Evidence that acetaminophen and N-hydroxyacetaminophen form a common arylating intermediate, N-acetyl-p-benzoquinoneimine.

TLDR: Observations provide strong evidence that the reactive intermediate formed in microsomes from acetarninophen and in solution from N-hydroxyacetaminophen is N-acetyl-p-benzoquinoneimine.

Abstract: Adding ascorbic acid to microsomal incubations containing acetaminophen inhibited covalent binding of the reactive metabolite. Adding ascorbic acid to incubations containing acetaminophen and cysteine markedly decreased acetaminophen-cysteine adduct formation. Ascorbic acid addition to aqueous incubations containing N-hydroxyacetaminophen and cysteine similarly inhibited the nonenzymatic formation of an acetaminophen-cysteine adduct. Therefore, the chemical reactions responsible for the nonenzymatic decomposition of N-hydroxyacetaminophen to yield acetaminophen-cysteine adducts were examined. In aqueous solutions above pH 7, N-hydroxyacetaminophen rapidly dehydrated to N-acetyl-p-benzoquinoneimine. In the absence of reducing compounds N-acetyl-p-benzoquinoneimine reacted with another molecule of N-hydroxyacetaminophen to give equal amounts of nitrosophenol and acetaminophen. The addition of cysteine or ascorbic acid slowed the decomposition of N-hydroxyacetaminophen and inhibited the formation of nitrosophenol. Cysteine effected these changes through decreasing the concentration of N-acetyl-p-benzoquinoneimine, primarily by reducing it to acetaminophen at low pH (5.5-7.0) or by conjugating with it to yield an acetaminophen-cysteine adduct at high pH (7.5-11.0). Ascorbic acid produced its effects only through reduction of the N-acetyl-p-benzoquinoneimine intermediate; thus acetaminophen was the only product. These observations provide strong evidence that the reactive intermediate formed in microsomes from acetarninophen and in solution from N-hydroxyacetaminophen is N-acetyl-p-benzoquinoneimine.