The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.

The toxic and carcinogenic effects of many compounds depend on their activation to reactive molecules in the cytochrome P-450 system of the endoplasmic reticulum of cells. Changes in dietary input alter P-450 levels in different tissues and so alter toxicity. In this way, low protein diets protect against carbon tetrachloride poisoning. Fats, proteins, and nonnutrients such as flavones, antioxidants, and contaminants like DDT all affect P-450 levels. The activated molecules may be diverted from their target sites by inactivation processes, such as epoxide hydratase or glutathione trappin. This is also under nutritional control. Low protein diets render animals sensitive to acetaminophen by reducing glutathione levels. In the sequence of events leading from initial contact of toxin with organism to eventual cell injury or neoplasm, nutritional factors are of import at every stage. Assessments of the toxicity of chemicals that do not take into account the nutritional variable in man are likely to be incorrect.

The experiments described in this paper have demonstrated that hepatocytes cultured on floating collagen membranes for periods of 10 days retain their ability to respond to the inducers of drug-metabolizing enzymes, phenobarbital and methylcholanthrene, by increases in cytochromes of the cytochrome P-450 complex. Since the regulation of these cytochromes is the rate-controlling factor in the metabolism of drugs and carcinogens in hepatocytes, such experiments indicate that hepatocytes cultured on floating collagen membranes retain those functions of the liver cell responsible for the metabolism and "activation" of carcinogenic substances. The data support this hypothesis and further indicate that this system may have potential application both in the investigation of hepatocarcinogenesis by chemicals in vitro and as a screening system for the detection of substances truly carcinogenic for the mammalian organism.