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Whole Spectrum of Cytochrome P450 Genes and Molecular Responses to Water-Accommodated Fractions Exposure in the Marine Medaka Jae-Sung Rhee,†,# Bo-Mi Kim,‡,# Beom-Soon Choi,‡,§ Ik-Young Choi,§ Rudolf S. S. Wu,∥ David R. Nelson,⊥ and Jae-Seong Lee*,‡ †

Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, South Korea Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea § National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-742, South Korea ∥ School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, China ⊥ Department of Microbiology, Immunology and Biochemistry, University of Tennessee, Memphis, Tennessee, United States ‡

S Supporting Information *

ABSTRACT: Water-accommodated fractions (WAFs) of crude oil include chemicals that are potent toxicants in fish. Increasing oil pollution thus demands a better understanding of molecular mechanisms for detoxification, metabolism, toxicity, and adaptation of fish. Previous studies with fish show modulation of expression of key genes in relation to stress response against WAF exposure, but there is still a lack of studies on responses of cytochrome P450 (CYP) genes and changes in biotransformation upon WAF exposure. In this study, we used the full spectrum of CYP genes of the marine medaka, Oryzias melastigma, to understand their potential mode of action on WAFs-triggered molecular mechanisms. We also analyzed further CYP-involved detoxification and endogenous steroidogenic metabolism after exposure to different concentrations of WAFs over different time courses in the marine medaka. Also, detoxification- and antioxidant-related enzymes’ activities were analyzed with different concentrations of WAFs. As a result, the WAF exposure induced CYP-involved detoxification mechanism but reduced CYPinvolved steroidogenic metabolism in the marine medaka. These data suggest that whole CYP profiling can be a way of understanding and uncovering the mode of action particularly with respect to emerging chemicals such as WAF exposure with the new finding that WAFs have dual functions on CYP-involved metabolisms.

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INTRODUCTION

Cytochrome P450 (CYP) enzymes play a crucial role in the metabolism of endogenous and exogenous chemical compounds such as multiple substrates, drugs, chemical carcinogens, and toxic compounds.5 CYP enzymes are ubiquitous enzymes and form a multigene superfamily comprising more than 6000 individual enzymes in many different organisms.6 In addition to those general functions, each CYP subfamily has a potentially specific role toward certain substrates in endogenous metabolism and animal physiology.7−9 Based on the function of metabolism and the substrate specificity of each CYP enzyme, the interaction and the mode of action of a certain chemical may be better understood by screening the whole spectrum of CYP gene expression. Thus, the application of whole CYP enzyme profiling could be a useful approach for diverse research areas as well as a biomarker discovery for

Recently, heavy oil spillage and oil exposure to the aquatic environment has been prominent but it is still difficult to use model species to assess resulting effects.1 It is well-known that extensive oil spills raise detrimental effects to most marine ecosystems that remain for several decades. Thus, oil pollution could provide serious damage to marine animals, and the persistence of certain components may also give diverse sublethal effects to marine species on aspects of physiology, behavior, and reproduction. Despite these concerns, the molecular effects of its components or water accommodated fractions (WAFs) are virtually unknown, even though toxicities of crude oil have been studied in marine animals. Crude oil is a mixture of polycyclic aromatic hydrocarbons (PAH) including diverse soluble hydrocarbons and alkylated PAHs as the most toxic components.2,3 In general, all PAHs are considered as hydrophobic and lipophilic compounds, and therefore, in fatty tissues of animals including fish, they can be readily dissolved and accumulated.4 © 2013 American Chemical Society

Received: Revised: Accepted: Published: 4804

January 17, 2013 April 2, 2013 April 10, 2013 April 10, 2013 dx.doi.org/10.1021/es400186r | Environ. Sci. Technol. 2013, 47, 4804−4812

Environmental Science & Technology

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for 24 h. For the time-course experiment, fish were exposed to 5% WAF, and sampled at 6, 12, 24, 48, 72, and 96 h. The artificial seawater was exchanged daily and chemicals were renewed immediately after each water change. Measurement of Transcript Modulations and Enzyme Activities. This information can be found in the SI as described in our previous study.14 Statistical Analysis. Data are expressed as means ± SD. Significant differences between the observations of control and exposed groups were analyzed using one-way comparison ANOVA followed by Tukey’s test. Any difference showing P < 0.05 was considered significant.

