Nair Pranav, Barrett Holly, Tanoto Kaylin, Xie Linna, Sun Jianxian, Yang Diwen, Yao Han, Song Datong, Peng Hui
Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada.
School of the Environment, University of Toronto, Toronto, ON M5S 3J1, Canada.
Environ Sci Technol. 2025 Apr 22;59(15):7474-7484. doi: 10.1021/acs.est.4c11823. Epub 2025 Apr 8.
Distinct from other nontoxic phenyl--phenylenediamine (PPD) quinones, -(1,3-dimethylbutyl)-'-phenyl--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 C tertiary carbon (C-OH-6PPD-Q). The formation of C-OH-6PPD-Q was only observed in microsomal but not in cytosolic fractions of rainbow trout () 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 C-OH-6PPD-Q, due to the weak C-H bond on the C tertiary carbon. Surprisingly, while cytotoxicity was observed for 6PPD-Q and C-OH-6PPD-Q in a coho salmon () embryo (CSE-119) cell line, no toxicity was observed for C-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 C-OH-6PPD-Q. In summary, this study highlighted the C 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.
与其他无毒的苯基 - 对苯二胺（PPD）醌不同，α - （1,3 - 二甲基丁基） - γ - 苯基 - γ - 对苯二胺 - 醌（6PPD - Q）最近被发现能在虹鳟鱼中区域选择性地代谢为烷基羟基化代谢物（烷基 - OH - 6PPD - Q）。目前尚不清楚独特的烷基 - OH - 6PPD - Q是否会导致6PPD - Q的毒性。为了验证这一点，我们在此合成了烷基 - OH - 6PPD - Q异构体的化学标准品，并研究了它们的代谢形成机制和毒性。主要的烷基 - OH - 6PPD - Q被确认为在叔碳（C - OH - 6PPD - Q）上发生羟基化。仅在虹鳟鱼肝脏S9的微粒体部分而非胞质部分中观察到C - OH - 6PPD - Q的形成。一种通用的细胞色素P450（CYP450）抑制剂氟西汀抑制了6PPD - Q羟基化代谢物的形成，这支持了CYP450催化羟基化反应。由于叔碳上的C - H键较弱，这很好地解释了C - OH - 6PPD - Q的化合物和区域选择性形成。令人惊讶的是，虽然在银大麻哈鱼胚胎（CSE - 119）细胞系中观察到6PPD - Q和C - OH - 6PPD - Q具有细胞毒性，但未观察到C - OH - 6PPD - Q的毒性。为了在生理相关条件下进一步证实这一点，我们对虹鳟鱼肝脏微粒体中形成的6PPD - Q代谢物进行了分离。观察到6PPD - Q部分具有细胞毒性，但C - OH - 6PPD - Q部分没有。总之，本研究强调了叔碳是6PPD - Q代谢和毒性的关键部分，并证实烷基羟基化是6PPD - Q的解毒途径。
