Abstract：
The tire rubber antioxidant N-(1,3-dimethylbutyl)-N\'-phenyl-p-phenylenediamine (6PPD) and its quinone product (6PPDQ) are prevalent emerging contaminants, yet their biotransformation profiles remain poorly understood, hampering the assessment of environmental and health risks. This study investigated the phase-I metabolism of 6PPD and 6PPDQ across aquatic and mammalian species through in vitro liver microsome (LM) incubations and in silico simulations. A total of 40 metabolites from seven pathways were identified using the highly sensitive nano-electrospray ionization mass spectrometry. Notably, 6PPDQ was consistently detected as a 6PPD metabolite with an approximate 2% yield, highlighting biotransformation as a neglected indirect exposure pathway for 6PPDQ in organisms. 6PPDQ was calculated to form through a facile two-step phenyl hydroxylation of 6PPD, catalyzed by cytochrome P450 enzymes. Distinct species-specific metabolic kinetics were observed, with fish LM demonstrating retarded biotransformation rates for 6PPD and 6PPDQ compared to mammalian LM, suggesting the vulnerability of aquatic vertebrates to these contaminants. Intriguingly, two novel coupled metabolites were identified for 6PPD, which were predicted to exhibit elevated toxicity compared to 6PPDQ and result from C-N oxidative coupling by P450s. These unveiled metabolic profiles offer valuable insights for the risk assessment of 6PPD and 6PPDQ, which may inform future studies and regulatory actions.
摘要：
轮胎橡胶抗氧剂N-(1,3-二甲基丁基)-N'-苯基-对苯二胺(6PPD)及其醌产物(6PPDQ)是普遍存在的新兴污染物,然而，他们的生物转化概况仍然知之甚少，妨碍对环境和健康风险的评估。这项研究通过体外肝微粒体（LM）孵育和计算机模拟研究了水生和哺乳动物物种中6PPD和6PPDQ的I期代谢。使用高灵敏度的纳米电喷雾电离质谱鉴定了来自七个途径的总共40种代谢物。值得注意的是,6PPDQ始终检测为6PPD代谢物，产率约为2％，强调生物转化是生物体中6PPDQ被忽视的间接暴露途径。6PPDQ是通过容易的两步苯基羟基化6PPD计算形成的，由细胞色素P450酶催化。观察到明显的物种特异性代谢动力学，与哺乳动物LM相比，鱼类LM表现出6PPD和6PPDQ的生物转化速率延迟，表明水生脊椎动物对这些污染物的脆弱性。有趣的是，两种新的偶联代谢物被鉴定为6PPD，与6PPDQ相比，预测其毒性升高，并且是由P450的C-N氧化偶联引起的。这些公布的代谢概况为6PPD和6PPDQ的风险评估提供了有价值的见解，这可能会为未来的研究和监管行动提供信息。
