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Female rats were dosed orally with one-fifth the LD50 of either 1-nitrosopiperidine (a carcinogen), cyclohexylamine, piperidine, 4-carboxy-1-nitrosopiperidine, 4-cyclohexyl-1-nitrosopiperidine or 2,6-dimethyl-1-nitrosopiperidine at 21 and 4 hr before they were killed. The five noncarcinogenic compounds had no effects on any experimental variables examined [hepatic DNA damage, ornithine decarboxylase (ODC) activity, serum alanine aminotransferase (SGPT) activity, cytochrome P-450 and glutathione content]. After administration of 40 mg/kg of 1-nitrosopiperidine, marked hepatic DNA damage and a 3- to 7-fold increase in the activity of hepatic ODC were observed. 1-Nitrosopiperidine (120 mg/kg, 3/5 LD50) caused DNA damage in rat liver and esophagus but not in leukocytes. This higher dose of 1-nitrosopiperidine also increased hepatic ornithine decarboxylase activity by 9-fold. Thus, this hepatic biochemical assay system correctly identified the one carcinogen and the five noncarcinogens in this series of six nitrogen-containing heterocycles.

Using the coupled microsomal activating/embryo culture system, in vitro experiments were performed to establish the role of metabolism in the embryo toxicity and teratogenicity of cyclophosphamide. Cyclophosphamide in the coupled microsomal activating/embryo culture system produced characteristic morphological lesions as well as a general inhibition of embryo and yolk sac growth. Increasing concentrations of NADPH in the presence of microsomes and cyclophosphamide produced progressively greater responses. These effects did not occur when microsomes and NADPH were present in the serum medium for the first 2 hours of incubation followed by one washing and then culturing of the conceptuses from hr 2 to hr 48 in a medium containing cyclophosphamide alone. Cytochrome P-450-depleted microsomes did not bioactivate cyclophosphamide to teratogenic or toxic metabolites. The results indicate that cytochrome P-450-dependent microsomal metabolism of cyclophosphamide is required for the embryotoxic and teratogenic effects observed in vitro.

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N -demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo- p -dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[ a ]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[ a ]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.

An NADPH-dependent microsomal-activating system has been coupled to a rat embryo culture in vitro . No embryonic morphological abnormalities or decreases in final yolk sac or embryo DNA and protein contents occurred when 0.2 mM NADPH was used in this coupled system. In contrast, 1.0 mM NADPH alone, or 0.2 mM NADPH in the presence of microsomes and a glucose-6-phosphate dehydrogenase-based NADPH-generating system, greatly reduced embryo and yolk sac growth in vitro . The toxicity of NADPH was not due to lipid peroxidation. Only minor decreases in final yolk sac protein levels occurred when embryos were grown in media containing male rat microsomes and 1.0 mM NADPH. The protective effect of rat hepatic microsomes on NADPH toxicity does not seem to have been due to the oxidation of NADPH to the less toxic NADP. Although cyclophosphamide alone was not toxic to rat embryos cultured in vitro , in the coupled microsomal-activating/embryo culture system, cyclophosphamide reduced yolk sac and embryo growth and caused abnormal embryonic differentiation. The uses of the coupled microsomal-activating/embryo culture system to study mechanisms in anomalous development, as well as its possible use in embryo toxicity and teratogenicity testing, are discussed.