Article pubs.acs.org/est
Identification of the Full 46 Cytochrome P450 (CYP) Complement and Modulation of CYP Expression in Response to Water-Accommodated Fractions of Crude Oil in the Cyclopoid Copepod Paracyclopina nana Jeonghoon Han,†,# Eun-Ji Won,†,# Hui-Su Kim,† David R. Nelson,‡ Su-Jae Lee,§ Heum Gi Park,∥ and Jae-Seong Lee*,† †
Department of Biological Science, College of Science, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea Department of Microbiology, Immunology, and Biochemistry, University of Tennessee, Memphis, Tennessee 38163, United States § Department of Life Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea ∥ Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung, Gangwon-do 210-702, South Korea ‡
S Supporting Information *
ABSTRACT: The 46 cytochrome P450 (CYP) gene superfamily was identified in the marine copepod Paracyclopina nana after searching an RNA-seq database and comparing it with other copepod CYP gene families. To annotate the 46 PnCYP genes, a phylogenetic analysis of CYP genes was performed using a Bayesian method. Pn-CYP genes were separated into five different clans: CYP2, CYP3, CYP20, CYP26, and mitochondrial. Among these, the principal PnCYP genes involved in detoxification were identified by comparing them with those of the copepod Tigriopus japonicus and were examined with respect to their responses to exposure to a water-accommodated fraction (WAF) of crude oil and to the alkylated forms of two polycyclic aromatic hydrocarbons (PAHs; phenanthrene and fluorene). The expression of two PnCYP3027 genes (CYP3027F1 and CYP3027F2) was increased in response to WAF exposure and also was upregulated in response to the two alkylated PAHs. In particular, Pn-CYP3027F2 showed the most notable increase in response to 80% WAF exposure. These two responsive CYP genes (Pn-CYP3027F1 and CYP3027F2) were also phylogenetically clustered into the same clade of the WAF- and alkylated PAH-specific CYP genes of the copepod T. japonicus, suggesting that these CYP genes would be those chiefly involved in detoxification in response to WAF exposure in copepods. In this paper, we provide information on the copepod P. nana CYP gene superfamily and also speculate on its potential role in the detoxification of PAHs in marine copepods. Despite the nonlethality of WAF, Pn-CYP3027F2 was rapidly and significantly upregulated in response to WAF that may serve as a useful biomarker of 40% or higher concentration of WAF exposure. This paper will be helpful to better understand the molecular mechanistic events underlying the metabolism of environmental toxicants in copepods.
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INTRODUCTION
bile acids, vitamin D, retinoic acid, eicosanoids, fatty acids, and some heme breakdown products.5,6 In particular, CYP gene expression in response to polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene has been reported in diverse vertebrate and invertebrate organisms.7−9 Thus, induction of CYP genes has been considered as a prominent biomarker for heavy oil pollution that includes PAHs.10−12 In vertebrates, CYP enzymes also have been used for studies to understand the metabolism of emerging chemicals such as polybrominated
The cytochrome P450 (CYP) enzymes are a diverse and large group of enzymes associated with phase I biotransformation and play an important role in the detoxification of xenobiotics in diverse organisms.1−3 In biotransformation, CYP enzymes transform nonpolar compounds into polar metabolites by oxidation, reduction, dealkylation, or hydrolysis and increase their solubility to facilitate subsequent metabolism.1 They also catalyze exogenous compounds, such as drugs and carcinogenic chemicals, and endogenous compounds, including steroids, fatty acids, and hormones.4 Modulation of the transcription and translation of CYP genes and the CYP-mediated metabolites is considered to be a potential bioindicator for the detection of xenobiotic compounds and a means of regulating endogenous systems that are associated with mainly lipids including steroids, © 2015 American Chemical Society
Received: Revised: Accepted: Published: 6982
March 11, 2015 April 29, 2015 May 5, 2015 May 5, 2015 DOI: 10.1021/acs.est.5b01244 Environ. Sci. Technol. 2015, 49, 6982−6992
Article
Environmental Science & Technology
P. nana Pn-CYP3027F2 gene expression can be useful as early warning signals in response to oil exposure during heavy oil spills in a marine environment.
diphenyl ethers (PBDE) and some pharmaceutical compounds (diclofenac, fluoxetine, and gemfibrozil).13,14 Influxes of crude oil stemming from accidental heavy oil spills wreak disaster on marine ecosystems.15−19 Crude oil is a complex mixture of diverse compounds such as PAHs, BTEX (benzene, toluene, ethylbenzene, and xylene), phenols, and small amounts of nonhydrocarbons (e.g., sulfur, metals).20 Of these, PAHs are the most carcinogenic and persistent compounds and can cause DNA damage in organisms.21 Thus, adverse effects such as growth inhibition and reduction of hatching rate have been observed in marine fauna in response to oil spills.20,22,23 Deleterious effects in aquatic organisms have been observed at the individual organism level, as PAHs are generally persistent and accumulate in the food chain, persisting for a long period.24 Heavy oil spillage can cause acute toxicity by resulting in exposure to highly soluble low molecular weight PAHs.25,26 To monitor the effects of oil pollution on marine organisms, a suitable experimental animal is required to examine biological effects in response to the water-soluble components of crude oil in an aqueous phase.27 Wateraccommodated fraction (WAF) is an example of such a substance, representing the water-soluble portion of crude oil, with high bioavailability toward marine organisms.25,26 The components of WAF are mainly alkylated PAHs, which comprise the main components of weathered crude oil in oil spill areas.19 CYP genes have previously been found to be significantly induced by B[a]P and diethylhexylphthalate, as is seen in the marine medaka Oryzias melastigma and the rainbow trout Oncorhynchus mykiss.7,9 In O. melastigma, the induction of overall CYP gene expression in response to exposure to the water-accommodated fraction (WAF) of crude oil showed that CYP gene expression profiling can be useful to reveal chemicals’ modes of biological action.3 In particular, the mining of CYP genes in fish using next generation sequencing (NGS) greatly enabled the study of specific CYP genes in the context of the modes of biological action of chemicals, as CYP genes comprise several families and have different functions.9,28 However, little information on profiles of CYP gene expression in response to WAFs is available for marine invertebrates. Copepods are marine invertebrates that play important roles in the marine ecosystem as a link between producers and high trophic consumers and have characteristics that render them suitable as model species for ecotoxicological studies.29 The marine cyclopoid copepod Paracyclopina nana in particular is suitable for monitoring the aqueous phase of heavy oil spills as it is a planktonic copepod that can easily be exposed to dissolved forms of toxicants in the water phase.30 Most of all, the susceptible response to environmental stresses (e.g., heavy metals including Cu and Cd, γ radiation, and UV radiation) compared to Tigriopus japonicus makes this species a promising model species.31−35 They are also highly sensitive to environmental conditions (e.g., temperature, salinity, pollutants)30,36 and are of small size (
