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Characterization and expression of cytochrome p450 cDNA (CYP9AT2) in Chironomus riparius fourth instar larvae exposed to multiple xenobiotics Prakash M. Gopalakrishnan Nair a,b , Sun Young Park a , Jinhee Choi a,∗ a

School of Environmental Engineering and Graduate School of Energy and Environmental System Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743, Republic of Korea b Department of Applied Biosciences, College of Life and Environmental Sciences, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea

a r t i c l e

i n f o

a b s t r a c t

Article history:

We identified and characterized a CYP9 family gene, CrCYP9AT2, from Chironomus riparius,

Received 3 March 2013

an eco-toxicologically important model organism. The 1978 base pair (bp) length CrCYP9AT2

Received in revised form

cDNA has an open reading frame of 1587 bp encoding a putative 528 amino acid protein.

21 August 2013

There was 267 bp 5 and 123 bp 3’ untranslated region with a polyadenylation signal site

Accepted 24 August 2013

(AATAAA). The putative heme-binding cysteine at position 471 and the typical p450 signature

Available online 31 August 2013

sequence of 463-FGIGPRNCIG-473 were also present. The CrCYP9AT2 transcript was present in all life stages with the highest expression in larvae. The modulation of CrCYP9AT2 was

Keywords:

studied using real-time polymerase chain reaction after 24 h exposure to cadmium chloride,

C. riparius

benzo(a)pyrene; bisphenol A; nonylphenol; chlorpyrifos and ethinylestradiol. Significant up-

CYP gene

regulation of CrCYP9AT2 gene was observed after exposure to Cd, B(a)P and CP. However,

Gene expression

CrCYP9AT2 was significantly down-regulated after exposure to BPA, NP and EE.

Molecular biomarker

1.

Introduction

The cytochrome p450 (CYP450) system comprises a super family of Phase I, heme-containing monooxygenase enzymes found in a diverse array of organisms including bacteria, plants and animals playing a central role in the oxidative metabolism or biotransformation of a wide range of xenobiotic substances (Guengerich, 1999; Scott, 1999). Because of their roles in the detoxification or bio-activation of foreign compounds, studying the alteration of the expression of CYP genes by environmental pollutants is important since the induction mechanism is regulated at the transcriptional level (LaBella, 1991; Batard et al., 1997; Williams et al., 1998). Transcriptional



Corresponding author. Tel.: +82 2 6490 2869; fax: +82 2 6490 2859. E-mail address: [email protected] (J. Choi). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.08.011

© 2013 Elsevier B.V. All rights reserved.

activation of CYP genes and subsequently p450 proteins by various environmental pollutants has already been reported (Feyereisen, 2005). Several CYP genes have been identified from insects and were classified into six types viz. CYP4, 6, 9, 12, 18, and 28 (Feyereisen, 1999). Aquatic organisms are exposed to various types of environmental pollutants having different modes of action viz. heavy metals, pesticides and xenoestrogens, released from various anthropogenic sources which can disrupt many biological functions. Heavy metal cadmium chloride (Cd) is a widespread environmental pollutant and accumulates in superficial sediments in aquatic systems through various ways (Korte, 1983; Nriagu et al., 1998). Toxicity and endocrine disrupting effects of Cd to several aquatic organisms has been reported from

