ABSTRACT
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.
Subject(s)
Carboxy-Lyases/antagonists & inhibitors , Environmental Pollutants/toxicity , Enzyme Inhibitors/toxicity , Methylmercury Compounds/toxicity , Models, Molecular , Nerve Tissue Proteins/antagonists & inhibitors , Neurons/drug effects , Absorption, Physiological , Agmatine/antagonists & inhibitors , Agmatine/metabolism , Animals , Arginine/metabolism , Binding Sites , Biocatalysis/drug effects , Carboxy-Lyases/chemistry , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Coordination Complexes/antagonists & inhibitors , Coordination Complexes/chemistry , Coordination Complexes/metabolism , Decarboxylation/drug effects , Environmental Pollutants/antagonists & inhibitors , Environmental Pollutants/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Ethylmercuric Chloride/antagonists & inhibitors , Ethylmercuric Chloride/metabolism , Ethylmercuric Chloride/toxicity , Gene Expression Regulation, Enzymologic/drug effects , Mercuric Chloride/antagonists & inhibitors , Mercuric Chloride/metabolism , Mercuric Chloride/toxicity , Methylmercury Compounds/antagonists & inhibitors , Methylmercury Compounds/metabolism , Molecular Dynamics Simulation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/enzymology , Neurons/metabolism , Phenylmercury Compounds/antagonists & inhibitors , Phenylmercury Compounds/metabolism , Phenylmercury Compounds/toxicity , RatsABSTRACT
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.
