Acute and Sublethal Effects of Ethylmercury Chloride on Chinese Rare Minnow (Gobiocypris rarus): Accumulation, Elimination, and Histological Changes.

Ethylmercury (EtHg) has been widely observed in the environment due to anthropogenic contamination and/or environmental ethylation of inorganic mercury. Herein, the acute and sublethal effect of EtHg chloride on Chinese rare minnow (Gobiocypris rarus) as a fish model was studied. EtHg chloride showed an obvious toxicity to 4-month-old Chinese rare minnow (LC50 24.8 µg L-1 (as Hg) at 24 h). Histological analysis revealed that acute EtHg exposure can induce necrosis, telangiectasis and exfoliation of epithelial cells in the gill, as well as edema, vacuoles, and pyknotic nuclei in hepatocytes. Sublethal dose exposure revealed a very high accumulation of EtHg in fish, which is subsequently metabolized to inorganic mercury and eliminated after depuration. A new mercury species, possibly diethylmercury, was also observed as the metabolite of EtHg in rare minnow. The present study provides useful information for assessing the risks of EtHg and understanding its bioaccumulation in aquatic organisms.

Arginine decarboxylase: A novel biological target of mercury compounds identified in PC12 cells.

Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl d-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg2+) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC50=7.96nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg>EtHg>PhHg>HgCl2), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity.

Comparative study on methyl- and ethylmercury-induced toxicity in C6 glioma cells and the potential role of LAT-1 in mediating mercurial-thiol complexes uptake.

Various forms of mercury possess different rates of absorption, metabolism and excretion, and consequently, toxicity. Methylmercury (MeHg) is a highly neurotoxic organic mercurial. Human exposure is mostly due to ingestion of contaminated fish. Ethylmercury (EtHg), another organic mercury compound, has received significant toxicological attention due to its presence in thimerosal-containing vaccines. This study was designed to compare the toxicities induced by MeHg and EtHg, as well as by their complexes with cysteine (MeHg-S-Cys and EtHg-S-Cys) in the C6 rat glioma cell line. MeHg and EtHg caused significant (p<0.0001) decreases in cellular viability when cells were treated during 30min with each mercurial following by a washing period of 24h (EC50 values of 4.83 and 5.05µM, respectively). Significant cytotoxicity (p<0.0001) was also observed when cells were treated under the same conditions with MeHg-S-Cys and EtHg-S-Cys, but the respective EC50 values were significantly increased (11.2 and 9.37µM). l-Methionine, a substrate for the l-type neutral amino acid carrier transport (LAT) system, significantly protected against the toxicities induced by both complexes (MeHg-S-Cys and EtHg-S-Cys). However, no protective effects of l-methionine were observed against MeHg and EtHg toxicities. Corroborating these findings, l-methionine significantly decreased mercurial uptake when cells were exposed to MeHg-S-Cys (p=0.028) and EtHg-S-Cys (p=0.023), but not to MeHg and EtHg. These results indicate that the uptake of MeHg-S-Cys and EtHg-S-Cys into C6 cells is mediated, at least in part, through the LAT system, but MeHg and EtHg enter C6 cells by mechanisms other than LAT system.

Potentiating effects of organomercuries on clastogen-induced chromosome aberrations in cultured Chinese hamster cells.

Mercury compounds are among the most serious environmental pollutants. In this communication, the potentiating effects of organic and inorganic mercuries on clastogen-induced chromosome aberrations were studied in Chinese hamster CHO K1 cells. Post-treatment with monoalkylated mercuries--methyl mercuric chloride (MeHgCl) and ethyl mercuric chloride (EtHgCl)--increased the number of breakage- and exchange-type aberrations induced by 4-nitroquinoline 1-oxide (4NQO) and methyl methanesulfonate. With the DNA crosslinking agents mitomycin C (MMC) and cisplatin, MeHgCl enhanced both types of aberrations while EtHgCl enhanced breakage-type aberrations only. Since these monoalkylated mercuries did not show clastogenic effects by themselves under the present experimental conditions, the increases in chromosome aberrations were not additive. Dialkylated mercuries (dimethyl mercury and diethyl mercury) and inorganic mercuries (HgCl and HgCl2) did not show any potentiating effects. When MMC- or 4NQO-treated cells were post-treated with MeHgCl during the G1 phase, both breakage- and exchange-type aberrations were enhanced. Treatment with EtHgCl during the G1 phase also enhanced both types of aberrations induced by 4NQO. With MMC, however, G1 treatment with EtHgCl did not show any potentiating effect. MeHgCl and EtHgCl treatments during the G2 phase enhanced breakage-type aberrations only. Based on these results, the following possible mechanisms for potentiation of clastogenicity by monoalkylated mercuries were suggested; (1) they interfere with repair of base lesions induced by 4NQO and MMS during the pre-replicational stage, thereby increasing unrepaired DNA lesions which convert into DNA double-strand breaks in S phase, (2) MeHgCl (but not EtHgCl) also inhibits repair of crosslinking lesions during the pre-replicational stage, and (3) their G2 effects enhance breakage-type aberrations only.

