of primary motor cortex and prelimbic cortex. Western blotting revealed that MeHg exposure increased tau phosphorylation at Thr-205, Ser-396 and Ser-422 in the cerebral cortex, consistent with the phosphorylation patterns noted in Alzheimer's disease and frontotemporal dementia. Immunohistochemical analyses revealed that the distribution of tau-phosphorylated (Thr-205) neurons corresponded with the areas showing considerable neuropathological changes. Among the kinases and phosphatases related to tau hyperphosphorylation, the activation of mitogen-activated protein kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) was recognized. Neither neuropathological changes nor tau hyperphosphorylation was detected in the hippocampus in this study although the mercury concentration here was twice that in the cerebral cortex. These findings suggest that MeHg exposure induces tau hyperphosphorylation at specific sites of tau mainly through the activation of JNK pathways, leading to neuropathological changes in the cerebral cortex selectively, but not in the hippocampus of mouse brain.

Methylmercury exposure downregulates the expression of Racl and leads to neuritic degeneration and ultimately apoptosis in cerebrocortical neurons.

Methylmercury (MeHg) has been recognized as a neurotoxicant targeted on the central nervous system including cerebellum and cerebral cortex. Some molecular targets of MeHg have been identified using cerebellar neuronal cells, but little is known in the cerebrocortical neuronal cells. In this study, the molecular mechanism underlying MeHg-induced cell death in cerebrocortical neurons was investigated using a primary culture of embryonic rat cortical neuronal cells. The cultured cells exhibited apoptosis 3 days after exposure to 100 nM MeHg, suggesting the involvement of caspase-dependent apoptotic pathways. We demonstrated for the first time that neuritic degeneration precedes MeHg-induced apoptotic death in neurons exposed to 100 nM MeHg. Immunocytochemical and ELISA analyses for neurite-specific proteins namely, tau and MAP2, showed that injury to tau-positive axons was first induced followed by damage to the dendrites and cellular bodies. To further investigate the factors responsible for neuronal death, we investigated the expression levels of Rho-family proteins (Rac1, Cdc42, and RhoA), which regulate neuritic functions and apoptosis in neurons. Western blot analysis demonstrated that MeHg downregulated the expression levels of Rac1 and Cdc42 but did not affect RhoA. The exposure concentration and time course studies confirmed that Rac1 is targeted during an early stage of MeHg-induced cytotoxicity. The results indicate that neuritic degeneration, in particular axonal degeneration triggered by the downregulation of Rac1 expression, contributes to MeHg-induced apoptotic cell death in cultured cerebrocortical neurons.

Feeding mice with diets containing mercury-contaminated fish flesh from French Guiana: a model for the mercurial intoxication of the Wayana Amerindians.

BACKGROUND: In 2005, 84% of Wayana Amerindians living in the upper marshes of the Maroni River in French Guiana presented a hair mercury concentration exceeding the limit set up by the World Health Organization (10 microg/g). To determine whether this mercurial contamination was harmful, mice have been fed diets prepared by incorporation of mercury-polluted fish from French Guiana. METHODS: Four diets containing 0, 0.1, 1, and 7.5% fish flesh, representing 0, 5, 62, and 520 ng methylmercury per g, respectively, were given to four groups of mice for a month. The lowest fish regimen led to a mercurial contamination pressure of 1 ng mercury per day per g of body weight, which is precisely that affecting the Wayana Amerindians. RESULTS: The expression of several genes was modified with mercury intoxication in liver, kidneys, and hippocampus, even at the lowest tested fish regimen. A net genetic response could be observed for mercury concentrations accumulated within tissues as weak as 0.15 ppm in the liver, 1.4 ppm in the kidneys, and 0.4 ppm in the hippocampus. This last value is in the range of the mercury concentrations found in the brains of chronically exposed patients in the Minamata region or in brains from heavy fish consumers. Mitochondrial respiratory rates showed a 35-40% decrease in respiration for the three contaminated mice groups. In the muscles of mice fed the lightest fish-containing diet, cytochrome c oxidase activity was decreased to 45% of that of the control muscles. When mice behavior was assessed in a cross maze, those fed the lowest and mid-level fish-containing diets developed higher anxiety state behaviors compared to mice fed with control diet. CONCLUSION: We conclude that a vegetarian diet containing as little as 0.1% of mercury-contaminated fish is able to trigger in mice, after only one month of exposure, disorders presenting all the hallmarks of mercurial contamination.

