AH RECEPTOR REGULATION OF CYP1B1 EXPRESSION IN PRIMARY MOUSE EMBRYO-DERIVED CELLS

Cytochrome P4501B1 is highly active in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic metabolites, The C3H mouse embryo fibroblast cell line, C3H10T1/2, and primary mouse embryo fibro blasts (MEFs) express CYP1B1 as the predominant cytochrome P-450 form, This is constitutively expressed and induced by 2,3,7,8-tetrachlorodi benzo-p-dioxin (TCDD) but also, to a greater extent, by PAH, To establ ish the role of the aryl hydrocarbon receptor (AhR) in the induction r esponses to PAH acid TCDD in MEFs, we measured CYP1B1 expression in pr imary MEFs generated from congenic C57b/6 mice that differ only at the AhR locus. These MEF cells express either b-1 or d-type Ah receptors, with high and low affinities for AhR agonists, respectively. Both typ es of MEFs express constitutive CYP1B1 to a similar extent, as measure d by expression of mRNA, protein, and activity (PAH metabolism), Induc tion of CYP1B1 in responsive b-1 MEFs exhibited a 5-fold lower EC50 fo r TCDD, as compared with MEFs expressing d-type AhR, This is fully con sistent with AhR mediation of the induction. Maximal induction of CYP1 B1 by 10(-8) hr TCDD was comparable in each MEF type, Very low levels of CYP1A1 mRNA, detected by reverse transcriptase-PCR, show a similar dependence on AhR phenotype in these MEFs, The expression of CYP1B1 wa s highly dependent upon the passage number of the primary MEF cultures . Paralleling decreased proliferation of these cells after passage 7 a re changes in cell morphology, the appearance of increasing numbers of differentiated cell types, and the complete loss of CYP1B1 expression . Colonies of these MEFs, which have escaped senescence, proliferate a t much later passages, regain CYP1B1 expression, and exhibit the same AhR-dependent CYP1B1 induction responses, as observed in early-passage MEFs and the C3H10T1/2 cell line. We conclude that CYP1B1 expression, including induction via the AhR, parallels a proliferative state for MEF cells.