Search

We have investigated the effect of paraquat (methyl viologen) on lipid peroxidation in bovine adrenal cortex mitochondria. Incubation of a buffered aerobic mixture of mitochondria in the presence of Fe2+ or NADPH resulted in the formation of lipid peroxides whose accumulation could be followed at 532 nm as malondialdehyde. Fe2+ stimulates lipid peroxidation in normal mitochondria and those in which enzymes have been inactivated with heat. In contrast, NADPH has a stimulatory effect only in normal mitochondria, but not in heat-treated mitochondria. These results indicate that NADPH-dependent lipid peroxidation is an enzymatic process. Paraquat strongly inhibits this enzymatic lipid peroxidation, but has no effect on the non-enzymatic Fe2+-dependent process. The chemiluminescence that accompanies the NADPH-dependent lipid peroxidation is also markedly decreased in the presence of paraquat. Superoxide dismutase, which removes Superoxide anion efficiently, does not inhibit malondialdehyde production. The mechanism of the inhibition of the lipid peroxidation by paraquat has been examined. Paraquat has no effect on NADPH-2,6-dichlorophenolindophenol reductase and on NADPH-cytochrome c reductase activities in bovine adrenal cortex mitochondria. However, paraquat strongly inhibits the NADPH-dependent reduction of cytochrome P-450. These results suggest that the inhibitory effect of paraquat on NADPH-dependent lipid peroxidation in adrenal cortex mitochondria is due to a decrease in the level of reduced cytochrome P-450 probably by diverting electrons from cytochrome P-450. Cytochrome c, which can compete with P-450 for available electrons from adrenodoxin, like paraquat had an inhibitory effect on NADPH-dependent lipid peroxidation. Lipid peroxidation was also strongly inhibited by steroid hydroxylase inhibitors e.g., amphenone B, aminoglutethimide and metyrapone.

1. 1. The interrelationship between progesterone (from cholesterol) biosynthesis and oxidative phosphorylation in human placental mitochondria was examined. 2. 2. ADP and ATP stimulated the malate, succinate and α-ketoglutarate-supported progesterone biosynthesis probably via the energy-dependent pyridine nucleotide transhydrogenase activation. The effect of ADP was abolished by rotenone and antimycin in the presence of malate or α-ketoglutarate. 3. 3. In the non-energized state of mitochondria malate may supported progesterone biosynthesis by the malic enzyme-dependent pathway. 4. 4. The inhibitory effects of antimycin or cyanide, and the stimulatory effect of rotenone on the succinate-supported progesterone biosynthesis indicate that the succinate to malate conversion is a necessary condition for the stimulation of progesterone biosynthesis from cholesterol. 5. 5. α-Ketoglutarate plus malonate did support progesterone biosynthesis also in the presence of ADP or ATP and to a lesser degree in the presence of DNP and rotenone. Arsenate in the presence of α-ketoglutarate, malonate, dinitrophenol and rotenone did not affect significantly progesterone biosynthesis. These results indicate that NADPH may be generated also by a non-energy-dependent transhydrogenation in placental mitochondria.

Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.