Cytotoxicity of menadione and related quinones in freshly isolated rat hepatocytes: effects on thiol homeostasis and energy charge

Abstract

The cytotoxic events in freshly isolated rat hepatocytes following exposure over 2 h to menadione (2-methyl-1,4-naphthoquinone) and two closely related quinones, 2,3-dimethyl-1,4-naphthoquinone (DMNQ) and 1,4-naphthoquinone (NQ), were examined. These quinones differ in their arylation capacity (NQ > menadione ≫ DMNQ) and in their potential to induce redox cycling (NQ ≈ menadione ≫ DMNQ). The glutathione status (reduced and oxidized glutathione) of the hepatocytes was determined using HPLC after derivatization with monobromobimane. Protein thiols were measured spectrophotometrically and the energy charge of the cells was determined with HPLC using ion pair chromatography. The leakage of lactate dehydrogenase was used as a marker for cell viability. All three quinones caused alterations of the glutathione status of the exposed cells but the effects were markedly different. Exposure to DMNQ resulted in a slow decrease of reduced glutathione and an increase of mixed disulfides. The other two quinones caused an almost complete depletion of reduced glutathione within 5 min. Hepatocytes exposed to NQ accumulated oxidized glutathione whereas menadione-exposed hepatocytes showed increased levels of mixed disulfides. We did not find any effects of DMNQ (200 μM) on protein thiols, energy charge or cell viability. There was a clear difference in the effects of menadione and NQ on protein thiols, energy charge and cell viability: exposure to NQ resulted in a more extensive decrease of protein thiols and energy charge and an earlier onset of lactate dehydrogenase leakage. From our results we conclude that the arylation capacity of a quinone is a determining factor in the cytotoxic potential of such compounds and that the decrease of protein thiols and of the energy charge are critical events preceding loss of cell viability.