[Pathological and biochemical studies of 30 Niigata autopsy cases related to Minamata disease].

OBJECTIVES: To reevaluate pathologically and biochemically 30 autopsy cases related to Minamata disease (MD) in Niigata Prefecture (NP) and compare the findings with those of autopsy cases related to MD in Kumamoto Prefecture (KP). METHODS: Recently, a set of pathological materials of these 30 autopsy cases has been sent from the Brain Research Institute at the University of Niigata to the National Institute for Minamata Disease (NIMD). The materials from each autopsy case were reexamined at the NIMD. RESULTS: There were no postnatal and fetal cases of MD in the NP autopsy materials. The contents of total mercury (T-Hg), methylmercury (Me-Hg), inorganic mercury (I-Hg) and selenium were measured in the organs of cerebrum, cerebellum, liver and kidney. The contents of T-Hg, Me-Hg and I-Hg were much higher in two cases than in controls. The pathological findings leading to the diagnosis of MD in the NP cases were essentially the same as those in KP, including the peripheral nerve lesions. In the most severely affected case of MD in NP, formation of multiple vacuoles of various sizes was observed in the cerebellar cortex, which was never encountered in the KP cases. The KP lesions were similar to that observed in an acute case of Me-Hg-treated common marmoset studied in the NIMD. CONCLUSION: The pathological features were essentially the same between the adult cases of MD in NP and KP.

Behavioral changes in metallothionein-null mice after the cessation of long-term, low-level exposure to mercury vapor.

The neurobehavioral changes in wild-type and metallothionein (MT)-null mice after the cessation of long-term, low-level exposure to Hg0 were investigated. MT-null and wild-type females were continuously (24 h/day) exposed to mercury vapor (Hg0) at 0.055 mg/m3 (range: 0.043-0.073 mg/m3), which was similar to the current threshold limit value (TLV), for 29 weeks. The effects on behavior, such as locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) were examined immediately and 12 weeks after the cessation of exposure. Immediately after the exposure had ceased, total locomotor activity in OPF was decreased in the both strain of mice, although the MT-null mice appeared to show more distinct effect. In the PA test, the exposed animals of both strains showed learning impairment as compared to un-exposed mice. Twelve weeks after the cessation of exposure, the locomotor activity in OPF was elevated in the exposed mice of both strains, while the learning ability in the PA test appeared normal in both strains. Spatial learning ability was not affected at all. Immediately after the exposure had ceased, the brain mercury concentration of the exposed wild-type mice was 1.75 microg/g, twofold of that in the MT-null mice. In 12 weeks, brain mercury levels decreased to approximately 1/20 of those in immediately after the exposure in both of the strains. These results for the first time indicated that long-term, low-level exposure to Hg0 could exert neurobehavioral effects, which were not reversible even after a long exposure-free period. Whereas the effects on learning ability were presumably transient, the effects on spontaneous behavior as evaluated in OPF were persistent. Finally, the MT-null mice seemed more susceptible to Hg0-induced neurotoxicity than the wild-type mice, confirming our previous results.

Changes in extracellular neurotransmitters in the cerebrum of familial idiopathic epileptic shetland sheepdogs using an intracerebral microdialysis technique and immunohistochemical study for glutamate metabolism.

