Pharmacy and Pharmaceutical Sciences
N-acetyl-m-aminophenol (3’-hydroxyacetanilide, AMAP) is the meta isomer of acetaminophen (4’-hydroxyacetanilide, APAP), the widely used analgesic that is safe at therapeutic doses but is hepatotoxic at larger doses. Unlike APAP, AMAP does not cause hepatotoxicity in mice even though AMAP and its metabolites covalently bind to hepatic proteins at levels comparable to APAP. Therefore, comparative studies with APAP and AMAP have been used in order to investigate mechanisms of toxicity and structure-toxicity relationships. However, the relationship between AMAP and CYP2E1, the enzyme generally implicated in the amplification of APAP-induced hepatotoxicity after ethanol ingestion, has not been fully elucidated. The microsomal metabolism of AMAP to reactive metabolites has been studied however, the identity of the reactive metabolite(s) of AMAP that bind to CYP2E1 has not been unequivocally determined. Therefore, we hypothesized that AMAP would covalently bind to and inhibit CYP2E1 in a reconstituted system and that mass spectral analysis would provide structural information for the reactive metabolite. Deconvoluted mass spectra indicated that a reactive metabolite of AMAP forms mono- and diadducts with CYP2E1 apoprotein (experimentally measured masses = 54622.4 ± 8.9 Da, 54791.3 ± 6.1 Da, and 54451.7 ± 5.5 Da, respectively) but not to other incubation components (i.e., heme, cytochrome b5, or cytochrome P450 reductase). NADPH was required for adduct formation while glutathione prevented it. The data indicated that reactive metabolite formation probably involves the addition of one oxygen atom to AMAP (MWAMAP = 151.2 Da; MWoxidized AMAP = 151.2 + 16.0 = 167.2 Da; experimentally determined mass of the small molecule adducted to CYP2E1 = 167.5 ± 7.1 Da. Therefore, the reactive metabolite of AMAP that covalently binds to CYP2E1 is likely formed from aromatic oxidation (quinone formation).
Harrelson, John and Nelson, Sidney D., "The Meta Isomer of Acetaminophen Is A Time Dependent Inhibitor of Human CYP2E1" (2010). Faculty Scholarship (PHRM). 40.