emerging and unforeseen chemicals in environmental studies. This kind of approach may shed light on the effects of pollutants and toxicants in human and animal ecosystems. To date, many CYP enzymes have been identified from diverse animals, and their potential roles also have been continuously investigated.7,10 In teleosts, CYP enzymes have primarily been studied as potential biomarkers of pollution of the aquatic environment. However, most previous studies have focused on the expression of the CYP1A enzyme in isolation. To validate the usefulness of whole CYP enzyme profiling, gene mining and annotation is essential prior to appropriate application. Regarding the CYP superfamily identified in teleosts, to date extensive CYP gene information was published in pufferfish and zebrafish but an in-depth toxicogenomic approach using whole CYPs has not yet been established.11,12 Our interest is to check the modulatory effect of certain environmental pollutants on the spectrum of CYP genes of the marine medaka that is one of several potential marine model species in ecotoxicology and molecular environmental research. Subsequently, we cloned all the CYP genes in the genome of the marine medaka, and tested the usefulness of the whole CYP gene profiling in application to marine environment monitoring. The overall objective of the present study is to check the usefulness of transcript profiling of whole Oryzias melastigma CYPs (Om-CYPs) upon exposure to WAF of Iranian heavy oil. The specific objectives were to (1) identify the Om-CYP superfamily as a first report in this species; (2) examine whether dispersed oil fraction modulates mRNA expression of the spectrum of Om-CYP genes; and (3) suggest whole CYP gene profiles as reliable molecular biomarkers for environmental studies. Finally, these results will be useful to obtain a better understanding on the mode of action of diverse environmental pollutants on CYP gene regulation in fish species.

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RESULTS Annotation and Nomenclature of Whole CYP Genes in the Marine Medaka, O. melastigma. A total of 65 OmCYP genes were obtained by extensive NR blast search and local blast analysis in the marine medaka, O. melastigma, genome database. Whole Om-CYP genes were registered at GenBank database (SI Table S1). To date, various CYP gene families have been identified: 17 of fugu, 18 of zebrafish, and 18 of human, even though there are differences in numbers of genes. In this species, Om-CYP genes fell into 17 distinctive CYP gene families (Table S2). Those genes were separated by two major functional groups, a group for oxidation of xenobiotics (CYP families 1 to 3) and a group with endogenous functions (CYP families 5 to 51). A description of different clans for each CYP family followed the criteria of fugu and zebrafish as clan 2, clan 3, clan 4, mitochondrial (MT) clan, and others.11,12 In addition, phylogenetic analysis supported the annotation information of the marine medaka whole CYP genes based on congruent separations according to different clans (Figure S1). WAF Composition. In the WAF employed in this study, naphthalene and its alkylated form (75%) were highly detected, followed by fluorene (10%) and phenanthrene (12%) (Table S3). Concentrations of 16 PAHs, alkylated PAHs, and GCTPHs were 4, 44, and 3956 ng/L, respectively. No mortality or alterations in behavior were observed in any group of WAFexposed fish throughout the experiments. In vivo Effects of WAF Exposure. Different concentrations of WAF exposure did not show any significant detrimental effect on embryo hatching rate. However, 100% WAF caused a pronounced increase in cardiac edema (≈ 42%) during embryogenesis (Table S4). In newly hatched larvae and juvenile fish (≈ 3 weeks old), mortality was induced by WAF exposure concentration dependently for 96 h in both groups. However, different concentrations of WAF exposure did not induce cardiac edema for 96 h. Cardiac edema was not observed until 1 month in surviving fish of both groups (Table S4). Overall, newly hatched larvae showed sensitivity in WAF treatment compared to juvenile fish. In juvenile fish, they did not show any significant mortality up to 20% of WAF exposure for 96 h. WAF exposure significantly decreased the level of 17βestradiol (E2) in 50% and 100% WAFs for 96 h (Figure S2). However, different concentrations of WAF treatment did not modulate the level of 11-ketotestosterone (11-KT) for 96 h in juvenile fish. Transcript Profiling of Whole Om-CYP Genes in Response to WAF Exposure. Analysis of the overall effect of WAF across the four exposure conditions (2.5, 5, 10, and 20%) relative to an unexposed control group revealed altered

