In the present study we used a new luminescent
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. In vertebrates, PAH-AhR-CYP1A axis has been well established. AhR-dependent transcriptional modulation is caused by the xenobiotic response Cycloheximide (XRE) located on its promoter sequences (Jeong and Kim, 2002). In few invertebrates, presence of AhR has been reported in arthropods, nematodes, molluscs, and chordates, assuming that AhR existed approximately 550millionyears ago when the first chordate appeared (reviewed by Hahn, 2002). However, unlike that of vertebrates, invertebrate AhR lacks specific binding with ligand (Butler et al., 2001, Hahn, 2002). Hahn (2002) suggested that AhR existed during early evolution and acted as a transcriptional activator without ligand binding capacity. In ciliates, presence of AhR is not known yet, indicating that CYP-mediated mechanisms of ciliates could be different with normal PAHs-AhR-CYP signaling pathways or may be induced by AhR-independent pathway. The mRNA expression of E. crassus CYPs was highly induced after exposure to both chemicals in a concentration - dependent manner (Fig. 6). Similar expression pattern was observed in the rotifer Brachionus koreanus, in which genes belonging to CYP2 and CYP3 families were upregulated after exposure to B[a]P (Kim et al., 2013). A significant increase in the expression of three CYP genes was also observed in a polychaete, Perinereis nuntia exposed to water accommodated fraction (WAF) of Iranian crude oil and B[a]P (Won et al., 2013). Taken together, our finding indicates that CYP-mediated phase I pathway may be involved in metabolisms of B[a]P and β-NF in this species. In particular, the apparent low viability observed after B[a]P exposure as shown in Fig. 1A suggests that high expressions of CYP genes might be associated with metabolisms of B[a]P, and production of reactive metabolites may affect to the viability of E. crassus (Shimada, 2006). Generally, AhR-ligand translocates to the nucleus, forms AhR/ARNT (aryl hydrocarbon receptor nuclear translocator) complex, and finally activates the target genes, such as CYP, by binding to dioxin response element (DRE) on the promoter sites of genes. In E. crassus, short intron and lack of putative transcription factor binding sites in promoter regions show requirement of further studies on CYP signaling pathways in detoxification mechanisms in the ciliate. A recent study on the ciliate Tetrahymena genome also demonstrated that high contents of A/T, more than 78%, make comparison of homologies with well-characterized promoter sequences difficult (Fu et al., 2009). In conclusion, in the present, we identified five CYP genes in marine ciliate E. crassus (CYP5680A1, CYP5681A1, CYP5681B1, CYP5682A1, and CYP5683A1) using sequence and phylogenetic analysis. We also investigated the effect of B[a]P and β-NF on the enzyme activity and mRNA expression of CYP genes. Sensitivity to B[a]P make E. crassus a potential model species for monitoring of PAHs toxicity. E. crassus CYP genes are typical CYPs because they have conserved domain/motifs. Although CYP activity showed low induction in response to B[a]P and β-NF exposure, transcriptional modulation of five CYPs indicates that these CYPs may be involved in detoxification of these xenobiotics as a cellular protective mechanism in E. crassus. The molecular responses also demonstrate that CYP genes could be potential molecular biomarkers for monitoring PAHs toxicity in this species. Further research on B[a]P metabolites, and molecular and biochemical approaches towards CYP gene regulation is required to better understand the detailed function of CYP in the detoxification pathway of xenobiotics in the ciliate.