School of Environment and Spatial Informatics, China University of Mining and Technology, Xuzhou 221116, China.
School of Environment and Spatial Informatics, China University of Mining and Technology, Xuzhou 221116, China; Key Laboratory of Pollution Exposure and Health Intervention of Zhejiang Province, Zhejiang Shuren University, Hangzhou 310015, China.
J Hazard Mater. 2024 Dec 5;480:136273. doi: 10.1016/j.jhazmat.2024.136273. Epub 2024 Oct 24.
N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) as a rubber antioxidant has attracted global concern, since its ozone-oxidation product 6PPD-quinone (6PPDQ) was found to be the primary toxicant responsible for urban runoff mortality syndrome in coho salmon. However, the biotransformation fate and associated toxicological mechanism of 6PPD have not received much study yet. In this work, the in vitro assays showed 6PPD can be transformed into 6PPDQ by cytochromes P450 (CYP450) in human liver microsomes (HLMs) with 0.98 % production rate, and the adducts of 6PPDQ with calf thymus DNA and the N-N coupling product between 6PPD and 6PPDQ were further identified after 6PPD incubation in HLMs. Further evidence for the 6PPDQ formation can be obtained from the in vivo assays that the 6PPDQ-DNA adducts and 6PPD-N-N-6PPDQ dimer were detected in mice by oral gavage with 6PPD, and the latter dimer species was detected as well in 6PPD exposure to zebrafish larvae. Especially, the bioaccumulation property and high reactivity of 6PPDQ result in the continuous formation of the significant DNA adducts and 6PPD-N-N-6PPDQ dimer even in case of low production rate of biotransformation of 6PPD to 6PPDQ, which may provide potentially effective biomarkers for such process. DFT computations revealed the formation mechanism of 6PPDQ is the (N)H-abstraction of 6PPD by CYP450, followed by amino radical rebound at the nearby ortho-carbon, yielding a quinol intermediate due to spin delocalization, that might readily undergo further oxidation by CYP450 into 6PPDQ.
N-(1,3-二甲基丁基)-N'-苯基-对苯二胺（6PPD）作为一种橡胶抗氧化剂，引起了全球关注，因为其臭氧氧化产物 6PPD-醌（6PPDQ）被发现是导致银大麻哈鱼城市径流死亡率综合征的主要毒性物质。然而，6PPD 的生物转化命运和相关的毒理学机制尚未得到广泛研究。在这项工作中，体外试验表明 6PPD 可以在人肝微粒体（HLMs）中被细胞色素 P450（CYP450）转化为 6PPDQ，其生成率为 0.98%，并且在 HLMs 孵育 6PPD 后，进一步鉴定出 6PPDQ 与小牛胸腺 DNA 的加合物以及 6PPD 和 6PPDQ 之间的 N-N 偶联产物。从体内试验中也可以得到 6PPDQ 形成的进一步证据，即通过口服灌胃 6PPD 后，在小鼠中检测到 6PPDQ-DNA 加合物和 6PPD-N-N-6PPDQ 二聚体，并且在 6PPD 暴露于斑马鱼幼虫中也检测到后一种二聚体。特别是，6PPDQ 的生物累积特性和高反应性导致即使在 6PPD 生物转化为 6PPDQ 的生成率较低的情况下，也会持续形成显著的 DNA 加合物和 6PPD-N-N-6PPDQ 二聚体，这可能为该过程提供潜在有效的生物标志物。DFT 计算揭示了 6PPDQ 的形成机制是 CYP450 对 6PPD 的（N）H 抽取，随后是附近邻位碳上的氨基自由基反弹，由于自旋离域产生醌中间体，该中间体可能容易地被 CYP450 进一步氧化成 6PPDQ。
