Stable Expression of MouseCyplaland HumanCYP1A2cDNAs Transfected into Mouse Hepatoma Cells Lacking Detectable P450 Enzyme Activity

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Abstract
Using the mouse hepatoma Hepa-lclc7 c37 mutant cell line that exhibits negligible benzol[a]pyrene hydroxylase (Cyplal) and acetanilide 4-hydroxylase (Cypla2) enzyme activities, we developed stable transfectants of plasmids containing the murine Cyplal (cytochrome P1450) and the human CYP1A2 (P3450) cDNAs. We show that the assay measuring metabolism of ethoxyfluorescein ethyl ester (EFEE) was invaluable in screening large numbers of individual cell lines for high Cyplal enzyme activity. Nine different plasmid constructs containing various combinations of promoter and enhancer sequences were compared, including: the Drosophila heat shock promoter, the mouse mammary tumor virus long terminal repeat (MMTV LTR) carrying the glucocorticoid-responsive element (GRE), enhancer sequences from simian virus 40 (SV40) and herpes simplex virus type 1 (HSV-1), and the aromatic hydrocarbon-responsive domain (AhRD) of the murine Cyplal gene. Interestingly, only those constructs containing the AhRD produced high levels of Cyplal enzyme activity. In contrast, high levels of CYP1A2 activity were obtained with plasmids carrying the HSV-1 enhancer, as well as the AhRD. These studies suggest that the AhRD, which responds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), provides a post-transcriptional signal necessary for the induction of functional Cyplal enzyme activity. Although untransfected c37 cells exhibit markedly elevated levels of endogenous Cyplal mRNA, the expression of exogenous Cyplal or CYP1A2 enzyme activity in these cells decreases the concentration of this endogenous Cyplal mRNA to negligible levels and restores Cyplal mRNA inducibility by TCDD; these data indicate that the functional product of either the Cyplal gene or the CYP1A2 gene might have a role in an autoregulatory loop controlling the constitutive expression of the Cyplal gene. The cell lines described herein should be valuable in assessing the contribution of these two P450 enzymes to the processes of cytotoxicity, mutagenesis, and carcinogenesis.