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

Distinct from other nontoxic phenyl-p-phenylenediamine (PPD) quinones, N-(1,3-dimethylbutyl)-N′-phenyl-p-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 (O. mykiss) 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 (O. kisutch) 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.