A group of monooxygenase systems collectively known as cytochrome P-450 catalyzes the oxidation of many xenobiotics, procarcinogens and steroids. Because of importance of these enzymes in drug inactivation and toxicity, a long term goal of the Laboratory has been to develop methods for predicting the metabolic profile of a new drug or xenobiotic for the various cytochrome P-450's. The development of predictive methods would involve: a) an understanding of the factors that govern the binding orientations of the substrates and b) the susceptibility of various functional groups of the substrates to be oxidized. Studies in the laboratory have developed a model to predict the relative tendency of a functional group to undergo oxidation. In order to test this model experimentally it was necessary to have a source of P-450 isozymes. In collaboration with the Laboratory of Frank Gonzales in NCI, we have employed preparations of P-450IIA expressed in Hepatoma G2 cells using Vaccinia virus as a vector. However, the maximum level or expression achieved with this system was about 0.2% of the total cellular protein. In addition, P-450IIAl was also expressed in Spodoptera frugiperda cells infected with the recombinant baculovirus containing the cDNA for P-450IIA1. The activity of these preparations could be increased nearly six-fold by the addition of hemin, mesoporphyrin IX or deuteroporphyrin IX. P-450IIA2 has also been expressed in hepatoma G2 cells using Vaccinia virus as a vector. Although P-450IIAl and P-450IIA2 are 90% identical in their amino acid sequences, their patterns of testosterone metabolites are markedly different. P-450IIA1 forms predominantly 7alpha-hydroxytestosterone, whereas P-450IIA2 forms predominantly 15alpha- hydroxytestosterone and a hither to unidentified metabolite. During the past year we have identified the unknown metabolite as 12alpha-hydroxytestosterone.