INHIBITION OF SUCCINATE-UBIQUINONE REDUCTASE AND DECREASE OF UBIQUINOL IN NEPHROTOXIC CYSTEINE S-CONJUGATE-INDUCED OXIDATIVE CELL INJURY

The role of complex II in the cellular protection against oxidative st ress was investigated in freshly isolated rat renal proximal tubular c ells (PTC) with the use of the nephrotoxin S-(1,2-dichlorovinyl)-L-cys teine (DCVC). DCVC caused oxidative stress in PTC as determined by flo w cytometry with dihydrorhodamine-123; this fluorescent probe is readi ly oxidized by primary hydroperoxides such as those formed during lipi d peroxidation. The oxidative stress could be prevented by inhibition of the P-lyase-mediated formation and covalent binding to cellular mac romolecules of reactive DCVC metabolites, with amino oxyacetic acid (A OA), or by the antioxidant N,N'-diphenyl-p-phenylenediamine. Both AOA and DPPD also prevented cell death. The DCVC-induced oxidative stress was associated with a decrease in the succinate:ubiquinone reductase ( SQR) activity of complex II, whereas NADH:ubiquinone reductase activit y of complex I remained unaffected. AOA prevented the effect on SQR ac tivity, whereas N,N'-diphenyl-p-phenylenediamine did not. Inhibition o f SQR activity with thenoyl trifluoracetone (TTFA) potentiated the DCV C-induced oxidative cell injury, suggesting the involvement of SQR act ivity in an antioxidant pathway. To investigate this in greater detail , PTC were treated with an inhibitor of cytochrome-c-oxidase, KCN, in a buffer containing glycine, which prevents cell death by KCN. Glycine did nor affect cell death by DCVC. KCN prevented the DCVC-induced oxi dative stress and cell death. KCN cytoprotection could be prevented by inhibition of SQR activity with oxaloacetate or TTFA, whereas inhibit ion of either complex I or III with rotenone and antimycin, respective ly, did not prevent it. The effect of DCVC on complex II was associate d with a decrease in the cellular amount of reduced ubiquinone (QH(2)) ; the KCN-mediated cytoprotection was related to a 60% increase of cel lular QH(2). Rotenone almost completely inhibited ubiquinone reduction even in the presence of KCN, whereas oxaloacetate in combination with KCN resulted in QH(2) levels comparable to control. This suggests tha t the SQR activity by complex II rather than the cellular content of r educed ubiquinone (QH(2)) is important as a part of the cellular antio xidant machinery in the cytoprotection against oxidative stress.