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  • Among the CYP enzymes CYP D

    2021-07-31

    Among the CYP450 enzymes, CYP2D has been considered a crucial protein that involves in the metabolism of xenobiotics and drugs (Zanger and Schwab, 2013). Some isoforms of the CYP2D subfamily have been well characterized, such as CYP2D6 due to its clinical importance and CYP2D14 in black cattle (Hamamoto et al., 2016; Feng and Liu, 2018). However, there are very few reports available on the characterization of CYP2D in bivalves, particularly their mechanistic role. In our study, we found that the expression of CYP2D14-like in digestive gland and gill tissue significantly altered after exposure to P. lima, suggesting that CYP2D14-like might be involved in the metabolism of OA. Besides, CYP2C8 in gill and CYP3L3 in digestive gland also exhibited altered expression, implying their potential role in DSP toxins metabolism which is in accordance with the previous report in mammals (Naraharisetti et al., 2010), and bivalves (de Boissel et al., 2017). Together, these findings uncovered the novel regulatory role of CYP450 on OA metabolism and provided the research topic for further study. CYP3A enzymes exemplify a first line of defense for mammals against the bioaccumulation of lipophilic substances by detoxifying the drugs, insecticides and carcinogens (Hegelund and Celander, 2003; James et al., 2005). Amongst, CYP3A4 is considered a potential candidate in metabolizing a variety of xenobiotics and clinical drugs (Kumagai et al., 2016). It has been reported that human CYP3A4 could efficiently hydrolyze OA into four metabolites in an in vitro experiment (Guo et al., 2010). CYP3A4 and CYP3A5 have been shown to mainly catalyze the metabolism of OA into oxygenated metabolites (Kittler et al., 2014; Kolrep et al., 2016). Interestingly, previous study showed that inhibition of CYP3A4 activity by ketoconazole could enhance the toxic effects of these toxins despite OA, DTX-1 and DTX-2 did not increase CYP450 (1A2, 3A4, 2C9, 2C19) activities in HepaRG PD 325901 (Ferron et al., 2016). Collectively, our findings demonstrated that the expression of CYP3A4 was significantly up-regulated after exposure to P. lima, thereby suggesting the crucial role of CYP3A4 in the metabolism of OA which is in line with the aforementioned reports. To further verify the role of CYP3A4 in the metabolism of OA in bivalves, we observed changes in CYP3A4 activity and OA content in the digestive gland after addition of KTZ, a specific inhibitor of CYP3A4 (Bazin et al., 2010; Darnell et al., 2011). Given the optimized concentration of KTZ (10 μМ) to inhibit CYP3A4 activity in human Cao-2 cells (Gibbs et al., 2000) and liver HepaRG cells (Kittler et al., 2014), we chose 10 μМ KTZ to investigate its inhibitory effect on CYP3A4 activity of P. viridis. We found that the CYP3A4 activity in P. lima-exposed mussels with KTZ (P. lima + MeOH + KTZ group) was significantly lower than that of P. lima-exposed mussels without KTZ (P. lima + MeOH group) at 2 h and 12 h, which corroborating the CYP3A4 inhibitory effect of KTZ in bivalve. Interestingly, further analysis on OA content in digestive gland showed that OA content was lower in the presence of KTZ than without KTZ at 2 h and 6 h, which corroborate the role of CYP3A4 in OA metabolism. To exclude the potential effect of methanol, we also used DMSO to dissolve KTZ which yielded similar results to that of methanol (data not shown). These results come to show that decrease in OA in digestive gland is resulted from KTZ, which inhibit the activity of CYP3A4 in bivalves. However, our result is in contrast with previous findings of unaltered OA metabolism under KTZ treated and un-treated group HepaRG cells (Kittler et al., 2014). This discrepancy might be associated with the intricate in vivo metabolism and species-dependent varying metabolic regulatory nodes. Unlike in vitro experiments, CYP450 and other metabolic enzymes might be involved in the OA metabolism in vivo experiment, which complicates the results interpretation. Except for CYP450, II-phase detoxifying enzymes such as glutathione transferase (GST) and ABC transporters such as P-glycoprotein (P-gp) has been shown to involved in OA metabolism in bivalves (Huang et al., 2015; Zou et al., 2015). Besides, previous reports have shown that KTZ could reduce the transport of digoxin, a P-gp substrate, however, could increase the MRP-2/BCRP-mediated ochratoxin A transport in Caco-2 cells (Sergent et al., 2009).