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Expression and Inducibility of Drug-metabolizing Enzymes in Novel Murine Liver Epithelial Cell Lines and Their Ability to Activate Procarcinogens¹

Istituto di Mutagenesi e Differenziamento, C.N.R., Via Svezia 2A, 56124 Pisa, Italy

Four novel nontransformed epithelial cell lines, isolated from fetal or adult mouse liver, were tested: (a) to determine the profile of xenobiotic metabolizing enzymes; (b) to evaluate the inducibility of the polysubstrate (cytochrome P-450-dependent) monooxygenase system by various classes of inducers; and (c) to assess the capacity of the cells to metabolize structurally different procarcinogens. With regard to the phase I pathway, the cells expressed various P-450 (class IA, IA2, IIB, IIE1, IIIA) and flavin adenine dinucleotide-containing monooxygenase-dependent biotransformation enzyme activities at levels (in lines C2.8 and C6) comparable with those present in murine adult liver preparations. The expression of various P-450s was demonstrated also by immunoprecipitation assays using rabbit polyclonal antibodies. For the phase II pathway, cells expressed substantial levels of glutathione S-transferase, glutathione S-epoxide transferase, and UDP-glucuronosyltransferase. Low expression of epoxide hydrolase was observed. Induction of P-450 function by sodium phenobarbital, ß-naphthoflavone, isosafrole, ethanol, and pregnenolone 16-carbonitrile, monitored using specific P-450-linked activities, was considerably elevated (over 5-fold in class IIB with the C2.8 and C6 cell lines). The most competent C2.8 and C6 cell lines were able to activate benzo(a)pyrene, cyclophosphamide, dimethylnitrosamine, diethylstilbestrol, and 2-naphthylamine as shown by the significantly increased frequencies of mitotic gene conversion, mitotic crossing-over, and point [reverse] mutation in the diploid D₇ strain of Saccharomyces cerevisiae after 4 [cyclophosphamide], 24 [benzo(a)pyrene,2-naphthylamine, dimethylnitrosamine] or 48 [diethylstilbestrol], h of exposure in the presence of 3 x 10⁶ cells/flask. The degree of conservation and the inducibility of representative oxidative and postoxidative reactions in the novel epithelial cell lines C2.8 and C6, together with their ability to activate a wide spectrum of procarcinogens, offers a means to study the potential of chemicals for inducing DNA damage in short-term genotoxicity testing. In addition the cells may be suitable for analyzing the metabolic disposition of compounds and the multistage process of carcinogenesis.

¹ This investigation was supported in part by C.N.R. (National Research Council of Italy), special project "Environment," and by a grant from SIGMA-TAU, Pomezia, Italy. E. B. is the recipient of a fellowship from the SIGMA-TAU Foundation.

² Present address: Dipartimento di Farmacologia, Sezione di Biochimica Tossicologica, Università degli Studi, Via Irnerio 48, 40126 Bologna, Italy.

³ To whom requests for reprints should be addressed.

Received 1/17/90. Accepted 10/ 5/90.

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