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.

Interaction of alkylmercuric compounds with sodium selenite. III. Biotransformation, levels of metallothioneinlike proteins and endogenous copper in some tissues of rats exposed to methyl or ethylmercuric chloride with and without sodium selenite.

The biotransformation efficiency of alkylmercurial compounds was studied in rat liver, kidneys, blood, and brain after 2-week administration of methylmercuric chloride (MeHg) and ethylmercuric chloride (EtHg) at doses of 0.25 or 2.5 mg Hg/kg, alone or in combination with sodium selenite (Se) at a level of 0.5 mg Se/kg. Simultaneously, the level of metallothioneinlike proteins (MTP) and endogenous copper (Cu) was monitored in tissues of control rats and intoxicated rats. Regardless of the dose, the highest concentrations of inorganic mercury from both the alkylmercurials was found in the rat kidneys. Sodium selenite had a variable effect on the amount of inorganic mercury liberated, depending on the organ and the molar ratio of Hg:Se administered. A statistically significant increase in the levels of MTP and endogenous Cu, compared with control group, was found only in the kidneys of intoxicated rats. This increase was dependent on the concentration of inorganic mercury liberated by biotransformation of alkylmercurials. The observed changes appeared when the level of inorganic mercury exceeded 10 micrograms Hg/g tissue and reached a plateau at about 40 micrograms Hg/g tissue. In the presence of selenium the plateau of MTP and Cu levels were no observed in the kidneys, regardless of the amount of inorganic mercury liberated.

Interaction of alkylmercuric compounds with sodium selenite. I. Metabolism of ethylmercuric chloride administered alone and in combination with sodium selenite in rats.

The effect of sodium selenite administered intragastrically in repeated doses to rats receiving ethylmercuric chloride po in various repeated doses (0.25 or 2.5 mg Hg/kg) on the excretion, whole-body retention, and organ distribution of mercury was studied. Selenium was found to affect the distribution of ethylmercury among tissues and subcellular fractions of the kidneys and liver as well as its binding to proteins of soluble fractions in these organs. Similarities and differences between the effect of interaction of sodium selenite with ethylmercuric chloride and methylmercury as well as inorganic mercury are also discussed.

Studies on the interaction and distribution of selenite, mercuric, methoxyethyl mercuric and methyl mercuric chloride in rats. I. Analysis of brain, liver, kidney and faeces.

The interaction of selenium with methyl mercury, methoxyethyl mercury and mercuric chloride was studied in a 37 days' experiment with rats. Liver, kidney, brain and faeces were analysed for mercury and seleneium at the end of the experimental period. Selenium supplementation increased the retention of mercury in liver, when mercuric chloride was given, in liver and brain when methyl mercury was given, and in all tissues examined when methoxyethyl mercury was given. The Hg/Se molar ratios in the tissues have been calculated and are found to vary considerably.

Studies on the interaction and distribution of selenite, mercuric, methoxyethyl mercuric and methyl mercuric chloride in rats. II. Analysis of the soluble proteins and the precipitates of liver and kidney homogenates.

In a 5 weeks' study rats were given mercuric, methoxyethyl mercury and methyl mercury alone or together with sodium selenite. The distribution of mercury and selenium among the soluble proteins of liver and kidneys has been investigated by gel chromatography. Estimates of the ratios of the concentration of mercury (selenium) of the soluble proteins to that of the precipitates of liver and kidney homogenates are reported. The ratios seem to decrease when selenium is given concomitant with mercury. When mercuric chloride was given alone, 50% of the mercury content in the soluble kidney proteins was found in the fractions of proteins with a molecular weight similar to that of metallothionein. No mercury could be detected in these fractions when both mercuric chloride and sodium selenite were given.

Preliminary study of the effects of feeding ethyl mercury chloride on four breeds of chickens.

Different breeds of chickens namely Single Comb White Leghorn (S.C.W.L.), New Hamsphire (N.H.), Iraqi (IRQ) and a cross (CRS.) S.C.W.L. X N.H. X IRQ. were housed in small pens (20 females and 2 males each) and given, in the diet, 40% wheat treatmed with ethyl mercury chloride, for 88 days. Throughout the whole experiment all birds remained active and showed no symptoms of toxicity. The Iraqi breed was significantly higher than the other breeds with respect to egg production. The results also indicated that mercury in egg white is almost three times as much as that in the yolk, although there was no significant difference between the breeds. The liver and kidney of the four breeds tended to accumulate the highest amount of mercury. Significant differences appeared between sexes according to liver and kidney. White Leghorn and local breeds behaved the same, but N.H. had the highest concentration of mercury in most tissues.

Purification and properties of an enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62 strain. I. Splitting enzyme 1.

An enzyme (S-1) which catalyzes the splitting of carbon-mercury linkages of organomercury compounds was purified about 24-fold from the cell-free extract of mercury-resistant Pseudomonas K-62 strain by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-150, DEAE-Sephadex, and DEAE-cellulose. A purified preparation of the enzyme showed a single band on polyacrylamide gel electrophoresis, and was colorless. The molecular weight of the enzyme was estimated to be 19,000, and Km was 5.3 X 10(-5) M for p-chloromercuribenzoic acid (PCMB). The temperature and pH optimum for the reaction were 50degrees and 7.0, respectively. The enzyme was capable of catalyzing the decomposition of methylmercuric chloride (MMC), ethylmercuric chloride (EMC), phenylmercuric acetate (PMA), and PCMB in the presence of a sulfhydryl compound to form a mercuric ion plus methane, ethane, benzene, or benzoic acid, respectively. The mercuric ion thus formed was reduced to metallic mercury by metallic mercury-releasing enzyme (MMR-enzyme).

Effects of feeding ethyl mercury chloride to chickens.

Four groups, 0, 5, 10 and 20%, of Single Comb White Leghorn chickens (30 males plus 30 females each) were fed a diet which contained either 0, 5, 10 or 20% ethyl mercury chloride dressed wheat for a period of 88 days. The wheat was dressed with the organic mercury compound at the rate of 500 gm. ethyl mercury chloride per metric ton of wheat. Therfore, the diets contained respectively 0, 25, 50 and 100 mg. organic mercury compound/kg. With average daily feed consumption of 101, 102, 101 and 98 gm. by the individual birds of the respective groups, the birds did not show any symptoms of disease during the course of the study. Egg production, egg quality and mortality of the treatment groups were comparable with those of the control group. The amount of residual mercury in egg white and yolk was determined at intervals. The residual mercury of egg white of the treatment groups was about three times as much as that of egg yolk, and made its significant appearance in the 20% group on the third day of the trial. The concentration was increasing with time in both white and yolk and was parallel to the concentration of the organic mercury in the diet. The liver followed by the kidney of both sexes accumulated the highest amounts of mercury. Tissues of female birds accumulated less mercury than tissues of male birds did probably due to the passage of some of the ingested mercury with the egg white and yolk. The results were discussed on the basis that the kind of mercury compound, daily intake and duration of treatment play major roles in the determination of induced effects.