OXIDATION OF ACETAMINOPHEN TO ITS TOXIC QUINONE IMINE AND NONTOXIC CATECHOL METABOLITES BY BACULOVIRUS-EXPRESSED AND PURIFIED HUMAN CYTOCHROMES P450 2E1 AND 2A6

Acetaminophen (APAP), a widely used analgesic and antipyretic agent, i s bioactivated by cytochromes P450 to cause severe hepatotoxicity. APA P is oxidized by two pathways to form a toxic intermediate, N-acetyl-p -benzoquinone imine (NAPQI), and a nontoxic catechol metabolite, 3-hyd roxy-APAP (3-OH-APAP). We investigated the role of P450 2E1 and 2A6 in APAP oxidation by using baculovirus-expressed and highly purified for ms of human P450 2E1 and 2A6. An electrochemical HPLC assay was develo ped to quantify both oxidative metabolites simultaneously. For the fir st time, it was demonstrated that human P450 2E1 selectively oxidized APAP to NAPQI (assayed as its glutathione conjugate, GS-APAP), whereas human P450 2A6 selectively oxidized APAP to 3-OH-APAP. At 1 mM APAP, the relative ratio for the formation of GS-APAP vs 3-OH-APAP with huma n P450 2E1 was approximately 6:1, whereas the ratio with human P450 2A 6 was 1:3. Apparent K-m and V-max values for the formation of GS-APAP by human P450 2E1 were 1.3 mM and 6.9 nmol/min/nmol of P450, respectiv ely, whereas they were 4.6 mM and 7.9 nmol/min/nmol of P450 for P450 2 A6. Apparent K-m and V-max values for the formation of 3-OH-APAP by hu man P450 2E1 were 4.0 mM and 2.5 nmol/ min/nmol of P450, respectively, whereas they were 2.2 mM and 14.2 nmol/min/nmol of P450, respectively , for P450 2A6. Thus, although at toxic doses of APAP P450 2E1 is the more efficient catalyst for the formation of the toxic metabolite NAPQ I, P450 2A6 also can contribute significantly to NAPQI production.