Structure and Toxicity Characterization of Alkyl Hydroxylated Metabolites of 6PPD-Q

Distinct from other nontoxic phenyl-<i>p</i>-phenylenediamine (PPD) quinones, <i>N</i>-(1,3-dimethylbutyl)-<i>N</i>'-phenyl-<i>p</i>-phenylenediamine-quinone (6PPD-Q) was recently discovered to be regioselectively metabolized to alkyl hydroxylated metabolites (alkyl-OH-6PPD-Q) in rainbow trout. It remains unknown whether the unique alkyl-OH-6PPD-Q contributes to the toxicity of 6PPD-Q. To test this, we herein synthesized chemical standards of alkyl-OH-6PPD-Q isomers and investigated their metabolic formation mechanism and toxicity. The predominant alkyl-OH-6PPD-Q was confirmed to be hydroxylated on the C₄ tertiary carbon (C₄-OH-6PPD-Q). The formation of C₄-OH-6PPD-Q was only observed in microsomal but not in cytosolic fractions of rainbow trout (<i>O. mykiss</i>) liver S9. A general cytochrome P450 (CYP450) inhibitor fluoxetine inhibited the formation of hydroxylated metabolites of 6PPD-Q, supporting that CYP450 catalyzed the hydroxylation. This well-explained the compound- and regio-selective formation of C₄-OH-6PPD-Q, due to the weak C-H bond on the C₄ tertiary carbon. Surprisingly, while cytotoxicity was observed for 6PPD-Q and C₃-OH-6PPD-Q in a coho salmon (<i>O. kisutch</i>) embryo (CSE-119) cell line, no toxicity was observed for C₄-OH-6PPD-Q. To further confirm this under physiologically relevant conditions, we fractionated 6PPD-Q metabolites formed in the liver microsome of rainbow trout. Cytotoxicity was observed for the fraction of 6PPD-Q, but not the fraction of C₄-OH-6PPD-Q. In summary, this study highlighted the C₄ tertiary carbon as the key moiety for both metabolism and toxicity of 6PPD-Q and confirmed that alkyl hydroxylation is a detoxification pathway for 6PPD-Q.