Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity.

Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radicals are believed to be initiators of peroxidative cell damage. This paper focused on the use of 2',7'-dichlorofluorescein-diacetate (DCFH-DA) to quantitate cerebral ROS as an index for neurotoxicity. This technique employs an assay of dichlorofluorescein (DCF), the fluorescent product of dichlorofluorescein (DCFH). Data from studies using various free radical generating systems, several iron chelators and hydroxyl radical scavengers suggest that DCFH oxidation may result in several reactive intermediates. In a biological system (synaptosomes isolated from untreated rats) DCF fluorescence was stimulated by ascorbate or FeSO4, while deferoxamine inhibited the ascorbate/FeSO4-induced stimulation of DCF formation. Two organometals, methylmercury (MeHg) and trimethyltin (TMT), known to produce neurotoxicity were tested. In vitro exposure to MeHg (10-20 microM) increased the rate of formation of ROS while TMT (5-40 microM) had no effect. In vivo, 48 hr and 1 week after a single injection of MeHg (1 mg/kg, i.p.) in mice and 1 week after a single injection of MeHg (5 mg/kg, i.p.) in rats, the rate of formation of ROS in both rat and mouse cerebellum was significantly increased. Pretreatment with deferoxamine, a potent iron-chelator, prevented MeHg-induced increase of ROS. In hippocampus and frontal cortex, ROS formation rates were also elevated 48 hr after TMT injection (3 mg/kg, i.p.) in mice. These results demonstrate that DCF fluorescence provides a good measure of overall ROS formation in synaptosomes of both in vitro as well as in vivo systems. Since ROS formation was selectively increased in areas known to be specifically vulnerable to organometals (cerebellum in the case of MeHg and hippocampus in the case of TMT), these studies further support that oxidative damage may be the primary mechanism underlying the neurotoxicity induced by these organometals.