APPLICANT'S ABSTRACT: There is much current interest in the role of oxidative stress and ethanol generation of reactive radical species in the mechanism(s) by which ethanol is toxic. It has been difficult to establish direct linkage between CYP2El, oxidative stress, and ethanol toxicity. Our laboratory has established a HepG2 cell line which constitutively expresses the human CYP2El. Ethanol or a polyunsaturated fatty acid (PUFA) was toxic to the E9 cells which express CYP2El, but not to control cells. Toxicity by ethanol and PUFA was prevented by antioxidants. These cells appear to be a valuable model to assess the role of CYP2El-dependent formation of reactive species such as the 1-hydroxyethyl radical (HER) in the hepatotoxic actions of ethanol. SPECIFIC Aim I is designed to study the role of HER, lipid, and other free radicals in the toxic actions of ethanol and PUFA to cells expressing CYP2El. The major analytical technique to be used is ESR spectroscopy. Studies are planned to study the mechanism of HER formation by CYP2El, and the role of HER and lipid radicals in the toxicity exerted by ethanol and PUFA; the interaction of HER with cellular constituents including adduct formation with CYP2El will be evaluated; activation of the transcription factor, NF-kB, by ethanol or CYP2El derived HER and other radical species will be determined. These studies should identify production of HER and lipid radicals in cells expressing CYP2El and assess the role of these radicals in the toxicity by ethanol and PUFA. Aim II will characterize NADPH- and NADH-dependent formation of HER, 02-, OH, and other reactive intermediates by microsomes from cell lines which express only one human P450 isoform. Toxicity by ethanol or PUFA to these cells will be evaluated, to indicate if human CYP2El is uniquely reactive in activating ethanol to HER, in formation of free radicals, and in promoting ethanol toxicity via a free radical, oxidative stress type of mechanism. The effect of NO on CYP2El catalytic activity and generation of HER and other free radicals will be evaluated in Aim Ill. If NO inhibits CYP2El, NO may prove to be useful as a protectant against the toxicity of ethanol and other toxins which are activated by CYP2El; this will be directly determined with the E9 cells. These studies will provide new information on the ability of NO to modulate CYP2El catalytic activity, generation of reactive intermediates, and ethanol and PUFA toxicity.