Identification and molecular characterization of cytochrome P450 (CYP450) family genes in the marine ciliate Euplotes crassus: The effect of benzo[a]pyrene and beta-naphthoflavone.
Marine ciliate Euplotes crassus, a single-cell eukaryote, and has been considered as a model organism for monitoring of environmental pollutions in sediments. Cytochrome P450 (CYP450) monooxygenase are phase I enzyme involved in detoxification of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs). However, little information on CYP450 family genes in ciliate is available. In the present study, acute toxicity of PAH, benzo[a]pyrene (B[a]P) and PAH-like model compound, beta-naphthoflavone (β-NF), was investigated; full-length cDNA sequences and genomic structure of five CYP450 genes (CYP5680A1, CYP5681A1, CYP5681B1, CYP5682A1, and CYP5683A1) were analyzed; and finally their activities and transcriptional changes were measured after exposure to PAHs for 48h. According to the results, B[a]P exposure showed a negative effect on E. crassus survival, whereas β-NF exposure showed no significant effect. The 8h-LC50value of B[a]P was determined to be 2.449μM (95%-C.L., 7.726-3.619μM). Five genes belonging to the CYP450 family had conserved domains and clustered with those of ciliate group, as revealed in phylogenetic analysis. CYP activity did not change after exposure to B[a]P, whereas it was slightly, but significantly, induced after exposure to β-NF. The mRNA expression of five CYP450 genes was significantly modulated in a concentration- and time-dependent manner after exposure to both the chemicals. Our findings suggest that CYP450 genes in E. crassus may be involved in detoxification of B[a]P and β-NF. This study would give a better understanding about the mode of action of B[a]P and β-NF in marine ciliates at the molecular level.
Department of Life Science, College of Natural Sciences, Sangmyung University, Seoul 03016, Republic of Korea.
Bora
B
Yim
Department of Life Science, College of Natural Sciences, Sangmyung University, Seoul 03016, Republic of Korea.
Hokyun
H
Kim
Department of Life Science, College of Natural Sciences, Sangmyung University, Seoul 03016, Republic of Korea.
Jisoo
J
Kim
Department of Life Science, College of Natural Sciences, Sangmyung University, Seoul 03016, Republic of Korea.
Haeyeon
H
Kim
Marine Chemistry & Geochemistry Research Center, Korea Institute of Ocean Science & Technology, Ansan 15627, Republic of Korea.
Eun-Ji
EJ
Won
Department of Life Science, College of Natural Sciences, Sangmyung University, Seoul 03016, Republic of Korea. Electronic address: ymlee70@smu.ac.kr.
Young-Mi
YM
Lee
D000595:Amino Acid Sequence; D059001:Aquatic Organisms / Q000187:drug effects* / Q000201:enzymology; D001564:Benzo(a)pyrene / Q000633:toxicity*; D002274:Carcinogens, Environmental / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016812:Euplotes / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007438:Introns; D007700:Kinetics; D007928:Lethal Dose 50; D010802:Phylogeny; D000072417:Protein Domains; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012989:Soil Pollutants / Q000633:toxicity; D023382:Toxicity Tests, Acute; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D019324:beta-Naphthoflavone / Q000633:toxicity*
D002274:Carcinogens, Environmental; D012333:RNA, Messenger; D012989:Soil Pollutants; D014874:Water Pollutants, Chemical; D001564:Benzo(a)pyrene; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System
Benzo[a]pyrene; Beta-naphthoflavone; Ciliate; Cytochrome P450; Euplotes crassus; Quantitative real-time RT-PCR