The present study investigated in vitro and in vivo biological responses associated with phase I biotransformation using the rainbow trout (Oncorhynchus mykiss) liver cell line RTL-W1 and juvenile fish after exposure to binary mixtures of benzo(a)pyrene (BaP), and either PFOA or PFOS. Our results indicate that exposure to PFAS/BaP binary mixtures altered CYP-like activity in vivo; however, those alterations were not observed in vitro. Previous studies have indicated that mixtures of PFAS and PAHs have the potential to alter biological processes in fish (Dale et al., 2020; Horvli, 2020; Khan et al., 2019). For instance, Khan et al. (2019) demonstrated female Atlantic cod (Gadus morhua) exposed to mixtures of PFAS and PAHs in vivo had altered dopaminergic signaling, including the modulation of dopamine biosynthesis, catabolism, and its receptor expression. Notably, the CYP19A1B gene expression was significantly decreased in the fish exposed to both low and high PAH doses and combined exposure to low PAH/high PFAS. In addition, Dale et al. (2020) evaluated CYP1A gene and protein expression in Atlantic cod liver microsomes following intraperitoneal exposures to PFAS/PAH mixtures and observed a significant increase in CYP1A expression in the low PAH/high PFAS exposure group relative to the control groups. However, no significant changes in gene or protein expression were observed for any of the other groups in the study. Similarly, Horvli (2020) demonstrated that PFAS were able to induce activation of CYP1A1 on their own and produced apparent synergistic effects in combination with BaP through in vitro/in vivo exposures in Atlantic cod.
Our results from the in vivo approach indicate that exposure to PFOA alone does not alter the CYP1A1-like activity in trout, but PFOS may be capable of inducing minimal activity. Trout exposed to either PFOS or PFOA alone also demonstrated that CYP1A2-like activity was similar to basal levels in PFOA-exposed fish but that the activity was higher in PFOS-exposed individuals. However, these increases were much lower than the activity that resulted from exposures to BaP alone, an AhR-ligand that has been previously shown to significantly induce CYP1A1 and CYP1A2 in trout (Jönsson et al., 2004). In contrast, we observed significant decreases in CYP3A4-like activity for the PFAS-only treatment groups, suggesting that PFAS may be capable of inhibiting CYP3A4 in trout. Although in humans, these observations were also shown in (Franco et al., 2020), when PFAS effects on biotransformation, primarily CYP3A4 expression and activity, were explored using the HepaRG cell line.
Furthermore, our results indicate that exposure to PFAS-BaP binary mixtures altered CYP1A1, -1A2, and. -3A4 activity in the isolated trout liver microsomes. The activity associated with these CYP isoforms significantly decreased to similar levels to unexposed controls. Considering that the exposure to BaP alone induced activity of two of these enzymes (CYP1A1/2), the decreases in activity following co-exposures may suggest that PFAS could impair the ability of CYP1A isoforms to function properly in exposure scenarios where biotransformation of PAHs may be necessary, leading to significant potential for PAH bioaccumulation. In addition, if the observed PFAS effects on the CYP isoforms evaluated in the present study are similar to other biotransformation pathways, aquatic organisms that require these pathways as protective mechanisms for other types of exposure (e.g., pharmaceuticals) (Connors et al., 2013) may experience impaired ecological stability if inhabiting ecosystems simultaneously contaminated with PFAS and other substances of emerging concern.
In terms of general toxicity, it has been suggested that PFOS is more toxic compared to PFOA in fish (Zheng et al., 2012) and that these effects may be attributed to the tendency of perfluorinated sulfonic acids (e.g., PFOS) to bioaccumulate to a greater extent compared to perfluorinated carboxylic acids (e.g., PFOA) (Liu et al., 2019). With the exception of the single-exposure CYP1A1-like activities, we found that, generally, PFOS and PFOA similarly altered CYP-like activities, implying that the effects we observed may not be directly associated with the functional groups and accumulation potential but rather the chain length. Several studies in fish have found that the carbon chain length may be a determinant of PFAS toxic effects (Chambers et al.; Conder et al., 2008; Gaballah et al., 2020; Hagenaars et al., 2011). In humans, Amstutz et al. (2022) demonstrated that CYP inhibition by PFAS decreases as the carbon chain length increases. Though this relationship has not yet been explored in fish, future research is needed to determine the involvement of altered CYP activity in the hepatotoxicity in fish exposed to PFAS of varying chain lengths.
Our findings using the in vitro approach, the RTL-W1 rainbow trout liver cell line, were not quite consistent with our findings in vivo. Although there is significant evidence that CYP1A expression is inducible in this cell line (Heinrich et al., 2014), it seems that RTL-W1 monolayers were not reflective of the observed effects in vivo in our study. However, it should be noted that RTL-W1 has enhanced hepatotypic functions and, subsequently, increased CYP1A expression and activity when cultured as spheroids (Lammel et al., 2019). Further studies utilizing RTL-W1 in alternative systems (e.g., spheroids) to assess CYP1A activity following exposure to PFAS-containing mixtures should be performed to gather further insight into potential PFAS effects in fish and to enhance the prediction of such alterations in vivo.