Induction and mRNA expression patterns of CYPs

Induction and mRNA EIPA patterns of CYPs have been considered as molecular markers for risk assessment of xenobiotics, such as PAHs metabolized by CYP–mediated pathway. Regarding β-NF and B[a]P exposure, many studies have already shown that AhR-mediated CYP genes play a prominent role in regulation of xenobiotics in vertebrates and invertebrates, such as nematode, bivalves, fish, and pigs (Menzel et al., 2001, Chirulli et al., 2007, Zanette et al., 2013, Kim et al., 2014a, Kim et al., 2014b). A previous study on species differences of CYP-mediated drug metabolism and induction conducted with five vertebrates suggested that low conserved CYP sequences among species could lead to different response to same substrates (Martignoni et al., 2006). With regard to CYP activity assay, many studies have been conducted to optimize the measurement conditions for CYP in aquatic invertebrates with limited information on substrates for indirect methods (Gagnaire et al., 2009, Gottardi et al., 2016). In general, a well-known method for measurement of CYP activity is 7-ethoxy-resorufin-O-dellthylase (EROD), which is a strong marker of CYP1 family, in particular 1A1 and 1A2 activity upon exposure to xenobiotics. James (1989) suggested that microsomal preparation is required for EROD assay, and endogenous inhibitors present in marine invertebrates made measurement of their CYP activities difficult. Furthermore, no CYP functions in PAHs metabolism like mammalian CYP1A has been identified in invertebrates (Livingstone et al., 1997, Chaty et al., 2004). Nevertheless, EROD assay has been used in aquatic invertebrates, such as crab (Fossi et al., 2000), clam, and polychaete (Pérez et al., 2004). Herron (2004) suggested that CYP responsible for B[a]P metabolism may be different and unique in invertebrates. In the present study, we used a new luminescent method to measure CYP activity with Luciferin-ME as a substrate which is used for wide ranges of mammalian CYPs, including CYP1A2, CYP2C8, CYP2C9, CYP2J2, CYP4A11, and CYP4F3B. As a result, no significant modulations of CYP activities in B[a]P exposure and low induction in β-NF were observed in E. crassus (Fig. 5). One explanation is that ciliate CYP seems to have low affinities to the substrate used. On the other hands, no induction of CYP activities in response to xenobiotics has been reported in aquatic invertebrates including blue crab (Singer et al., 1980) and lobster (James and Little, 1984). Similar to our results, Monari et al. (2009) also showed a low or decreased induction of CYP activity in clam, Chamelea gallina. There is a report revealing that amount of total CYP proteins and their activities are 10-fold lower in marine invertebrates than in vertebrates (Livingstone, 1991). Koenig et al. (2012) reported that differences in CYP activity using six substrates between fish and crustacea are related to their species–specific polychlorobiphenyls (PCBs) accumulation patterns, demonstrating that xenobiotic metabolisms are different quantitatively and qualitatively between vertebrates and invertebrates.