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  • CYP s are heme containing monooxygenases constituting

    2020-02-08

    CYP450s are heme-containing monooxygenases constituting a conserved gene superfamily of heme-thiolate proteins, and are found in prokaryotes and eukaryotes (Martignoni et al., 2006). They play a key role in detoxification of endogenous (e.g., steroids) and exogenous compounds (e.g., drug, insecticides, PAHs, and polychlorinated biphenyls (PCBs)) (Werck-Reichhart and Feyereisen, 2000), and are involved in steroidogenesis. Among them, CYP 1–4 families are known to vitamin k3 synthesis be mainly involved in detoxification of xenobiotics, and are transcriptionally activated by xenobiotic-AhR complex (Waxman, 1999). In particular, the CYP1 family (CYP1A, CYP1B, CYP1C, and CYP1D) has been extensively studied and considered as a reliable biomarker in aquatic vertebrates to evaluate metabolisms and/or biological effects of various environmental pollutants, including PAHs (reviewed by Guengerich, 2008, Iwamoto et al., 2012). However, little information on drug metabolism pathways in non-vertebrate is available (Solé and Livingstone, 2005, Rewitz et al., 2006). Until now, several CYP genes are identified in human (57 CYPs), mouse (108 CYPs), nematode (75 CYPs, Caenorhabiditis elegans), sea urchin (120 CYPs, Strongylocentrotus purpuratus), water flea (83 CYPs, Daphnia magna), and yeast (2–3 CYPs) (Nelson, 2013). In the phylum Ciliophora which is one of the major evolutionary lineage that constructs the Alveolates (Parfrey et al., 2006), CYPs from only freshwater ciliate Tetrahymena thermophila (40 CYPs) are identified (Fu et al., 2009), and one of these CYP genes (CYP5013C2) was characterized in response to dichlorodiphenyltrichloroethane (DDT) (Feng et al., 2014). Thus, identification and expression of CYP in response to xenobiotics are further required in ciliates to determine the function of CYPs in ciliates and to better understand vitamin k3 synthesis of CYPs in eukaryotes. Single-celled eukaryotic ciliate protozoa are common in terrestrial, freshwater, and marine environments (Germond and Nakajima, 2016). As they have a small size enough to consume bacteria and small phytoplankton, ciliates play an important role in the planktonic and benthic microecosystems as transmitters of energy from producer to primary consumers in the aquatic environment (Fenchel, 1987). In fact, protozoans are responsible for the consumption of 10 to 50% of the carbon fixed by a producer in seawater (Libes, 1992). In ecotoxicological aspects, since unicellular ciliate protozoa respond more sensitively to environmental stressors than multicellular organisms do (Gutierrez et al., 2003, Dayeh et al., 2005) and share characteristics with eukaryotic cells, their biological response to various stressors can be used to predict the response of higher organisms (Trielli et al., 2007). Marine ciliate, Euplotes crassus, has further advantages, such as small size, short generation time (~10h) (Roth et al., 1985), genetically homogeneous progeny by asexual reproduction, and easy maintenance under laboratory conditions. Therefore, it is considered to be a potentially suitable eukaryotic model species for evaluating the effects of contaminants on aquatic communities (Gomiero et al., 2012, Gomiero et al., 2013, Kim et al., 2014b, Kim et al., 2017). Furthermore, since sediments serve as a reservoir of PAHs, marine benthic organisms that inhabit these sediments, such as ciliates, can be the main target for ecotoxicological research. In the present study: 1) acute test of B[a]P was performed. To enable a comparative study, beta-naphthoflavone (β-NF) was also used as a positive control for assessing acute toxicity and CYP expression; 2) the full-length cDNAs of five CYP 450 genes (CYP5680A1, CYP5681A1, CYP5681B1, CYP5682A1, and CYP5683A1) marine ciliate E. crassus were sequenced and characterized for the first time; and 3) CYP activities and transcriptional modulation of these genes in response to B[a]P and β-NF were investigated. The study aimed to identify E. crassus CYP genes and reveal their roles under PAH exposure conditions (B[a]P and β-NF); to highlight the usefulness of CYP genes as potential molecular biomarkers for monitoring PAHs; and finally to understand mode of action of B[a]P- and β-NF-mediated toxicity at the molecular level in this species.