Biotransformation of 6PPD-quinone In Vitro Using RTL-W1 Cell Line

Abstract

Urban stormwater runoff contains the tire-derived transformation product N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone), which poses significant environmental risks due to its high toxicity toward certain salmonids. 6PPD-quinone biotransformation has been investigated to explain some of the stark interspecies differences in sensitivity across different fishes; however, the primary mechanisms of 6PPDquinone biotransformation remain unclear. This work aimed to explore the toxicokinetics of 6PPD-quinone in immortalized rainbow trout (Oncorhynchus mykiss) liver cells (RTL-W1) to identify transformation products, using coexposure with different enzyme inhibitors and inducers. Using high-resolution mass spectrometry, we identified three phase I 6PPD-quinone transformation products, with phenyl ring hydroxylation dominating, followed by hydroxylation of the alkyl side chain, and an unknown transformation product after 4 h of exposure. Co-exposing RTL-W1 cells with α-naphthoflavone and quercetin greatly inhibited the biotransformation of 6PPD-quinone, revealing that CYP1A is primarily involved in phase I biotransformation. Hepatic clearance predicted from in vitro results was further verified based on isolated perfused trout liver experiments. Further studies are necessary on the biotransformation and kinetics of 6PPD-quinone and the detoxification pathways involved in a wide phylogenetic space in fishes.

Xenometabolome of Early-Life Stage Salmonids Exposed to 6PPD-Quinone

“…The two monohydroxylated TPs ( m/z = 315.17) included an alkyl sidechain hydroxylation (TP-OH1, RT = 13.45) and a phenyl ring hydroxylation (TP-OH2, RT = 14.56), both of which have been previously described. 11 TP-OH1 was only detected in the more sensitive fish species for the 2-2.5 µg/L exposure bin (Figure 1D), with BRT only having TP-OH2 present. The dihydroxylated TP ( m/z = 331.17) showed identical fragmentation patterns for all species, indicating a phenyl ring hydroxylation and alkyl sidechain hydroxylation, and was found to be greatest in RBT (averaged sum; 3.30x10 4 arbitrary units, au), followed by LKT (5.74 × 10 2 au) and BRT (4.31 × 10 2 au) (Figure 1D).…”

“…This provides additional evidence that the preferred phenyl ring monohydroxylation occurs at the para position due to increased availability for CYP biotransformation and decreased steric hindrance. 11 Phase I to phase II normalized relative abundance ratio values indicated that RBT had the lowest capacity to conjugate hydroxylated TPs (Figure 1C), followed by LKT. BRT had the greatest ability to biotransform hydroxylated TPs further through phase II conjugation, which could have important implications for the differences in the reported species-specific sensitivities to 6PPD-Q.…”

“…The one sulfonate-conjugated dihydroxylated TP was likely O -sulfonated at the phenyl ring hydroxylation, with an MS 2 fragment of m/z = 337.05, indicating that the other hydroxylation event occurred on the methyl carbon adjacent to the amine, as previously predicted (Figure 2, Supplementary Figure S4). 11…”

“…29 Several new TPs were observed and identified using Biotransformer 3.0 and CFM-ID for spectral verification, along with review of literature. 11 SIRIUS GUI was used to analyze the feature data using the SIRIUS algorithm with Orbitrap-based default settings. Features were additionally analyzed using CSI:FingerID with Fingerprint Prediction using Fallback Adducts of [M+H]+, [M]+, [M+K]+, and [M+Na]+, and Structure Database Search using Bio Database, HMDB, and KEGG.…”

“…9 Zebrafish embryos have also been shown to have multiple biotransformation pathways, including sulfonate-, N-acetylcysteine-, and glutathione-conjugation, that could reduce sensitivity to 6PPD-Q by detoxifying the chemical. 10 Hydroxylated TPs are a result of phase I biotransformation, including an alkyl sidechain monohydroxylated TP (TP-OH1), a phenyl ring monohydroxylated TP (TP-OH2), 11 and a dihydroxylated TP (TP-2OH). 11,12 Additional phase II TPs occurring in 6PPD-Q-exposed salmonids must be better characterized to understand differences in detoxification upon aqueous exposure.…”