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-HETE) in a homogenate of mouse epidermal cells. When the epidermal homogenate was preincubated with scavengers of reactive oxygen species (ROS), catalase or superoxide dismutase, significantly larger amounts of 12-HETE were produced as compared to untreated controls, suggesting that 12-lipoxygenase is quite prone to inactivation by ROS and peroxides. Mouse epidermal homogenate was then exposed to nine different ROS-generating systems to study the effects of superoxide, hydrogen peroxide, singlet oxygen, hypochlorite, peroxyl radicals, and alkyl hydroperoxides on the enzyme activity. Analysis by chiral phase high performance liquid chromatography demonstrated that the 12-HETE biosynthesized from arachidonic acid by mouse epidermal homogenate was the 12 (S)-enantiomer and excludes oxidation of arachidonic acid by ROS in a nonspecific free radical mechanism which leads to racemic 12-HETE. ROS generated by the interaction of xanthine with xanthine oxidase strongly inhibited 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 epidermal 12-lipoxygenase activity. The decrease in 12 (S)-HETE biosynthesis appeared to involve both superoxide and hydrogen peroxide. The efficacy of the latter species was also documented by exposure of mouse epidermal 12-lipoxygenase to glucose and glucose oxidase, which resulted in similar inhibitory effects on 12 (S)-HETE biosynthesis. The presence of the iron chelator diethylenetriaminepentaacetic acid during incubation of epidermal 12-lipoxygenase with both the xanthine/xanthine oxidase or the glucose/glucose oxidase systems partially protected the enzyme against inhibition, indicating that hydroxyl radical contributes to the overall inhibitory effect. Also, organic hydroperoxides inhibited epidermal 12-lipoxygenase, whereas singlet oxygen, hypochlorite, and 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.