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previous studies (Pascoe et al., 1989; Postma and Davis, 1995). Alkylphenols, such as nonylphenol (NP), is widely used in many industrial applications as well as household cleaning products and are released to aquatic environments (Ying et al., 2002). The eco-toxicological effect of NP to aquatic organisms has been reported from previous studies (Nimrod and Benson, 1996; Lee and Choi, 2006). Polycyclic aromatic hydrocarbons such as benzo(a)pyrene [B(a)P] is widely present in aquatic systems and reported to cause many adverse effects on organisms (Juhasz and Naidu, 2000; Nogami et al., 2000; Shaw et al., 2004). Bisphenol A (BPA) is an intermediate in the production of polycarbonate and epoxy resins having endocrine disrupting properties and causes toxic effects to aquatic organisms (Staples et al., 1998). Ethynylestradiol (EE) is a synthetic estrogen used as a female contraceptive (Purdom et al., 1994) and is a well-known endocrine-disrupting chemical frequently found in aquatic environments (Gutendorf and Westendorf, 2001). Chlorpyrifos (CP) is a widely used organophosphorous insecticide which could enter to aquatic systems by many routs (Cowgill et al., 1991). The toxicity of CP to aquatic organisms has been reported from many studies (Giesy et al., 1999). The aquatic midge, Chironomus riparius, is widely used as an ecologically important bio-monitoring species due to their widespread distribution, tolerance to various environmental conditions, short life-cycle and easily identified developmental stages (EPA, 1996). Due to their association with benthic sediments, C. riparius larvae are also used for evaluating sediment toxicity (OECD, 2004). Survival tests, morphological changes and developmental parameters are used in most studies to evaluate toxicity responses (Callaghan et al., 2001; Watts et al., 2001, 2003). Even though, studies using classical end points give valuable information about the effect of chemicals, studying molecular level changes such as alteration of gene expression has become a major tool as early-warning biomarkers in animals exposed to environmental contaminants (Nuwaysir et al., 1999; de Longueville et al., 2004). Since genomic sequence information is scarce in C. riparius and is essential to study the effect of environmental pollutants at the molecular level, we identified and characterized the full length cDNA of a CYP gene, classified as CrCYP9AT2 from the previously developed expresses sequence (ESTs) data base (Nair et al., 2011). The transcriptional modulation of CrCYP9AT2 was studied upon exposure to environmental pollutants having different modes of action viz. Cd, NP, B(a)P, BPA, CP and EE.

2.

Materials and methods

2.1.

Animals and chemical exposure

C. riparius larvae provided by the Korea Institute of Toxicology (Daejeon, South Korea), were reared in a 2 L glass chamber containing aerated, dechlorinated tap water and acid washed sand. The larvae were fed with fish flake food (Tetramin, Tetrawerke, Melle, Germany) and exposed to 16 light plus 8 h dark photoperiod at a temperature of 20 ± 2 ◦ C. Exposure to Cd (0, 2, 10, 20 mg/L), NP (0, 10, 50 and 100 ␮g/L), B(a)P (0, 10, 100, 1000 ␮g/L), BPA (0, 1, 10, 100 ␮g/L), CP (0, 0.2, 1, 2 ␮g/L) and EE (0, 1, 10, 100 ␮g/L) were done for 24 h period. All the chemicals used in this study were obtained from Sigma–Aldrich, USA.

The exposure concentrations of the chemicals were selected based on previously conducted acute and chronic toxicity tests in our laboratory. Three independent sets, containing 15 fourth instar larvae in each exposure set, were maintained for all exposure conditions and for the controls in beakers containing 100 mL dechlorinated tap water. The controls were maintained without any exposure to chemicals for the different durations and concentrations along with the chemical exposed samples. After the exposure, the larvae were collected and immediately frozen in liquid nitrogen before being stored at −80 ◦ C.

2.2.

Identification and sequence analysis

An ESTs database was developed earlier by pyrosequencing of cDNA obtained from fourth instar larvae of C. riparius (Nair et al., 2011). In short, sequencing was performed following the protocols for the genome sequencer GSFLX system (Roche, Mannheim, GE). The reads were assembled using the GS De Novo Assembler (http://454.com/productssolutions/analysis-tools/gs-de-novo-assembler.asp). C. riparius CYP9AT2 cDNA sequence was retrieved from the ESTs database using BlastX searches of the NCBI GenBank database (http://blast.ncbi.nlm.nih.gov/). The deduced amino acid sequence of the CrCYP9AT2 cDNA was aligned with equivalent sequences from other species using ClustalW (Thompson et al., 1997).

2.3.

