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  • Cytochromes P CYP are a superfamily of hemoproteins containi


    Cytochromes P450 (CYP 450) are a superfamily of hemoproteins containing a heme cofactor, which are widely distributed in animals, plants and microbes (Hannemann et al., 2007). CYP450 exhibits a variety of biological functions including biotransformation of drugs, detoxification of endogenous and xenobiotic components through hydroxylation, bio-oxidation and reduction (Guengerich, 2001; Hannemann et al., 2007; Zanger and Schwab, 2013). Previous reports has demonstrated that the human enzyme CYP1A2 could metabolically activate OA (Hashizume et al., 2009). Congruently, biochemical assays showed that CYP3A4 and CYP3A5 converted OA into other derivatives (Guo et al., 2010). Furthermore, Kittler et al. (2014) reported that CYP3A4 detoxified OA in HepaRG 5'-Iodoresiniferatoxin sale by forming two hydroxylated metabolites. However, earlier studies have shown that CYP-mediated OA metabolism is species-specific. Kolrep et al. (2016) demonstrated that OA metabolism into oxygenated metabolites was mainly catalyzed by human CYP3A4 and CYP3A5, whereas the orthologous CYP3A1 and CYP3A2 did not play a predominant detoxification role in rat. Bivalve mussel Perna viridis is an economically important mussel with capability to tolerate a range of biotic and abiotic contaminants and hence, emerged as the suitable biological model organism to investigate physiological alterations in response to the transitional environments (Viarengo et al., 2007; Carrington et al., 2015; Galvao et al., 2015). The toxic dinoflagellate Prorocentrum lima has been studied extensively in toxicological applications as a known producer of DSP toxins (Bravo et al., 2001; Pinto-Silva et al., 2005; Romero-Geraldo and Hernández-Saavedra, 2014). The aim of the current study is to provide a foundation for more detailed characterization of CYP450 functions in DSP toxins detoxification in bivalves. For this, the expression of the five CYP genes (CYP3A4, CYP2C8, CYP2D14-like, CYP3L3 and CYP3A1) was analyzed in gill and digestive gland of P. viridis after exposure to P. lima, as well as OA accumulation. In addition, as described in other organisms (Darnell et al., 2011; Kittler et al., 2014), the inhibitory effect of ketoconazole on the CYP3A4 activity from digestive gland was evaluated.
    Materials and methods
    Discussion It has been well established that cytochromes P450 play a crucial role in detoxification and toxins metabolism in various systems (Uno et al., 2012). Previous studies have also reported the involvement of CYP1A, 2B, 2E, 3A and 4A in the detoxification of environmental pollutants in M. edulis (Peters et al., 1998). However, the potential role of CYP genes in the detoxification of DSP toxins in bivalves remains largely unexplored, despite their economical and health significance. We thus aimed to investigate the regulatory role of CYP450 genes in the detoxification mechanism. To this regard, we investigated the expression pattern of key CYP450 genes including CYP3A4, CYP2C8, CYP2D14-like, CYP3A1, and CYP3L3 by qPCR, which uncovered the distinct expression pattern of these genes in organ-specific manner. Particularly, expression level of CYP3L3 in gill and CYP2C8 in digestive gland was found to be low. However, CYP3A4, CYP3A1 and CYP2D14-like genes were found to have higher levels of expression both in gill and digestive gland, suggesting these genes possess different regulatory role in various tissues. Our finding is in line with the previous report which showed the differential expression level of CYP2C8 mRNA in various tissues (Klose et al., 1999). In contrast, Zanette et al. (2013) observed lower CYP3-like expression level in gill compared with other organs such as labial palps, digestive gland and outer margin of the mantle in M. edulis. These observed difference might be due to the analyzed subtypes of CYP3, which was shown to have 14 subtypes in oyster C. gigas (Meng et al., 2018). After exposure to P. lima, expression level of CYP3A4 and CYP2D14-like was found to be altered in gill and digestive gland, whereas OA content was found to be increased in gill and digestive gland which was in accordance with the previous report (Bauder et al., 2001; Rossignoli and Blanco, 2008; Huang et al., 2015). Similarly, Vidal et al. (2014) demonstrated a significant increment in cytochrome P450 reductase activity and OA accumulation in M. galloprovincialis exposed to the DSP toxins-producing microalgae D. acuminata and D. acuta. Huang et al. (2015) reported that CYP450 in the gills of bivalves exposed to P. lima significantly increased. These outcomes suggested that CYP450 could play a crucial role in the metabolism of DSP toxins in bivalve. However, the expression of CYP3A1 didn't show significant modulation in the mussels exposed to P. lima, demonstrating different regulations and responses of different types of CYP450 genes to specific chemicals. Actually, many papers have discussed these phenomena. After exposure to β-naphthoflavone (BNF) and 3,3′,4,4′,5-polychlorinated biphenyl (PCB126), CYP3-like-2 mRNA was significantly up-regulated in gill by BNF, and up-regulated in digestive gland by PCB126, whereas there was no significant change observed in the expression of CYP1-like in M. edulis (Zanette et al., 2013). CYP3A-like-1 transcript level significantly decreased after exposure to 17 beta-estradiol and tributyltin, but CYP3A-like-2 did not experience any changes in M. edulis (Cubero-Leon et al., 2012).