Changes in adrenocortical-pituitary activity in the catfish, Clarias batrachus (L.), after mercury treatment.

The toxic effects of mercuric chloride (HgCl2), emisan 6 (a methoxy-ethyl mercury fungicide), and methyl mercuric chloride (CH3HgCl) on the adrenocortical-pituitary activity of the catfish were investigated after 45, 90, and 180 days of exposure. The adrenocortical cells were highly stimulated in the 90-day HgCl2 group and became hyperplastic in the 180-day group. These cells were active in the 90-day emisan 6 group, with the infiltration of lymphocytes and fibrosis in the 180-day group. In the 90-day CH3HgCl gropu, the cells were active, with lymphocytic infiltration, fibrosis, and necrotic changes in some areas. In the 180-day CH3HgCl group, there was an extensive infiltration of lymphocytes, with localized sites of necrosis and hyperplasia. The ACTH cells in the pituitary were hypertrophied and degranulated in the Hg-treated groups, suggesting increased secretion of ACTH. The plasma cortisol level decreased significantly in the 90- and 180-day Hg-exposed groups in a time-dependent manner. Of the three mercurials, CH3HgCl is the most toxic in reducing the plasma cortisol level in all durations. These results suggest that Hg impairs the adrenocortical-pituitary activity of the catfish.

Behavioral effects of prenatal methoxy-ethyl-mercury chloride exposure in rat pups.

A number of neurotoxic drugs, when administered prenatally, induce neurobehavioral impairments and cause delay of the development of central nervous functions, without morphological malformations. Experiments were undertaken to clarify the behavioral teratogenicity of the fungicide methoxy-ethyl-mercury chloride (MEMC). CFY rat dams were treated with different doses of MEMC during 7th-15th days of gestation (2.0, 0.62 and 0.02 mg/kg daily), perorally. Development of gait, motor coordination, behavior patterns in an open field test, swimming, and conditioned avoidance learning were tested at different ages of rat pups. MEMC did not cause any mortality of dams, but there was a mild ataxia at the 2.0 mg/kg treatment. While birthweight, number of offspring, ear-eye opening and gait were normal, unexpectedly high mortality occurred perinatally. After weaning, open field behavior was nearly normal, there was a mild decrease of rearing, grooming and ambulation and an initial preference for the periphery of the open field decreased. Ambulation increased significantly in 90-day-old pups. Motor coordination on a rotorod decreased in 23- and 36-day-old pups, but increased in 90-day-old pups at the 2.0 mg/kg dose. There was no difference among groups in amphetamine sensitivity tested in a swim stress test. During avoidance conditioning, pups treated with the two higher doses performed poorly when compared to controls and the latency of the positive conditioned response was lengthened significantly. Our results show a dose-dependent behavioral teratogenicity of this organomercurial fungicide. The so called no effect level--as far as the neurobehavioral impairments due to prenatal exposure are concerned--is 0.02 mg/kg daily.

Toxic effects of three mercurial compounds on survival, and histology of the kidney of the catfish Clarias batrachus (L.).