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MATERIALS AND METHODS Detailed descriptions for all materials and methods have been incorporated in the Supporting Information (SI) as described in our previous studies.13,14 Fish. The marine medaka, O. melastigma, were reared and maintained at the aquarium facility of the Department of Chemistry, College of Natural Sciences, Hanyang University (Seoul, South Korea). Fish were maintained in glass aquaria (60 L capacity) and each aquarium housed 30 adult fish (both sexes). Retrieval and Annotation of Whole CYP Genes. Genomic DNA of O. melastigma was sequenced using several Next Generation Sequencing (NGS) technologies and bioinformatics. The obtained contigs after assembly were subjected to the BLAST analysis to the nonredundant (NR) database at GenBank. Annotation and nomenclature of all OmCYP genes was done according to David R. Nelson’s method,10 based on their sequence similarities compared to teleost CYPs. WAF Preparation and Experimental Design. The overall WAF preparation and exposure methods followed the CROSERF (The Chemical Response to Oil Spills: Ecological Research Forum) protocol that was established as a standardized method to prepare WAF of physically dispersed and chemically dispersed oil products in relation to laboratory exposures to aquatic organism and analytical chemistry measurement. Juvenile fish (≈ 3 weeks old; Stage 43; first juvenile stage; 10−12.5 mm; n = 20 for each group) were exposed to five different diluted WAFs (0, 2.5, 5, 10, and 20%) 4805

dx.doi.org/10.1021/es400186r | Environ. Sci. Technol. 2013, 47, 4804−4812


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expressions of 65 Om-CYP genes within 24 h (Figure 1, Figure S3). For each Om-CYP gene, we measured mRNA expression

genes for clan 2 (81%), 3 genes for clan 3, 1 gene for MT, and 2 genes for other clans. In the case of downregulated genes, 10 genes were separated into clan 2 (4 genes), MT (5 genes), and others (1 genes). To characterize whole Om-CYP expressions upon exposure to 5% WAF, a time-course analysis was further employed for 96 h. We used hierarchical clustering to generate a heat map to allow visualization of patterns of gene expression across timecourse exposures (Figure 2). All upregulated genes belonged to CYP families 1, 2, and 3 (clan 2 and 3), but downregulated genes were distributed within different CYP families. We analyzed the substrates of the various modulated genes based on metabolic functions of vertebrate CYP genes reviewed by several publications.7,10,15,16 Those genes were categorized according to their potential substrates, such as xenobiotics, sterol, vitamins, fatty acid, and eicosanoid (Figure 3). The vast majority of potential substrates were xenobiotics (61%) in the case of upregulated genes. In the downregulated genes, most of the predicted substrates are related to sterol metabolism (45%), followed by xenobiotics (15%) and vitamin metabolism (15%). Notably, Om-CYP1A and Om-CYP1B as well-characterized biomarkers of toxicants exposure were highly induced by WAF exposure in different concentrations and time-courses. CYP19 encodes P450 aromatase, an important enzyme involving estrogen biosynthesis. In this study, both CYP19 genes such as Om-CYP19A1 (ovarian type aromatase) and Om-CYP19A2 (brain type aromatase) were downregulated by WAF treatment. Transcript Profile and/or Enzyme Activity for Xenobiotic Metabolism-Related Genes. Transcript expressions of two aryl hydrocarbon receptors (AhRs) and three peroxisome proliferator-activated receptors (PPARs) were checked following different concentrations of WAF exposure (Table S6). Om-AhR2 transcript was drastically increased in a concentration-dependent manner, while Om-AhR1 transcript showed slight inductions at both 10% and 20% WAF. All three PPARs were also significantly induced upon 20% WAF exposure. Regarding other enzymes involved in the phase I detoxification system with CYPs, aldehyde dehydrogenase (Om-ALDH) and heme oxygenase (Om-HO) did not show a significant transcript change. In the case of phase II detoxification enzymes, 11 genes were significantly upregulated ≥2.0 fold relative to unexposed control (Table S6), including 3 GSTs (Om-GST alpha, OmGST omega, and Om-GST theta), 4 SULTs (Om-SULT1C1, Om-SULT3-like, Om-SULT4A1, and Om-SULT6B1), and 4 UDP-glucuronyltransferases (Om-UGT1B, Om-UGT2A, OmUGT2A2, and Om-UGT2A3). The expressions of total activities of GST (Figure S4A) and SULT (Figure S4B) were induced in a concentrationdependent manner, indicating that the protein level of both genes was also regulated compared to the level of those transcripts. We also observed significant inductions of antioxidant defense system-related genes (Figure S5), suggesting that WAF exposure could be a potential oxidative stress inducer in this species. In addition, we can assume that intracellular oxidative stress may be induced by WAF treatment based on the results of several heat shock protein (Hsp) expression through intracellular ROS-mediated oxidative stress (Table S6).17 Regarding the expression of phase III transporter proteins, transcripts of all genes tested in this study were slightly increased in response to 10−20% WAF treatment (Table S6).

