Impairment of the mitochondrial respiratory chain activity in diethylnitrosamine-induced rat hepatomas: possible involvement of oxygen free radicals.

Alterations in the energy metabolism of cancer cells have been reported for many years. However, the deleterious mechanisms involved in these deficiencies have not yet been clearly proved. The main goal of this study was to decipher the harmful mechanisms responsible for the respiratory chain deficiencies in the course of diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis, where mitochondrial DNA abnormalities had been previously reported. The respiratory activity of freshly isolated hepatoma mitochondria, assessed by oxygen consumption experiments and enzymatic assays, presented a severe complex I deficiency 19 months after DENA treatment, and later on, in addition, a defective complex III activity. Since respiratory complex subunits are encoded by both nuclear and mitochondrial genes, we checked whether the respiratory chain defects were due to impaired synthesis processes. The specific immunodetection of complex I failed to show any alterations in the steady-state levels of both nuclear and mitochondrial encoded subunits in the hepatomas. Moreover, in vitro protein synthesis experiments carried out on freshly isolated hepatoma mitochondria did not bring to light any modifications in the synthesis of the mitochondrial subunits of the respiratory complexes, whatever the degree of tumor progression. Finally, Southern blot analysis of mitochondrial DNA did not show any major mitochondrial DNA rearrangements in DENA-induced hepatomas. Because the synthetic processes of respiratory complexes did not seem to be implicated in the respiratory chain impairment, these deficiencies could be partly ascribed to a direct toxic impact of highly reactive molecules on these complexes, thus impairing their function. The mitochondrial respiratory chain is an important generator of noxious, reactive oxygen free radicals such as superoxide and H2O2, which are normally catabolized by powerful antioxidant scavengers. Nineteen months after DENA treatment, a general collapse of the antioxidant enzymatic system was demonstrated in the hepatomas, as recurrently observed in cancer cells. This oxidant versus antioxidant imbalance was characterized by the establishment of oxidative stress in the course of hepatocarcinogenesis, as partly shown by the important decrease of glutamine synthetase activity, an enzyme whose function is highly sensitive to oxidant reactions. This disequilibrium would result in a net increase of the steady-state concentration of superoxide generated between respiratory complexes I and III in the mitochondria. Once generated, superoxide would likely inactivate complexes I and III via oxidant reactions on their superoxide-sensitive [4Fe, 4S] clusters. The role of mitochondrial respiratory chain impairment in chemical carcinogenesis and/or the persistence of the cancerous state is further discussed.