Evidence for the formation of 6PPD-quinone from antioxidant 6PPD by cytochrome P450.

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.