Figure 1. Transcript expressions of the marine medaka whole CYP profiles in response to different concentrations of WAF exposure: (A) 2.5%, (B) 5%, (C) 10%, and (D) 20%, for 24 h. Values were distributed by fold change (X-axis) and P-value (Y-axis).

using the association between significance (P < 0.05) and fold change (>2 fold) in each WAF concentration. Overall transcript profiles revealed that 32 Om-CYP genes were upregulated and 10 Om-CYP genes were downregulated within a range of fold change (2-fold) and p value cutoff (P < 0.05) in a concentration-dependent manner (Table S5). In 20% WAFexposed group, 32 upregulated genes were composed by 26 4806



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Figure 2. Transcript expressions of the marine medaka whole CYP profiles after exposure to 5% WAF in different time courses. Hierarchical clustering analysis was employed in order to analyze mRNA expression pattern of whole CYPs by heat map.

expression upon 20% WAF exposure (Table S6), including 3β-hydroxysteroid dehydrogenase (Om-3β-HSD), 17β-hydroxysteroid dehydrogenase (Om-17β-HSD), 5α-reduatase (OmSRD5A), and steroidogenic acute regulatory protein (OmStAR). In fact, E2 level was decreased by relatively high concentrations of WAF at the same experimental conditions (Figure S2). Therefore, we suggest that WAF treatment is a potential disruptor of endogenous hormone homeostasis, particularly steroidogenesis mechanism. Finally, we propose a schematic diagram for possible mechanisms involved in the spectrum of modulation of CYPs in the WAFs-exposed marine medaka based on comparison

This result suggests that transporting activity can be induced by WAF exposure to eliminate hazardous components of WAFs. Taken together, the regulation of transcript expression of phase I, phase II, and phase III detoxification system would be the efficient way of protecting against the potential impact toward both metabolism homeostasis and elimination of WAFs in this species (Figure S6). Transcript Profile for Endogenous Metabolism-Related Genes. We further characterized the steroidogenesisrelated genes, as WAF exposure reduced transcripts of several CYPs involved in endogenous hormone regulation. Interestingly, 4 genes showed significant declining patterns of 4807



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Figure 3. Schematic pie charts for putative substrates of upregulated or downregulated CYPs by WAF exposure.

