Acute acrylonitrile toxicity: Mechanistic studies.

摘要

Acrylonitrile is an organic compound produced in large quantities by the chemical industry. It is used as a monomer in the production of various polymers. Acrylonitrile is an acute toxin. The long-term goal of this research is to identify the mechanism responsible for the acute lethality of acrylonitrile. Several mechanisms have been proposed to explain the toxicity of acrylonitrile: GSH depletion with subsequent oxidative tissue damage, metabolism by P450 followed by release of cyanide and covalent binding to tissue proteins. GSH depletion doesn't seem to be the only factor involved in lethality because diethylmaleate, which is a potent GSH depletor, is not particularly toxic. Cyanide, although toxic by itself, is not the only mechanism involved in the acute toxicity of AN either, because preventing CN formation by P450 inhibitors does not prevent AN induced lethality. Covalent binding to tissue proteins could alter the function of vital proteins and might prove to be a major component in AN-induced toxicity. This study addresses this last mechanism.; Previous work in our laboratory has shown that AN is highly reactive with cysteine residues in proteins. Glyceraldehyde-3-phosphate dehydrogenase is a critical glycolytic enzyme with Cys149 at its active site. AN irreversibly inhibits GAPDH with a second-order rate constant of 3.8 M-1s -1 at pH 7.4 and 25°C. A combination of MALDI-TOF and ESI-MS/MS was used to show that AN covalently binds to Cys149. Impairment of glycolytic ATP production by direct inhibition of GAPDH coupled with the inhibition of mitochondrial ATP synthesis by the AN metabolite cyanide could account partially for the acute toxicity of AN in vivo. We measured energy metabolites (PCr, ATP, ADP, and AMP) by HPLC and compared the levels of energy metabolites in brains of AN treated rats to controls. Results obtained to this date suggest that while phosphocreatine levels are considerably decreased in AN treated rats, ATP levels and Energy Charge are partially preserved.; In an effort to identify other protein targets of AN, brain proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Radiolabeled spots were in-gel digested and identified by peptide mass fingerprinting using matrix-assisted laser desorbtion/ionization mass spectrometry. To date 62 AN labeled proteins have been identified by MALDI-TOF in rat brain and 47 in rat liver.; Understanding the mechanism of toxicity of acrylonitrile will help design better treatments for AN intoxication, as well as for compounds with a similar mechanism of toxicity.