Expression analysis of the CrCYP9AT2

Total RNA was extracted from the control and exposed samples using TrizolTM (Invitrogen, USA) following the manufacturer’s instructions. Developmental expression of the CrCYP9AT2 gene transcript was investigated in eggs (two egg masses), fourth instar larvae, pupae and male and female adults (five animals for each stage). One microgram of total RNA from different developmental stages, control and chemical exposed larvae was used for making cDNA by reverse transcription using an oligo dT20 primer and RT-Premix (Bio-Rad, USA) in a 20 ␮L reaction, as per manufacturers’ instructions. CrCYP9AT2 and Chironomus actin gene-specific primers were designed using Primer 3.0 (http://frodo.wi.mit.edu/primer3/) (Table 1). The substrate specificity of CrCYP9AT2 primer was checked using the polymerase chain reaction (PCR) using conditions of 94 ◦ C for 4 min followed by 35 cycles at 94 ◦ C for 30 s, 55 ◦ C for 30 s and 72 ◦ C for 30 s with a final extension at 72 ◦ C for 10 min using PTC 100 thermal cycler (MJ Research, Lincoln, MA, USA). The PCR products were subsequently run on 1.5% agarose gels and verified that only one band is obtained. The qRT-PCR was performed using a CFX96TM Real-Time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and accompanying software (CFX Manager Software) with 10 ␮L 2X IQ SYBR super mix (Bio-Rad, USA), 0.2 ␮M of the forward and reverse primers and 1 ␮L of template cDNA in a final volume of 20 ␮L. Chironomus actin gene was used as a normalizing gene for quantitative real-time PCR (qRT-PCR). The qRT-PCR reactions were run with an initial denaturing at 95 ◦ C for 7 min followed by 44 cycles of 95 ◦ C for 15 s, 55 ◦ C for 1 min and extension of 72 ◦ C for 15 s. Melting curve quantitation was done from 65 to 95 ◦ C with a heating rate of 0.2 ◦ C for every second. The qRT-PCR was done

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Table 1 – Primers used in real time PCR study. Primer name

Sequence of primer (5 –3 )

CrCYP9AT2-F CrCYP9AT2-R Actin-F Actin-R

TGGGTCGCTCAAGATGCTGCC ACGACGCCATGCAGCACGA GATGAAGATCCTCACCGAACG TTCGAGTGAGGTTGATGCAG

using samples from three independent exposure and control sets.

2.4.

Data analysis of gene expression

Cycle threshold (Ct ) values were converted to relative gene expression levels by 2−Ct method using the gene expression analysis software provided with the Bio-Rad PCR machine (Bio-Rad, USA). Statistical analysis of the results obtained from control and treated larvae were done using one-way analysis of variance (ANOVA) using SPSS 12.0 KO (SPSS Inc., Chicago, IL, USA). Dunnett’s post-doc test was done to determine the effect of different exposures on gene expressions and p values less than 0.05, 0.01 and 0.001 was considered as statistically significant. All data were given as relative mRNA expression as means ± S.E.

3.

Results

3.1.

Sequence analysis of CrCYP9AT2

In this study, the full length cDNA encoding a CYP gene was identified from the C. riparius ESTs database and the expression of the corresponding mRNA was studied during distinct stages of development and following exposure to Cd, NP, B(a)P, BPA, CP and EE. Based on the nucleotide sequence analysis it was assigned to CYP gene, family 9 and subfamily AT2 and was named as CrCYP9AT2 (http://drnelson.uthsc.edu/CytochromeP450.html). The complete cDNA of CrCYP9AT2 has an open reading frame of 1587 bp, encoding 528 amino acid residues with a predicted molecular mass of 61.29 kDa. It has a 123 bp 5 and 267 bp 3’untranslated region with a polyadenylation signal (AATAAA) (Fig. 1). Alignment of the CrCYP9AT2 amino acid sequences with other insect species’ CYP9 genes indicated the presence of putative threonine residue at position 321 which is participating in proton delivery network in the enzymatic active site and heme-binding cysteine at position 471 in the typical P450 signature sequence of ‘FGIGPRNCIG’ at positions 463–473 (Fig. 2). Since CrCYP9AT2 contains conserved amino acids and motifs present in well characterized CYP genes from other species the nomenclature of CrCYP9AT2 is justified and the sequence CrCYP9AT2 is deposited in NCBI GenBank under the accession no. JN600619.

