Formation and Export of the Glutathione Conjugate of 4-Hydroxy-2,3-E-nonenal (4-HNE) in Hepatoma Cells

The cellular metabolism of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic and genotoxic product of oxidative stress-induced lipid peroxidation, was investigated in rat H35 hepatoma cells. Previous studies from our laboratory (1) have characterized the degree to which oxidative, reductive, and conjugative metabolic pathways function simultaneously during hepatocellular metabolism of 4-HNE to rapidly eliminate the compound from suspensions of freshly isolated rat hepatocytes. In the current studies, we have extended the investigation of 4-HNE metabolism to examine the pharmacokinetic parameters of 4-HNE elimination and export in a hepatoma cell line and determined that the ensuing oxidative and conjugative metabolites of 4-HNE are rapidly and efficiently transported out the cell. Low concentrations of 4-HNE (25 μM) were used in an attempt to simulate physiologically relevant conditions. The H35 hepatoma cell line studied was first evaluated for enzymes known to play important roles in the metabolism of 4-HNE and were found to possess activities for glutathioneS-transferase, aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase of 24.00 ± 1.12, 3.45 ± 0.17, and 6.44 ± 0.29 nmol min−1mg−1protein, respectively. Hepatoma cells were incubated with 25 μM 4-HNE and metabolites in intra- and extracellular fractions were quantitated by reversed-phase HPLC over the time course of treatment. Reduced glutathione (GSH) and the GSH metabolites of 4-HNE were quantitated by reversed-phase HPLC as the dinitrobenzene derivatives. Uptake of 4-HNE from the extracellular medium occurred with an estimated rate of 0.398 ± 0.181 min−1106hepatoma cells−1. The oxidative metabolite of 4-HNE, 4-hydroxy-2-nonenoic acid (HNA), produced by ALDH, appeared rapidly in the intracellular fraction achieving concentrations of 0.28 HNA nmol 106hepatoma cells−1and was efficiently eliminated with a first-order rate constant of 0.988 min−1. The GST-mediated conjugative metabolite, 3-glutathionyl-4-hydroxy-2-nonanal (4-HNE-SG), rapidly reached maximal intracellular concentrations of 1.88 ± 0.44 nmol 106hepatoma cells−1and was eliminated at a rate of 0.101 ± 0.033 min−1. Extracellular rates of formation, representing export, for HNA and 4-HNE-SG were 0.247 ± 0.045 and 0.044 ± 0.009 min−1106hepatoma cells−1, resulting in maximal extracellular concentrations for HNA and 4-HNE-SG of 0.70 ± 0.10 and 3.03 ± 0.84 nmol 106hepatoma cells−1. Approximately 75% of the administered concentration of 4-HNE was convert