https://hdl.handle.net/10388/15650

Montgomery, David James (2024) Biotransformation of 6ppd-quinone in vivo, ex situ, in silico, and in vitro: A potential toxicokinetic explanation for differences in species senstivity, USASK Harvest - Theses and Dissertations, https://hdl.handle.net/10388/15650

The ubiquitous toxicant, 6PPD-Quinone (6PPD-Q), originates from the environmental oxidation of 6PPD, an antioxidant found in rubber tires and other rubber products. 6PPD-Q is dispersed from roadways during rain events to nearby waterways such as rivers, creeks, streams, and lakes. Subsequently, species living in these environments are exposed to 6PPD-Q, which causes acute lethality in select species of fishes. While 6PPD-Q was determined as non-toxic to invertebrates such as rotifers or cladocerans, some species of fishes, including coho salmon, rainbow trout, and brook trout, display symptoms of urban runoff mortality syndrome (URMS) with a specifically rapid onset of mortality (4 hours for coho salmon). Peak concentrations of 6PPD-Q causing URMS are relatively short-lived due to the rapid degradation of 6PPD-Q in water. It is important to note that 6PPD-Q degradation may play a major role in determining if a species will have mass die-offs in the environment, although it is not the scope of this research. While the abovementioned species of fishes are sensitive to 6PPD-Q, other fishes appear to be insensitive at relatively high concentrations that are past currently measured environmental concentrations. While the reasons for these marked interspecies differences in sensitivity are presently unknown, it has been hypothesized that metabolic differences among fish species are responsible, i.e., their differential ability to biotransform 6PPD-Q and influence the amount of 6PPD-Q reaching the gills in recirculation of blood. The objectives of this thesis aimed to explain interspecific sensitivity to 6PPD-Q via quantifying toxicokinetic (TK) parameters. Assessments measured ex situ hepatic clearance and extraction fraction (in rainbow trout), in vivo biliary metabolite identification and abundance (in various fishes) in addition to in vitro intrinsic clearance (in rainbow trout and Arctic char). Lastly, ex situ hepatic transport, and cytochrome P450 identification (assessed in vitro) were also assessed. Semi-quantification of biliary metabolites after in vivo exposure pointed toward a possible TK explanation for the interspecies differences in sensitivity to 6PPD-Q, i.e., sensitive species exhibited a decreased ability to detoxify 6PPD-Q compared to insensitive species. Ex situ perfused liver experiments suggested that rainbow trout are moderate to rapid metabolizers of 6PPD-Q, suggesting even rapidly biotransformed 6PPD-Q may be insufficient to prevent the acute responses observed in vitro with gill cells, potentially leading to lethality in vivo at environmentally relevant concentrations. Results from in vitro experiments with fish hepatocytes were inconclusive due to low basal metabolic activity. Further studies are necessary to optimize the in vitro assays for the assessment of interspecies differences in biotransformation rates and activities of common cytochrome P450s. In summary, this thesis provided important insights into interspecies differences in 6PPD-Q detoxification. Phase I and II metabolite abundance were correlated with sensitivity or exposure concentration, indicating these compounds might be useful in predicting toxic responses in fishes and might be assessed as a potential biomarker of exposure. Furthermore, ex situ results indicate that rainbow trout, a species known to be sensitive to 6PPD-Q exposure, is a moderate to rapid detoxifier, which might be inconsistent with the in vivo results that suggest rainbow trout have a relatively low capacity to detoxify 6PPD-Q. Bile metabolite levels are a crude measurement of overall systemic detoxification, tissue-specific biotransformation rates might be another important driver of 6PPD-Q toxicity. Future studies should aim to explain the discrepancy between ex situ and in vivo results and other studies should include detoxification of 6PPD-Q as an included component of their studies if interspecific differences in sensitivity are of interest. Further assessment in vitro is necessary to develop assays which could significantly reduce animal use in the future.

https://harvest.usask.ca/bitstreams/6e198d86-5f7b-4ab1-82f6-3fe42771316b/download