EFFECTS OF REACTIVE OXYGEN SPECIES ON THE BIOSYNTHESIS OF 12(S)-HYDROXYEICOSATETRAENOIC ACID IN MOUSE EPIDERMAL HOMOGENATE

Arachidonic acid is converted to 12-hydroxyeicosatetraenoic acid (12-H ETE) in a homogenate of mouse epidermal cells. When the epidermal homo genate was preincubated with scavengers of reactive oxygen species (RO S), catalase or superoxide dismutase, significantly larger amounts of 12-HETE were produced as compared to untreated controls, suggesting th at 12-lipoxygenase is quite prone to inactivation by ROS and peroxides . Mouse epidermal homogenate was then exposed to nine different ROS-ge nerating systems to study the effects of superoxide, hydrogen peroxide , singlet oxygen, hypochlorite, peroxyl radicals, and alkyl hydroperox ides on the enzyme activity. Analysis by chiral phase high performance liquid chromatography demonstrated that the 12-HETE biosynthesized fr om arachidonic acid by mouse epidermal homogenate was the 12(S)-enanti omer and excludes oxidation of arachidonic acid by ROS in a nonspecifi c free radical mechanism which leads to racemic 12-HETE. ROS generated by the interaction of xanthine with xanthine oxidase strongly inhibit ed epidermal 12(S)-HETE biosynthesis. A flux of 0.7 nmol of superoxide /min/ml of reaction medium resulted in more than 50% inhibition of epi dermal 12-lipoxygenase activity. The decrease in 12(S)-HETE biosynthes is appeared to involve both superoxide and hydrogen peroxide. The effi cacy of the latter species was also documented by exposure of mouse ep idermal 12-lipoxygenase to glucose and glucose oxidase, which resulted in similar inhibitory effects on 12(S)-HETE biosynthesis. The presenc e of the iron chelator diethylenetriaminepentaacetic acid during incub ation of epidermal 12-lipoxygenase with both the xanthine/xanthine oxi dase or the glucose/glucose oxidase systems partially protected the en zyme against inhibition, indicating that hydroxyl radical contributes to the overall inhibitory effect. Also, organic hydroperoxides inhibit ed epidermal 12-lipoxygenase, whereas singlet oxygen, hypochlorite, an d peroxyl radicals were not effective. The results of this study lead to the proposal that 12-lipoxygenase activity may be regulated by ROS such as hydrogen peroxides, superoxide, and hydroxyl radicals. (C) 199 7 Elsevier Science Inc.