Phenanthrene (PA) is one of the most toxic and mutagenic polycyclic aromatic hydrocarbons (PAHs) in fossil fuels and a prominent PAH in byproducts of industries. Some substituted PAs such as methyl-, dimethyl- and cyclopenta- (PA) have pronounced mutagenic and tumor- initiating activity. Its lower molecular weight makes it more water soluble than the larger PAH compounds, contributing to its abundance in oil polluted water. PA requires stereoselective metabolic activation by cytochromes P450 (P450) and epoxide hydrolase (EH) to exert the mutagenic activity. Because PA is the simplest angular PAH and a known best substrate for microsomal P450, we utilized human lymphoblastoid cell lines (AHH-1) engineered to express human P450 1A1 or 1A2 and EH cDNAs, and eleven forms of P450s expressed from a vaccinia virus vector in Hep G2 cells to define the role of individual P450 in the regio- and stereoselective metabolism of PA. Turnover numbers in the metabolism of PA by 12 forms of P450s ranged from 0.2 to 55 nmol/min/nmol. Using vaccinia virus expression of P450 enzymes, mouse 1A1 (m1A1) and 1A2 (m1A2) were found to be the most active P450s. Of the human P450s, 1A2 and 2B6 have the highest activity and 2C9 has moderate activity. Human 1A1 in AHH-1 cell line had approximately twice the activity of human 1A2. The order of activity of P450s tested is similar to that for B[a]P metabolism reported recently. Regioselectivities for PA metabolism were found to be both isozyme and species dependent. Stereochemical analysis revealed that the P450s human 1A1, m1A1, m1A2, rat 2A1, rat 2B1, phenobarbital- and 3- methylcholanthrene-treated rat liver microsomes preferentially formed 3R,4R-diol enantiomer (88-97%), whereas rabbit 4B forms 1R,2R-diol enantiomer (80-96%). This is the same stereochemistry as the precursors to some diol epoxides (B[a]P diol-epoxides) that are potent carcinogens.