Intracerebral microdialysis combined with electroencephalographic recordings was performed on 4 dogs of a familial idiopathic epileptic Shetland sheepdog colony to identify the kinds of neurotransmitters responsible for seizure activity. Immunohistochemistry using glutamate (Glu), glutamate transporter (GLT-1 and GLAST), and glutamine synthetase (GS) antibodies was also carried out on the cerebrum of four familial dogs that died of status epilepticus (SE). High values for extracellular levels of Glu and aspartate (ASP) were detected in association with an increased number of spikes and sharp waves during hyperventilation in 3 of 4 the familial epileptic dogs. The values of other amino acids analyzed were not altered in any of the familial epileptic dogs. Immunohistochemically, Glu-positive granules were occasionally found in the perineuronal spaces of the cerebral cortex in 3 of the familial epileptic dogs that died of SE. Immunostains for GLT-1 antibody predominantly decreased in the cerebral cortex and lateral nucleus of the thalamus in all the dogs that died of SE, whereas there were no differences detected in immunolabellings for GLAST and GS antibodies between familial epileptic dogs and controls. These results suggest that an extracellular release of both Glu and Asp may play an important role in the occurrence of seizure activity in this epileptic colony, and that a decreased expression of astrocytic GLT-1 may be related to development of SE.

Acute pulmonary toxicity caused by exposure to colloidal silica: particle size dependent pathological changes in mice.

To compare the pulmonary toxicity between ultrafine colloidal silica particles (UFCSs) and fine colloidal silica particles (FCSs), mice were intratracheally instilled with 3 mg of 14 nm UFCSs and 230 nm FCSs and pathologically examined from 30 minutes to 24 hour postexposure. Histopathologically, lungs exposed to both sizes of particles showed bronchiolar degeneration and necrosis, neutrophilic inflammation in alveoli with alveolar type II cell swelling and particle-laden alveolar macrophage accumulation. UFCSs, however, induced extensive alveolar hemorrhage compared to FCSs from 30 minutes onwards. UFCSs also caused more severe bronchiolar epithelial cell necrosis and neutrophil influx in alveoli than FCSs at 12 and 24 hours postexposure. Laminin positive immunolabellings in basement membranes of bronchioles and alveoli of UFCSs treated animals was weaker than those of FCSs-treated animals in all observation times. Electron microscopy demonstrated UFCSs and FCSs on bronchiolar and alveolar wall surface as well as in the cytoplasm of alveolar epithelial cells, alveolar macrophages and neutrophils. Type I alveolar epithelial cell erosion with basement membrane damage in UFCSs treated animals was more severe than those in FCSs-treated animals. At 12 and 24 hours postexposure, bronchiolar epithelial cells in UFCSs-treated animals showed more intense vacuolation and necrosis compared to FCSs-treated animals. These findings suggest that UFCSs have greater ability to induce lung inflammation and tissue damages than FCSs.

Neurobehavioral changes in metallothionein-null mice prenatally exposed to mercury vapor.

We studied the neurobehavioral effects of prenatal exposure of MT-null and wild-type mice to elemental mercury vapor (Hg0). Pregnant mice of both strains were repeatedly exposed to Hg0 vapor at 0.50 and 0.56 mg/m3 for 6 h/day until the 18th day of gestation. The behavioral effects were evaluated with locomotor activity in the open field, learning ability in the passive avoidance response and spatial learning ability in the Morris water maze at 12 weeks of age. Hg0-exposed MT-null mice showed a significant decrease in total locomotor activity in males, and a learning disability in the passive avoidance response and a retarded acquisition in the Morris water maze in females as compared with the control. In contrast, Hg0-exposed wild-type mice did not differ from controls in the three behavioral measurements. The results indicate that MT-null mice would be more susceptible than wild-type mice to the behavioral neurotoxicity of prenatal Hg0 exposure. Mercury concentrations in the brain of both strains were slightly higher in the exposed group than in the control group, indicating the retention of residual mercury even 12 weeks after the cessation of the exposure. Brain concentrations of mercury were also significantly higher in the exposed-females than exposed-males in either strain. From these results, we suggest that the increased susceptibility of MT-null females to behavioral changes caused by prenatal Hg0 exposure is due to a greater retention of mercury and lack of MT-I,-II in the brain.

Anaplastic ependymoma in the cervical spinal cord of a maltese dog.

An 8-year and 6-month-old female Maltese dog showed a stoop with rigidity of her cervix and back. Neurologic examination showed loss of proprioception, and deficiency of pain response. Postmortem examination revealed the neoplastic mass replacing the central area in the cervical spinal cord at the level from 4th to 5th segments. Histologically, the mass was composed of neoplastic ependymal cells. The neoplastic cells showed marked atypism, and occasionally formed ependymal rosettes. Based on the morphologic features, the tumor was diagnosed as anaplastic ependymoma. Immunohistochemistry showed that the neoplastic cells were negative for glial fibrillary acid protein, and slightly positive for vimentin and cytokeratin.

