Menadione-induced oxidative stress in hepatocytes isolated from fed and fasted rats: the role of NADPH-regenerating pathways.
Isolated hepatocytes were prepared from fed and fasted rats and exposed to a range of menadione (2-methyl-1,4-naphthoquinone) concentrations. Menadione (300 microM) caused a rapid decline in the (NADPH)/(NADPH + NADP+) ratio from 0.85 to 0.39 within 15 min, with further decreases over the 90-min incubation period in cells isolated from fed animals. This decrease of NADPH resulted from oxidation to NADP+ since there was no loss of total pyridine nucleotide (NADP+ + NADPH) content. In addition, menadione (100 microM) caused a five-fold stimulation of the hexose monophosphate shunt by 30 min as indicated by the oxidation of [1-14C]glucose. LDH leakage was slightly but significantly elevated (30% of total) following exposure of cells to 300 microM menadione for 2 hr. Menadione caused a concentration-dependent GSH depletion: 100 microM menadione caused no depletion and 200 and 300 microM menadione caused a 75 and 95% decrease, respectively. Intracellular NADPH was significantly reduced within 30 min by 100 and 200 microM menadione but then returned to values equivalent to or greater than control by 60 min. In contrast, a sustained decrease of NADPH was produced by 300 microM menadione (5% of control after 2 hr). A marked potentiation of the oxidative cell injury produced by menadione was observed in hepatocytes prepared from 24-hr-fasted rats. LDH leakage was 50 and 95% when these cells were exposed to 100 and 200 microM menadione, respectively. Menadione (100 and 200 microM) also caused a marked GSH depletion (95% of control) by 90 min. In contrast to cells isolated from fed animals, menadione (100 and 200 microM) caused an 85% depletion of NADPH by 60 min in cells isolated from fasted rats. This potentiation of menadione-induced oxidative injury was not related to the decreased GSH content produced by fasting since menadione toxicity was not potentiated in control cells partially depleted of GSH by diethyl maleate. A further comparison was made between cells isolated from fasted rats and incubated either with or without supplemental glucose in order to determine a possible protective effect by glucose. In this comparison a significant (p less than 0.05) glucose effect was indeed observed in the direction of preventing GSH and NADPH depletion, as well as attenuating LDH leakage, when hepatocytes were exposed to either 50 or 100 microM menadione.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D004357:Drug Synergism; D005215:Fasting; D005502:Food; D005947:Glucose / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000187:drug effects*; D008297:Male; D008298:Maleates / Q000633:toxicity; D009249:NADP / Q000096:biosynthesis*; D010084:Oxidation-Reduction; D010427:Pentose Phosphate Pathway / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D014812:Vitamin K / Q000633:toxicity*
D008298:Maleates; D014812:Vitamin K; D009249:NADP; D007770:L-Lactate Dehydrogenase; C014476:diethyl maleate; D005978:Glutathione; D005947:Glucose