A liver microsomal protein, previously referred to as preneoplastic antigen, from hyperplastic nodules of rats fed a diet containing 2-acetylaminofluorene has been identified as the enzyme epoxide hydrase [glycol hydro-lyase (epoxideforming), EC 4.2.1.631. Purified preneoplastic antigen from hyperp astic nodules and purified rat liver microsomal epoxide hydrase are immunochemically identical on the basis of Ouchterlony double-diffusion analysis. In addition, the purified proteins have identical minimum molecular weights in sodium dodecyl Su crylamide gels, and both proteins catalyze the hydration o arene oxides to dihydrodiols. Chronic feeding of 2-acetylaminofluorene to rats results in a 5-to 7-fold increase in epoxide hydrase activity in rat liver. The induced level of the enzyme is maintained in developing hyperplastic nodules and hepatomas but not in the nontumor tissue after removal of the carcinogen from the diet. (9) showed by immunodiffusion and immunoelectrophoresis in the presence of deoxycholate that PN antigen was present in normal rat liver at approximately one-fourth the concentration found in hyperplastic nodules. The PN antigen has been purified (9) and the present report identifies the protein as the microsomal enzyme epoxide hydrase [glycol hydro-lyase (epoxide-forming), EC 4.2.1.63].MATERIALS AND METHODS Animals. Male Holtzman rats (150-170 g) were fed a basal diet (10) with or without 0.05% AAF for a period of 13-16 weeks according to the following protocol as originally described by Epstein et al. (3): 3 weeks on basal diet containing AAF, 1 week on basal diet, 2 weeks on diet containing AAF, 2 weeks on basal diet, and 3 weeks on diet containing AAF followed by removal of the carcinogen from the diet until termination of the experiments. Intermittent feeding of AAF produces a high incidence of large hyperplastic nodules in rat liver (3). Control rats received only the basal diet.Enzymes. Animals were killed by cervical dislocation and the livers were perfused with ice-cold isotonic saline. Hyperplastic nodules were dissected out of the liver, leaving behind a rim of nodular tissue to ensure a clean separation of nodular tissue from the surrounding liver. Microsomes were prepared from hyperplastic nodules and control liver as described (8). Purification of PN antigen from hyperplastic nodule microsomes was performed by the method of Griffin and Kizer (9). Liver microsomal epoxide hydrase was purified from immature male Long-Evans rats as described by Lu et al. (11). The method for the production of antibodies against PN antigen has been described (4, 9) and that for purified liver microsomal epoxide hydrase will be described elsewhere.Assay Procedures. Ten substrates for epoxide hydrase activity were used in the present study. The synthesis and specific activity of these 3H-labeled substrates and the assay for epoxide hydrase activity have been described (12

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