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  • br Materials and methods br Results br Discussion In the


    Materials and methods
    Discussion In the present study, as a trial to evaluate the marine ciliate E. crassus for further studies, its susceptibilities to B[a]P and β-NF were measured. Acute toxicity of B[a]P in aquatic organisms has been extensively studied, but not enough data is available in unicellular eukaryote. 48-h LC50 values of Caenorhabditis elegans (Sese et al., 2009) and Daphnia magna (Atienzar et al., 1999) were 174μg/L (0.69μM) and 200μg/L (0.79μM), respectively. In the marine diatom Thalassiosira pseudonana, 24-h EC50 value was 55.24μg/L (0.22μM) (Bopp and Lettieri, 2007). When red sea bream Pagrosomus major embryos and larvae were exposed to B[a]P, 48-h EC50 value was 3.40μg/L and 1.02μg/L, respectively, which was lower than that of other organisms (Zhao et al., 2016). In E. crassus, 8-h LC50 value was determined between 25–10μM of B[a]P (Gomiero et al., 2012), which was higher than our result showing 2.449μM in the same species. This difference could come from different culture and exposure condition, and different strain. Although the susceptibility of E. crassus to B[a]P cannot be easily compared with that noted in previous studies conducted in multicellular organisms with different exposure time, our findings suggest that E. crassus is sensitive to B[a]P exposure. In addition, sensitivity to B[a]P seems to be species-dependent because its toxicity is determined by various factors, such as adsorption affinity of surface (Meador et al., 1995) and difference in cellular membrane structure along with related enzyme system (Abdel-Shafy and Mansour, 2016), extent of B[a]P PF-670462 and lipid composition (Driscoll and McElroy, 1996). In particular, the small body size with a high surface area-to-volume ratio provides large contact and interaction chances with pollutants in the surrounding environment (Gomiero et al., 2012). Furthermore, the susceptibility of E. crassus to B[a]P is likely to be both a higher bioavailability and metabolisms of B[a]P. In a recent study, bioavailability of PAH fractions accounting for the toxic effects in organisms was reported (Yang et al., 2016). Besides, induction of CYP450 enzymes in PAHs metabolisms can lead to toxic results by increasing unstable and reactive intermediates (Shimada, 2006). Some studies suggested that degradation through CYP-mediated mechanisms is also responsible for resistance of compounds (Feng et al., 2014). Thus, the susceptibility to B[a]P allows the E. crassus to be an adequate species to evaluate its toxicity. Meanwhile, β-NF had no effect on survival of E. crassus at the maximum concentration of 40μM in the present study (Fig. 1B). Similarly, in recent comparative study on mutagenic effects of BaP and β-NF, similar mutagenic activation in the European eel Anguilla Anguilla L. was induced at BaP (0.15–0.75μM) and β-NF (3.8–33μM), respectively (Maria et al., 2002). Tolerance to β-NF were also reported in mouse Hepa 1c1c7 cells, the viability of which was retained up to approximately 80% at even 50μM β-NF (Elbekai et al., 2004). Many recent studies, however, showed repercussions of β-NF as an oxidative stress inducer in various conditions both in vitro and in vivo (Elbekai et al., 2004, Rhee et al., 2015). For example, in copepod, six different biomarkers that are associated with oxidative stress condition, including intracellular reactive oxygen species, glutathione concentration, residual glutathione S-transferase, glutathione peroxidase, glutathione reductase, and superoxide dismutase activities were significantly increased after β-NF exposure (1mg/L of β-NF for 12–24h). Thus, in this study, no significant difference in the ciliate viability after β-NF exposure demonstrate a relatively low bioavailability of β-NF through metabolic mechanisms compared to that of B[a]P. Generally, CYP450s have conserved domains, including a heme-binding region (FXXGXRXCXG), which has a conserved cysteine residue that acts as the fifth ligand to the heme and is completely conserved between CYP proteins (Vaccaro et al., 2007). Another domain is K-helix (EXXR), which is highly conserved and involved in stabilization of the core structure (Sirim et al., 2010). These domains were observed in all the five E. crassus CYP450s in this study (Fig. 2), indicating that these proteins are typical CYP450s.