In Clarias batrachus methylmercuric chloride (CH3HgCl) was 1.18 and 10.05 times more toxic than mercuric chloride (HgCl2) and emisan 6 (methoxyethyl mercury chloride), respectively, for 96 hr. A similar trend was observed at 24, 48, and 72 hr. Exposure of the catfish to sublethal concentrations of these mercurials for 14 and 28 days caused progressive changes in the kidney. The diameter of the proximal convoluted tubules was increased, the epithelial cells were hypertrophied, and the lumen was reduced and filled with secretory material and sloughed-off cells. In 14-day emisan 6-treated fish, some glomeruli were also degenerated. Exposure of the catfish to toxicologically safe concentrations of the mercurials for 90 days caused vacuolation, atrophy, and extensive damage of the tubules in HgCl2-and CH3HgCl-treated fish, and hypertrophy and vacuolation of epithelial cells of some tubules in the emisan 6-treated fish. Regeneration was noticed in the 90-day HgCl2-exposed group. After 180 days of exposure, however, histology of the kidney appeared normal in all the groups.

Effect of organomercurial poisoning on the peripheral blood and metabolite levels of a freshwater fish.

This work evaluated the hematological and biochemical changes in the fish, Puntius conchonius, under experimental organomercurial poisoning. Long-term (8 weeks) exposure to 3.63 and 6.03 mg/liter methoxyethyl mercuric chloride (MEMC) (0.2 and 0.33 fractions of 96-hr LC50) led to morphological aberrations in mature erythrocytes including nuclear and cytoplasmic deterioration, vacuolation, chromatin condensation, and hypochromia. Immature erythrocytes showing membrane leakage were also encountered. Erythrocyte count and hemoglobin (Hb) were significantly lowered after 1 and 3 weeks followed by a marginal rise persisting upto 8 weeks. Differential leucocyte counts revealed significant thrombocytopenia, lymphocytosis, and neutropenia. Collateral evaluation of blood glucose and tissue glycogen levels revealed significant hyperglycemia as well as glycogen depletion in liver and brain. Heart glycogen content evinced a substantial increase after 5 and 8 weeks exposure.

Investigations on the mutagenicity of two organomercurial pesticides, Ceresan and Agallol 3, in Drosophila melanogaster.

Two organomercurial fungicides, Ceresan containing phenyl mercury acetate and Agallol 3 containing methoxyethyl mercury chloride, were investigated for their mutagenicity in Drosophila melanogaster. The fungicides, administered by both larval and adult feeding techniques, were evaluated for their efficacy in inducing dominant lethals, sex-linked recessive lethals, and II-III translocations in the male germ cells. Both fungicides failed to induce significant dominant lethals, sex-linked recessive lethals, and II-III translocations when administered in the larval diet (P greater than 0.05). But Ceresan in the adult diet brought about a significant increase in the frequency of sex-linked recessive lethals (P less than 0.05). Broodwise analysis revealed that spermatids and spermatocytes were more sensitive than others to all the concentrations of Ceresan and Agallol 3 used. The mutagenic implications of such fungicides in the human environment are discussed.

Activity of chosen indicator enzymes in blood serum of guinea pigs exposed to ethyl- and phenylmercuric chloride alone or jointly with sodium selenite.

The effect of sodium selenite on the activity of the selected enzymes in blood serum and on mercury concentration in some tissues of guinea pigs exposed to ethyl- (EtHg) or phenylmercuric chloride (PhHg) was investigated. Every second day for a 3-month period animals were given intragastrically a solution of mercuric compounds (2.5 mg Hg/kg) with or without sodium selenite (1 mg Se/kg). The activity of malate dehydrogenase (MDH, EC 1.1.1.37), phosphohexoizomerase (PHI, EC 5.3.1.9), and gamma-glutamyltranspeptidase (GGTP, EC 2.3.2.2) in blood serum of control animals was ca. 3.8, 325, and 48 IU. After 10 weeks of exposure to EtHg and PhHg, the activities (IU) of the above enzymes were, respectively, 5.9 and 6.5 (MDH), 585 and 600 (PHI) and 211 and 86.5 (GGTP). Sodium selenite administered with mercuric compounds did not prevent in increases in enzyme activity. During the experiment the level of inorganic as well as organic mercury accumulated in kidneys and liver was estimated. After a 12-week exposure, sodium selenite decreased the level of total mercury in the liver (in the case of both EtHg and PhHg: from 47.0 to 31.8 and from 41.3 to 25.4 micrograms Hg/g tissue, respectively). It also slightly decreased the mercury level in the kidneys of animals exposed to PhHg (from 889 to 73.3 micrograms Hg/g tissue) but did not change the mercury concentration in the kidneys of guinea pigs exposed to ethylmercuric chloride.