3.2.

Expression analysis of CrCYP9AT2 mRNA

The mRNA expression of CrCYP9AT2 gene was studied during different developmental stages using RT-PCR and was found to be expressed during all stages of development with the highest expression being found in the larval stage (Fig. 3). To assess the transcriptional modulation of the CrCYP9AT2

Amplified product length (bp) 100 145

gene, the mRNA expression in C. riparius fourth instar larvae was analyzed using real-time PCR, under untreated and 24 h treatment conditions with different environmental pollutants having different modes of action. It was observed that, exposure to 2 mg/L of Cd did not change the mRNA expression of CrCYP9AT2 gene significantly. However, significant up-regulation of CrCYP9AT2 gene was observed after exposure to 10 and 20 mg/L of Cd as compared to the controls (p < 0.05; p < 0.01) (Fig. 4A). There was no significant effect after exposure to10 ␮g/L of NP as compared to the control conditions. Significant decrease in the mRNA level of CrCYP9AT2 was observed after exposure to 50 and 100 ␮g/L of NP (p < 0.05; p < 0.01) (Fig. 4B). Exposure to 10 ␮g/L of B(a)P resulted in no significant change in the expression of CrCYP9AT2 gene as compared to the control. However, exposure to 100 and 1000 ␮g/L of B(a)P has resulted in 4 and 6 fold increases in CrCYP9AT2 mRNA expression respectively (p < 0.001) (Fig. 4C). No significant change was observed after exposure to 1 and 10 ␮g/L of BPA but at 100 ␮g/L exposure conditions CrCYP9AT2 gene showed a significant decrease (p < 0.01) in the expression level as compared to the control (Fig. 4D). The results showed that, after exposure to 2 ␮g/L of CP, there was a 2.5 fold increase in the CrCYP9AT2 transcript level, which was significantly different from controls (p < 0.05). However, larvae which were exposed to 0.2 and 1 ␮g/L of CP exhibited no significant change in CrCYP9AT2 mRNA levels as compared to the controls (Fig. 4E). Down regulation of CrCYP9AT2 mRNA was observed after exposure to different concentrations of EE which was significant at 10 and 100 ␮g/L (p < 0.05; p < 0.01) as compared to the control (Fig. 4F).

4.

Discussion

In insects such as Anopheles gambiae and Aedes aegypti, several CYP9 gene families has been identified which were involved in detoxification of xenobiotics and metabolic resistance to insecticides (David et al., 2005; Strode et al., 2008). However, only limited sequence information of CYP genes, especially CYP9 genes, are available from the eco-toxicologically impor˜ et al., 2007; Park and tant aquatic organism C. riparius (Londono Kwak, 2008; Martínez-Paz et al., 2012). In the present study, we identified and characterized a CYP9 gene from C. riparius and studied its mRNA expression after exposure to a variety of environmental pollutants. The CrCYP9AT2 gene showed high homology with equivalent genes from different species and the presence of several conserved residues were also identified. Presence of the heme-binding motif (FxxGxxxCxG), which is known to be a highly conserved segment (Black and Coon, 1987; Porter and Coon, 1991; Tudzynski and Hölter, 1998), was conserved in deduced amino acid sequences of CrCYP9AT2 gene.

