Paraquat damage of rat liver mitochondria by superoxide production depends on extramitochondrial NADH.
Pure rat liver heavy mitochondrial fractions, in which the absence of significant microsomal contamination was confirmed by electron microscopy and by the lack of glucose-6-phosphatase activity, were used to demonstrate the effect of paraquat on mitochondrial ultrastructure in the presence of external NADH. Starved mitochondria (orthodox conformation) did not show O2 uptake or structural injury from either paraquat alone or NADH alone. Marked O2 uptake and structural breakage occurred only when paraquat and NADH were added in combination. These alterations were resistant to rotenone and malate plus glutamate or NADPH could not substitute for NADH. Paraquat was reduced anaerobically by the mitochondria in the presence of NADH, but not of NADPH. The addition of superoxide dismutase, ferricytochrome c or p-benzoquinone protected against the breakage of mitochondria caused by paraquat plus NADH. These results demonstrate that mitochondria may produce paraquat radicals in the presence of extramitochondrial NADH and thus generate superoxide anion radicals, resulting in structural injury to the mitochondria, by mechanisms that may involve the mitochondrial outer membrane rather than the electron transfer chain. These mitochondrial mechanisms in paraquat toxicity seemed to be more probable in vivo than are microsomal mechanisms; the latter are postulated to function in detoxication because phenobarbital diminished paraquat toxicity and SKF 525-A or cobaltous ions enhanced the toxicity.