differential expression patterns of whole Om-CYP genes using the real-time RT-PCR array to validate its potential for the risk assessment of WAF contamination with the further possibility of environmental monitoring. Typically, PAHs in WAFs are known to act as strong mutagens and/or carcinogens but the available information is limited on their molecular mechanism for gene modulation prior to the phenotypic appearance of physiological toxic effects. In the PAH composition of WAFs used in this study, naphthalene, fluorene, dibenzothiophene, phenanthrene, and their alkylated forms showed the highest level but there was difficulty in comparing our WAF profile with other reports or with environmental concentrations of WAFs from oil spills due to limited and unmatched analytical information on WAF composition. Regardless of this, we analyzed the molecular effect of WAF exposure on whole CYPs modulation as well as on CYP-triggered metabolism in the marine medaka. The CYPs are the major component in chemical defensomes of phase I oxidative metabolic enzyme. The most acutely upregulated Om-CYP genes identified in this study belonged to CYP families 1−3. Actually, CYP1−4 families have been considered as the most important defensome genes in vertebrates.7,10,15,16 In fish, CYP1 family consists of 4 subfamilies (CYP1A, CYP1B, CYP1C, and CYP1D) and is used as a strong biomarker to assess pollution of the aquatic environment. Regarding transcript profiles of CYP1 family in this species, Om-CYP1A1 and Om-CYP1B1 were significantly induced by different concentrations of WAF exposure over time-courses. Also, in the same way, 3 CYP1C subfamilies and Om-CYP1D1 showed upregulated expression patterns. Tran-

with well-characterized mechanisms in vertebrates and aquatic invertebrates (Figure 4).

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DISCUSSION In vivo effects of different concentrations of WAF exposure revealed that high concentrations (100%) of WAF induced cardiac edema without any detrimental effect on embryo hatching. However, significant mortality was observed in newly hatched larvae and juvenile fish upon WAF exposure for 96 h. Regarding WAF sensitivity of both group, we showed that a detoxification mechanism is gradually set during juvenile development after hatching. Since our research objective is to analyze the CYP-triggered whole detoxification mechanism in WAF-exposed marine medaka, we chose 20% WAF-exposed juvenile fish that showed no mortality and any detrimental physiological effect. WAF-induced cardiac edema and the examination of its induction mechanism during embryogenesis are in progress. Our results show that the application of whole CYP profiling is a valuable testing method for the study of exposure to WAFs, as each transcript of CYPs can respond to certain substrates of the mixed-pollutant via a specific response mechanism. The present investigation was initiated to obtain our knowledge of the potential, in environmental monitoring, of using transcript profiling of whole Om-CYP families as a novel approach for risk assessment of the marine aquatic environment. Based on the specific response of individual CYPs, the application using whole CYPs could be expanded to diverse research areas. For example, CYP profiles permit to examine the effects of generating a chemical specific profile. In this way, we analyzed 4808



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Figure 4. Schematic summary of CYPs-involved molecular responses by WAF exposure in the marine medaka.

scripts of a diverse family of CYP1 in fish would be induced by a number of organic or inorganic compounds, and several recent studies strongly suggested that those genes showed biomarker potential for the exposure of WAF, heavy oil, or weathered oil in fish.18−22 Our results are in accord with the previously published findings. Therefore, Om-CYP1 family is a promising biomarker for WAF exposure based on the clear correlations of transcript expressions in different concentrations and timecourse exposure series. In mammals, CYP2 and CYP3 families are believed to play crucial roles in eliminating a large number of diverse xenobiotics and drugs.6,8,23 A series of characterizations of CYP2/3 family genes on their responsive ability for diverse substrates conducted in several fish revealed that many CYP2/3 family genes are involved in the metabolism of xenobiotics and endogenous metabolites.24−29 Therefore, it can be concluded that WAF exposure could induce several genes of the CYP2/3 family to metabolize different components or mixed compounds of WAF in this species. Also, it may be that either the detoxification mechanism or further toxicity would be induced by modulating the CYP1−3 family in this species. To expand our knowledge on the effects of WAFs on detoxification metabolism, several key regulators or metabolizing enzymes involved in phase I, II, and III metabolism were further examined. In vertebrates, transcription factors regulate drug/detoxification mechanisms via the AhR and nuclear receptor family such as constitutive androstane receptor (CAR), PPARs, and pregnane X receptor (PXR).30,31 These major xenobiotic-sensing receptors are activated by diverse environmental pollutants including PAHs. In our study, both mRNA expression of AhRs and PPARs were significantly

