Vol. 31, Issue 4, 384-391, April 2003

Inhibition and Activation of the Human Liver Microsomal and Human Cytochrome P450 3A4 Metabolism of Testosterone by Deployment-Related Chemicals

Khawja A. Usmani, Randy L. Rose, and Ernest Hodgson

Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Cytochrome P450 (P450) enzymes are major catalysts involved in the metabolism of xenobiotics and endogenous substrates such as testosterone (TST). Major TST metabolites formed by human liver microsomes include 6-hydroxytestosterone (6-OHTST), 2-hydroxytestosterone (2-OHTST), and 15-hydroxytestosterone (15-OHTST). A screen of 16 cDNA-expressed human P450 isoforms demonstrated that 94% of all TST metabolites are produced by members of the CYP3A subfamily with 6-OHTST accounting for 86% of all TST metabolites. Similar K_m values were observed for production of 6-, 2-, and 15-OHTST with human liver microsomes (HLM) and CYP3A4. However, V_max and CL_int were significantly higher for 6-OHTST than 2-OHTST (~18-fold) and 15-OHTST (~40-fold). Preincubation of HLM with a variety of ligands, including chemicals used in military deployments, resulted in varying levels of inhibition or activation of TST metabolism. The greatest inhibition of TST metabolism in HLM was following preincubation with organophosphorus compounds, including chlorpyrifos, phorate, and fonofos, with up to 80% inhibition noticed for several metabolites including 6-OHTST. Preincubation of CYP3A4 with chlorpyrifos, but not chlorpyrifos-oxon, resulted in 98% inhibition of TST metabolism. Phorate and fonofos also inhibited the production of most primary metabolites of CYP3A4. Kinetic analysis indicated that chlorpyrifos was one of the most potent inhibitors of major TST metabolites followed by fonofos and phorate. Chlorpyrifos, fonofos, and phorate inhibited major TST metabolites noncompetitively and irreversibly. Conversely, preincubation of CYP3A4 with pyridostigmine bromide increased metabolite levels of 6-OHTST and 2-OHTST. Preincubation of human aromatase (CYP19) with the test chemicals had no effect on the production of the endogenous estrogen, 17-estradiol.