4-Hydroxynonenal Detoxification by Mitochondrial Glutathione S-Transferase Is Compromised by Short-Term Ethanol Consumption in Rats

Abstract

Background 4-Hydroxynonenal (HNE), a toxic lipid peroxidation product, has been implicated in mitochondrial damage in rat liver by ethanol consumption. The present study assessed the effects of short-term in vivo ethanol exposure on HNE detoxification by mitochondrial glutathione S-transferase (GST). Methods Male Sprague Dawley® rats were administered 5 doses of ethanol (4 g/kg) at 12 hr intervals by gavage. Pair-fed rats that received isocaloric dextrose instead of ethanol served as controls. Mitochondrial and submitochondrial fractions were prepared from the livers. Mitochondrial contents of HNE and HNE-glutathione conjugate were measured by high-performance liquid chromatography. GST isoforms were identified by Western blots in submitochondrial fractions. Results Whereas there was an 80% increase in mitochondrial HNE content after ethanol consumption, there was a 42% decrease in the content of HNE-glutathione conjugate, compared with controls (p < 0.05). After ethanol exposure, the GST activities toward HNE in intact mitochondria and in the membranous fraction were decreased by 37% and 45% (p < 0.05), respectively, whereas that in the aqueous fraction was unchanged. Kinetic analysis of HNE conjugation by the membrane-associated GST showed that ethanol decreased the Vmax nearly by half (p < 0.05), whereas it did not affect the Km. HNE conjugation by the aqueous GST demonstrated a higher Km than that of the membrane-associated GST, although its kinetics were not significantly altered by ethanol. Immunochemical analysis with Western blots demonstrated that both the membranous and the aqueous fractions of mitochondria contain GST-α and GST-μ isoforms, whereas GST-π was absent. Conclusions HNE detoxification by mitochondrial GST is compromised by short-term ethanol consumption, which may contribute to elevated mitochondrial HNE content and hence its toxicity in the ethanol-exposed liver.