Cytochrome P450 Enzymes Involved in Acetaminophen Activation by Rat and Human Liver Microsomes and Their Kinetics

Acetaminophen (APAP), a commonly used analgesic, is catalyzed by cytochrome P450 (P450) enzymes to a toxic intermediate which can be trapped by glutathione. Using this approach, involvement of enzymes in the activation of APAP and their kinetics were studied. With human liver microsomes, there were three apparent K_m values (approximately 10,474, and 13 000 µM) for the oxidation of APAP to its glutathione conjugate. With rat liver microsomes (control and ethanol induced) the kinetic data were best fit to a two-K_m model (approximately 30 and 1100 µM). Liver microsomes from dexamethasone (DEX)-treated female rats showed a single K_m of 56 µM and a V_max of 7500 pmol of product formed/(min·mg of protein). Antibodies specific for rat P450s 2E1 and 1A2 each inhibited approximately 40% of the APAP metabolism in control male rat microsomes. Only slight inhibition was observed with the P450 3A1/2 antibodies in control male or female rat liver microsomes. Antibodies against rat P450s 3A1/2 inhibited the activity in DEX microsomes by 80%. Antibodies inhibitory to human P450 3A4 inhibited 38% of the activity in human liver microsome sample HL107 and 76% in human microsome sample HL110. Human P450s (2A6, 2E1, 1A2, 3A4, 3A5, 3A3, 2D6, 2F1, 2C8, 2B6, and 2C9) expressed in Hep G2 cells using a vaccinia virus expression system were each tested for APAP metabolism. Of these, P450 2E1, 1A2, and 3A4 showed substantial activity, with respective K_m and V_max values of 680 µM. and 330 pmol/(min·mg) for P450 2E1 (with added cytochrome b₅), 3430 µM and 74 pmol/(min·mg) for P450 1A2, and 280 µM and 130 pmol/(min·mg) for P450 3A4. In the presence of α-naphthoflavone (α-NF), expressed P450 3A4 activity was increased 11-fold, expressed P450 2E1 activity was unaffected, and expressed 1A2 activity was inhibited 83%. With expressed P450 3A4, both the K_m and V_max for APAP oxidation were increased by α-NF. α-NF stimulated APAP oxidation 2- to 5-fold in human and control male rat liver microsomes. In the presence of α-NF, the P450 3A1/2 antibody became strongly inhibitory in control male rat liver microsomes, suggesting 3A2 activation by this flavone compound.