Mechanistic studies on the metabolic activation of acetaminophen in vivo

Three analogues of acetaminophen (APAP), labeled at specific positions with either oxygen-18 or deuterium, were administered by ip injection to male BALB/c mice at the moderately hepatotoxic dose of 200 mg kg-1 in order to probe the mechanism by which APAP undergoes metabolic activation in vivo. The thioether conjugates of APAP present in bile, urine, and feces, which are believed to derive from the electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI), were isolated following aqueous-phase derivatization, separated by HPLC, and converted to a common volatile derivative for analysis by GC-MS. The observed labeling patterns of these conjugates indicated that APAP undergoes metabolism to NAPQI by a process that does not involve the generation of a free oxygenated intermediate, but which more likely entails the sequential removal of two electrons from the substrate. On the basis of these findings, an integrated metabolic scheme is proposed which invokes initial cytochrome P-450 mediated generation of a caged oxygen-centered APAP radical species. Subsequent reactions of this intermediate may account for the formation of all known oxidative metabolites of APAP.