In this study it was
In this study, it was noted that two out of five E. crassus CYPs (CYP5681A1 and CYP5682A1) had no intron, and 3 others had one intron at different positions (Fig. 3). Number and length of introns are varied in diverse species. For example, most hypotricha genes characterized so far lack introns (Prescott, 1994). Among 44 T. thermophila CYP genes, 16 CYP genes lacked intron, whereas others had one to four introns (Fu et al., 2009). In 83 Drosophila CYPs, five had no intron, and 78 others had one to eight introns, with average length of 50–70bp (Tijet et al., 2001). It is known that certain organisms have abundance of very small introns (<100bp), such as >60% of introns from Schizosaccharomyces pombe (Prabhala et al., 1992), ≈50% from T. thermophila (Csank et al., 1990), and >62% from Euplotes sp. (Meyer et al., 1992). However, the shortest introns in most organisms range 35–50bp in length. Intron of E. crassus CYP5681B1 (37bp) is included in the general range, but CYP5680A1 (25bp) and CYP5683A1 (26bp) had extremely short introns. Similarly, intron less than 35bp was reported in Drosophila (31bp) (Tijet et al., 2001) and E. crassus (24bp) (Wang et al., 1992). Fu et al. (2009) reported a good correlation between the conserved intron position and phylogenetic relationship of T. thermophila P450 subfamily members, which can be caused by iso 1 events during evolution process. Regarding the evolution of introns in Alveolates, Nguyen et al. (2007) suggested that many gains and losses of introns have been noted in the evolution of Alveolates (≈800Myr). In the present study, phylogenetic analysis showed that E. crassus CYP genes were distinctly clustered from other species (Fig. 4). Tetrahymena, Oxytricha, Stylonychia, and Euplotes belong to Class Ciliophora in Alveolates. Euplotes are more close to Oxytricha and Stylonychia, which are included in Spirotrichae. Their phylogenetic relationship likely reflects the evolutionary distance of CYP genes. When even T. thermophila CYP genes were compared to those of Paramecium that are closely related to each other, most CYP genes were species-specific, suggesting that their genomes have evolved through different mechanisms (Chalker and Stover, 2007, Fu et al., 2009). These findings are supported by studies on a higher rate of evolution in the ciliates genome compared to other eukaryotes (Eisen et al., 2006, Zufall et al., 2006). In general, CYP family shares 40% amino acid identity, and subfamily shows >50% identity. Among CYP families, the identity is <30%. Low conservation of CYPs may be derived from different sequences, except conserved regions. Induction and mRNA expression 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.