Holey electron transport chain: aging increases mitochondrial superoxide production in rat liver

Abstract

Superoxide (O2·−) is produced by the one-electron reduction of O2 mediated by mitochondrial electron transport chains (ETC) and has been proposed to play a key role in the increased oxidative damage associated with aging. Electron paramagnetic resonance spectroscopy (EPR) was used to test the hypothesis that rat liver mitochondria produce more O2·−as a function of age. Mitochondria were isolated from livers of young (6 mo) and old (24 mo) rats (n=4). Incubations of mitochondria for determinations of O2·− production were done with ETC substrates in the presence or absence of inhibitors of Complex I or Complex III [i.e., Rotenone (ROT) or Antimycin A (AA), for I and III, respectively], which are known to increase O2·− production in isolated mitochondria. Using the spin trap DMPO, EPR analysis indicated that DMPO-OH signals were significantly greater (P<0.05) in freshly isolated mitochondria from old versus young rats when incubated with the Complex I substrate NADH alone, NADH + AA, and NADH + ROT. In contrast, the Complex II substrate succinate, succinate + AA, or succinate + ROT produced no apparent differences in DMPO-OH signals between age groups. Inclusion of superoxide dismutase in the incubation eliminated virtually all DMPO-OH signals from isolated mitochondria, confirming that the DMPO-OH signals can be attributed to mitochondrial formation of O2·−. These data suggest that O2·− arises from Complex I of isolated rat liver mitochondria and increases as a function of aging. Alteration in O2·− from the ETC could provide insight into the contribution of mitochondria to aging-associated increases in oxidative damage. Supported by NIH AG12350