Susceptibility of metallothionein-null mice to the behavioral alterations caused by exposure to mercury vapor at human-relevant concentration.

While recent human studies suggested adverse neurobehavioral outcomes of low-level exposure to mercury vapor (Hg0) as found among those having dental amalgam fillings and dental personnel, past animal experiments only dealt with exposure at much higher mercury concentrations. The present study aimed to examine neurobehavioral effects of prolonged, low-level Hg0 exposure in mice and to evaluate the protective role of metallothionein-I,II (MT-I,II) against Hg0-induced neurotoxicity, using a knock-out strain of mice. Adult female metallothionein-I,II-null (MT-null) and wild-type OLA129/C57BL6 mice were exposed to 0.06 mg/m3 of Hg0 for 8 h per day for 23 weeks. Neurobehavioral effects were evaluated at 12 and 23 weeks of exposure using open-field test and passive avoidance test. Subcellular distribution of mercury and the induction of MT were also assessed. The Hg0 exposure resulted in significantly enhanced locomotion in the open-field test and poorer performance in the passive avoidance test at a brain Hg concentration less than 1 ppm. These effects were slightly exaggerated in MT-null mice, which showed less induction of MT, lower brain Hg concentration, and lower calculated concentration of MT-unbound cytosolic Hg. The results showed, for the first time, that a concentration of Hg0 relevant to human exposure level could cause neurobehavioral effects in adult mice. The higher susceptibility of MT-null mice suggested that MT-I,II have protective roles in the metal-induced neurobehavioral toxicity, which cannot be entirely explained by kinetic mechanisms, thus suggesting an involvement of nonkinetic mechanisms.

B-cell immunoblastic lymphoma with multinucleated giant cells in a cat.

A 10-year-old male mixed breed cat died after six months history of intermittent vomiting and weight loss. At necropsy, large white-colored foci were found in both kidneys, and whitish thickening of the gastric wall was present at the pyloric part of the stomach. Histopathological examination revealed that both lesions consisted of proliferation of large-sized neoplastic lymphocytes intermingled with multinucleated giant cells. Immunohistochemically, the neoplastic cells were positive for both B-cell antigen receptor complex (CD 79 alpha cy) and MHC class II, although multinucleated giant cells were negative. The present case was diagnosed as B-cell immunoblastic lymphoma with multinucleated giant cells.

Neospora caninum antigens recognized by mouse IgG at different stages of infection including recrudescence.

Western blotting was performed to analyze Neospora caninum tachyzoite antigens recognized by mouse IgG at different stages of infection including recrudescence. At the early stage of infection, a 36-38 kDa antigen was clearly recognized by the mouse antisera. After day 48 postinoculation, the signal of the 36-38 kDa antigen gradually weakened. Meanwhile, a 43 kDa antigen was intensely and continuously recognized from 48 to 125 days postinoculation. This 43 kDa antigen was clearly detectable with the antisera from the mice under immunosuppression. Sera from naturally infected cattle strongly reacted with the 43 kDa antigen. Therefore, the 43 kDa antigen may be useful for immunological reactions to detect infected animals except in the early stage of the infection.

Olfactory neuroepithelioma in a dog: an immunohistochemical and electron microscopic study.

A case of olfactory neuroepithelioma was investigated electron microscopically and immunohistochemically. The tumor mass was found in the nasal cavities of a 10-year-old female dog, which showed epistaxis, nasal discharge and facial swelling. The tumor tissue consisted of tubular structure of cuboidal to columnar cells and compactly arranged nests of small cells surrounded by a fibrovascular stroma. Mitotic figures were frequently observed. Immunohistochemically, the tumor cells frequently showed positive for neurofilament protein, synaptophysin and/or carnosine in addition to keratin. Ultrastructurally, tight junction was observed between the tumor cells. No dense-cored secretory granules were shown in the tumor cells. These findings indicated that the present tumor had neuronal and epithelial features probably originating from the olfactory epithelium.