Cytochrome P-450 isozyme selectivity in the oxidation of acetaminophen

Abstract

Highly purified isozymes of cytochrome P-450 catalyzed the formation of 3-glutathion-S-ylacetaminophen (GS-APAP) and 3-hydroxyacetaminophen (3-OH-APAP) from acetaminophen (APAP). A major isozyme from untreated male rats (P-450UT-A) catalyzed the formation of ca. 2.0 nmol/nmol of P-450/10 min of 3-OH-APAP and approximately 7.2 nmol of GS-APAP/nmol of P-450/10 min. Antibodies specific for cytochrome P-450UT-A caused a decrease in the amounts of both metabolites produced in microsomal incubations. In contrast to these results, two other constitutive P-450 isozymes from rat liver, cytochrome P-450UT-F and the female specific isozyme P-450UT-I, produced less of both oxidative metabolites. Moreover, they produced significantly more of the catechol metabolite than the glutathione conjugate. These results are in accord with the observation that male rats are more susceptible to acetaminophen hepatotoxicity than female rats. Isozymes induced by phenobarbital also produced more of the catechol than the glutathione conjugate. Conversely, the major isozyme induced by .beta.-naphthoflavone, cytochrome P-450.beta.NF-B, produced a significantly greater amount of GS-APAP than 3-OH-APAP. When comparison was made to a major phenobarbital inducible form (cytochrome P-450PB-B) a definite isozyme specificity for the formation of the two metabolites was seen. The catechol was formed at rates of 2.21 and 0.53 nmol/nmol of P-450/10 min by cytochromes P-450PB-B and P-450.beta.NF-B, respectively. On the other hand, cytochrome P-450PB-B produced 1.62 nmol/nmol of P-450/10 min of GS-ASAP versus 4.26 nmol/nmol of P-450/10 min for cytochrome P-450.beta.NF-B. These results show that 3-OH-APAP and GS-APAP arise primarily from different intermediates. Furthermore, a reactive intermediate in the oxidation of APAP by cytochrome P-450, N-acetyl-p-benzoquinone imine (NAPQI), was directly detected in single turnover experiments with cytochrome P-450.beta.NF-B, whereas the amount formed by cytochrome P-450PB-B was below the limits of detection for the assay.