TRANSFECTION OF CDNAS FOR DRUG METABOLISM INTO MAMMALIAN CELLS

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To develop more efficient in vitro systems for the study of pro-mutagenic or pro-carcinogenic chemicals, we have developed transgenic C3H/10T 1/2 cell lines expressing human cytochrome P450 (CYP) 2A6. A retroviral vector containing the cloned CYP2A6 cDNA was packaged in psi-2 cells, and used to infect C3H/10T 1/2 cells. From approximately 100 G418 resistant clones, three cell lines were chosen for further study based upon their morphologies, growth rates, and CYP2A6-dependent coumarin 7-hydroxylase activities. The 10T 1/2 cells and infected clone 10T 1/2-04 had no detectable CPY2A6 enzyme activity, whole clones 10T 1/2-10 and 10T 1/2-19 had microsomal CYP2A6 activities within the range of those found in human liver microsomes. Western analysis was in agreement with the observed enzyme activities. By Southern analysis, clone 10T 1/2-04 had one vector copy per cell while clones 10T 1/2-10 and 10T 1/2-29 had 6 and 5 copies per cell, respectively. Southern analysis also indicated the presence of endogenous CYP2A6 hybridizable DNA in the four cell lines. All exhibited about equal sensitivity to the cytotoxicity of, and induction of ouabain resistance by the direct acting mutagen N-methyl-N'-nitro-N- nitrosoguanidine. The four lines were about equally sensitive to transformation by benzo(a)pyrene, a chemical requiring metabolic activation. However, only clones 10T 1/2-10 and 10T 1/2-29, which express CYP2A6 activity, were able to be mutated and morphologically transformed by the cigarette smoke carcinogen, 4-(methylnitroso)-1-(3-pyridyl)-1-butanone, NNK. In addition, studies performed on a subset of the original 100 clones to relate enzyme activity to copy number shoed that the copy number ranged from one to eight, and that enzyme activity varied linearly with the number of copies integrated. These data, coupled with the ease of the enzyme assay, indicate that CYP2A6 should make a good reporter gene in transgenic systems. AS52 cells expressing CYP2A6 have been derived. These cells have been treated with NNK and, in collaboration with Dr. Kenneth Tindall, the mutagenic spectrum in the resultant mutated clones is being studied. These studies should improve the usefulness of cultured mammalian cells for the identification and investigation of the biological effects of environmental chemicals.