induced by different concentrations of WAF exposure. Differences in transcript fold changes of Om-AhRs directly indicated the functional ability of AhR2 to bind PAHs as suggested previously in teleosts.32,33 Regarding significantly induced AhR battery such as CYP1A1 and CYP1B1, we suggest that AhR signaling plays a crucial role in both sensing and metabolizing the vast majority of WAFs in the marine medaka. To date, we have not identified orthologs of CAR and PXR in this species but several studies have suggested the potential roles of PPARs in the detoxification metabolism of diverse exogenous substrates;34−36 further study would be needed to clarify this. Using an in vitro system, Kim et al. suggested that PAHs would be a strong exogenous ligand for PPARs.37 Therefore, we would be able to consider a possibility of AhR/ NR-triggered transcript modulation of Om-CYP by WAF exposure in this species. Each superfamily of phase II metabolizing enzymes consists of subfamilies of genes, having different substrate specificity and inducibility and/or inhibitory by xenobiotics.38 As shown in Figure S2 and Table S6, total enzyme activities of GST and SULT and several transcripts of GSTs, UGTs, SULTs, and EH were significantly increased in this study, indicating that the WAFs-triggered activation of phase II detoxification mechanism occurred in this species. In fact, to eliminate xenobiotics containing oxidized functional groups, reactive electrophiles are further conjugated by phase II detoxification enzymes, such as GSTs, SULTs, and UGTs after phase I biotransformation.5 Moreover, multifaceted and complicated signal processes regulate transcriptional responses of phase II enzymes via several cis-acting regulatory elements.38 We also found antioxidant response elements (ARE) and xenobiotic response 4809



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to deoxycorticosterone), and CYP27 (vitamin D3 metabolism) are notable.12,50 In fact, PAHs, one of the major components of WAFs, significantly inhibited steroidogenic activities of such CYPs and their products with inhibitory effects of other important steroidogenic enzymes (e.g., 3β-HSD, 11β-HSD, 17β-HSD, SRD5A, and StAR) in fish with emphasis on a potential inhibitory role of WAFs in this species.51 Typically PAHs are known to interrupt innate endocrine function by either blocking or mimicking traits. Indeed, PAH components of WAFs would disrupt endocrine functions including steroidogenesis due to their competitive binding affinity on the active site of aromatase and interaction with the estrogen receptor (ER)-dependent pathway via activation of AhR.52,53 Regarding steroidogenic products and biomarkers, changes in fish steroid hormones have been known to be a biomarker upon PAH exposure. In fact, several PAHs and WAFs of diesel oil and gasoline affected physiological mechanisms regulated by estradiol or plasma cortisol level.54−56 Thus, we assumed that WAF treatment would modulate the innate steroid cascade of the marine medaka, although hormone levels of estradiol and cortisol were not measured in this species. In addition, upon cellular damage by WAF, the energy budget is limited. The consumed metabolic energy for increased detoxification activity in WAFs-exposed marine medaka might directly influence a decreased transcript expression of steroidogenic CYP genes. This explanation is congruent with findings from the literature reporting an increase of metabolic cellular fuels (e.g., glucose, lactate) and breakdown of steroidogenesis after exposure to several PAHs including WAFs in fish.55,57,58 Therefore, WAFs-induced toxic effects with modulation of hormone levels would produce changes in whole energy metabolism of the WAFs-exposed marine medaka. Taken together, our study suggests that the endogenous steroidogenesis pathway would be affected directly or indirectly by WAF exposure in this species. Physiological change and/or hormonal disruption should be analyzed by in vivo testing and modulation of gene expression. In conclusion, our study suggests that two different metabolic pathways of CYPs, such as detoxification mechanism and steroidogenic metabolism, are targets of WAFs. Some of our results are supporting the observation as demonstrated in mammalian studies in terms of the correlation of PAH effects with CYP induction and/or inhibition as well as CYP-involved pathways. Also, these data are in agreement with physiological response of teleosts associated with PAH treatment as discussed previously. Overall, further studies would be helpful to compare the role of each CYP to prove its biomarker potential in either laboratory or field studies, as different components of WAFs may have different modulatory effects. However, our study is supportive that overall CYP genes’ profiling can be a powerful method for better understanding of the mode of action of certain chemicals, particularly those that are emerging.

elements (XRE)/aromatic hydrocarbon response elements (AhRE) in 5′-flanking regions of several phase II enzymes of the marine medaka (data not shown). The results from this study indicate that metabolic activation of WAFs by a series of detoxification mechanism may occur in the marine medaka. This finding is novel in showing impacts of WAF exposure in the view of induction of conjugating enzymes in phase II xenobiotic metabolism. Regarding transcript induction of ABC transporters of the marine medaka, our results suggest the strong involvement of several transporters in terms of eliminating WAF to avoid toxicity. ABC transporters typically export a broad range of substrates including xenobiotics in the phase III detoxification system.39 In the marine medaka, one of the CYP3A subfamily, Om-CYP3A38 gene, and Om-P-gp significantly were induced by WAF exposure, as shown in mammals in which these are tightly coexpressed in a regulated manner for xenobiotic elimination.40,41 Co-induction of ABC transporters with phase II enzymes showed a synergistic role in mediating the elimination of xenobiotics in this species.42 Also, transcriptional activation of ABC transporters would be modulated by PAHs via the AhRE in their promoter region.43 In addition, mRNA expression of the cellular stress protein Hsps and enzyme activity of antioxidant-related genes were significantly induced by WAF exposure. Since the general mechanisms of stressrelated genes’ induction upon xenobiotic exposures including PAHs have been well-known in fish species, detailed discussion was excluded in this manuscript. Taken together, we concluded that the effects of WAFs were a function of induction in intracellular detoxification mechanism, regardless of property or mechanism of individual component of WAFs. Regarding the CYPs’ critical role in the series of detoxification mechanism, we suggest that CYP profiling would be a potential biomarker to predict the combinational mode of action of certain chemical and serve as strong early warning signal for chemical toxicity. Another interesting point revealed by our study is that intracellular stresses were induced by WAF exposure in this species. As several researchers suggested, induced enzyme activities of antioxidant-related proteins of the marine medaka in relation to antioxidant defense system is associated with detoxifying oxidative stress-triggered detrimental effects of PAHs in WAFs.44−46 As for Hsps expression, there are no studies available on the effect of WAFs in fish species, while in other aquatic animals cellular defense proteins such as molecular chaperones and several Hsps are induced as adaptive and protective mechanisms against environmental stresses.47−49 Thus, these observations highlight the potential effect of WAFs in the modulation of intracellular stress with CYP regulation, even though the toxicity mechanisms and a way of elimination of WAF-triggered intracellular stress are not fully understood. To date, contradictory results have been reported in fish. As one of other interesting findings, WAF exposure reduced the E2 level and transcript expressions of diverse endogenous metabolism-mediated Om-CYPs. These results were not found in previous studies, indicating that the activation of detoxification pathways may not be sole biomarkers for WAF exposure from the modulation of whole CYPs. Of these genes, steroidogenic genes such as CYP11 (subfamily for pregnenolone and aldosterone synthases; CYP11A, essential for cholesterol side-chain cleavage; CYP11C1, putative aldosterone synthase), CYP17A (catalyze dual functions of steroid-17α hydroxylase and steroid-17, 20-lyase), CYP19 (is important in the biosynthesis of estrogen), CYP21A (convert progesterone

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ASSOCIATED CONTENT

S Supporting Information *

Gene annotation, chemical preparation, RNA extraction, realtime RT-PCR, amplification conditions, measurement of enzyme activity, and technical details of relevant methods. This material is available free of charge via the Internet at http://pubs.acs.org. 4810



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AUTHOR INFORMATION

Corresponding Author

*Telephone: +82-2-2220-0769; fax: +82-2-2299-9450; e-mail: [email protected]. Author Contributions #

J.-S.R. and B.-M.K. contributed equally to this manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. J. K. Chipman and Dr. Hans-U. Dahms for their comments on the manuscript. We also thank Drs. Jong-Hyeon Lee and Chan-Gyoung Sung (Institute of Environmental Protection and Safety, NeoEnBiz Co., Bucheon, South Korea) for their excellent technical assistance on in vivo effects of WAF exposure. This study was supported by a grant from the Korean Ministry of Oceans and Fisheries (PM56380: Oil Spill Environmental Impact Assessment and Environmental Restoration) funded to Jae-Seong Lee.

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