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1 acgatttatacgtcacatcaaatactcgtattc 34 tgttaagttcttgatcataaattaactcaacaaaaaaactttaat 79 tttttttctcagcaaattaactggtatttcaaggtcatatcaatc 124 atgctgtatttggttctaattcttgccatcatctacttgatatac M L Y L V L I L A I I Y L I Y 169 aaatggggaacaaatactttcgattactttgaaaagagaggtatt K W G T N T F D Y F E K R G I 214 aagtataacaagccgttgttccttgttggttctcgattgagtatt K Y N K P L F L V G S R L S I 259 cttttgaagaactcaagtatggtcgactctgttcagaagacttat L L K N S S M V D S V Q K T Y 304 aaagagtttagaaatgagaaaatctcgggaatgtttgagtttaag K E F R N E K I S G M F E F K 349 cacccaacttattttattcgagatcccgacatcattaaacgttta H P T Y F I R D P D I I K R L 394 gccattaaagaatttgaccattttacggatcatcgtcttgttctt A I K E F D H F T D H R L V L 439 gacgaagaagttgaacctctctttgctaagggattatttggttta D E E V E P L F A K G L F G L 484 actggacagaaatggaaagacatgcgagcaactttatcgccggct T G Q K W K D M R A T L S P A 529 tttacaggtagtaaaatgcgcttgatgtttaacctaatgaataaa F T G S K M R L M F N L M N K 574 gttgggtcacagatgacagcttcaatcagaaatcaaatcgacagt V G S Q M T A S I R N Q I D S 619 ggaaagaataacgaagttgaattcaaggaattcgcgagaaatttt G K N N E V E F K E F A R N F 664 actttagatattattgctacttgcgcttttggaattgaggtcaat T L D I I A T C A F G I E V N 709 tcttttgaacatccagacaacgaattcattaagatcgctagaaaa S F E H P D N E F I K I A R K 754 gccacaaactttggtgacattccaatttataaattcattggcttc A T N F G D I P I Y K F I G F 799 tttttattccccaaattgatggcaaagctcaacattaaattcctt F L F P K L M A K L N I K F L 844 gataaggatttatatgtattttttgacgaagtgttgtcggatacg D K D L Y V F F D E V L S D T 889 attcagcaacgagagaaaaaaggaatagtccgaaatgacatgatt I Q Q R E K K G I V R N D M I 934 gatcttttactgcaagctaaaaaaggaagcctaactcatgatgaa D L L L Q A K K G S L T H D E

979 agtcaagaagaaacgttgtcaaacattggttttgcaacagttgag S Q E E T L S N I G F A T V E 1024 gaatcagacattggaaaacacaaagtcaaaagaacttggactgac E S D I G K H K V K R T W T D 1069 gaagatttaatggctcaggctttcattttcttctttgccggcttc E D L M A Q A F I F F F A G F 1114 gagactgtgtcaactgtcatgacctttatggcctacgaattacta E T V S T V M T F M A Y E L L 1159 cttaatcctgatgtccaaacaaaattacagaaggaaatcgatgaa L N P D V Q T K L Q K E I D E 1204 gtctacaaatcacttggtggaaaggaattgacatatgagcatgta V Y K S L G G K E L T Y E H V 1249 cagggtatgaagtatatggatatggttgtgtctgagacattgaga Q G M K Y M D M V V S E T L R 1294 aaatggccagctgctccagttgtcgatagaaattgcacaaaacct K W P A A P V V D R N C T K P 1339 tttactttggaatatgatgataaaaaaattgattttgaaattgga F T L E Y D D K K I D F E I G 1384 agaaacttttatgtgcctatttatgctattcatcatgatccactt R N F Y V P I Y A I H H D P L 1429 tactatgagaacccagagaaattcgaccctgaacgattcagtgat Y Y E N P E K F D P E R F S D 1474 gagaataaggataaaattgctaatgttctctacgctccattcggt E N K D K I A N V L Y A P F G 1519 ataggtccaagaaattgcattggaagcagatttgctcttctcgaa I G P R N C I G S R F A L L E 1564 gtcaaaacaattttctactatttattgttgaacttcagcttcgag V K T I F Y Y L L L N F S F E 1609 gcaaccagcaagacaaaaattccaattcagtttgttaagattcct A T S K T K I P I Q F V K I P 1654 tcaatcttccagattgaaggtggtttggacattgcattggctcca S I F Q I E G G L D I A L A P 1699 agatcaaagtaaattgaatgttaaagtaaattaaaattttattcc R S K * 1744 tataaagatgataataattatgaaaattaaagcttattgctgttg 1789 ttatatggcttggaaacgtcaaattttattttataaagaaagttc 1834 aagttcaatttcaaattcaactttaacttcaagtttggtttattt 1879 ttcaaactattgttaagaaaatgtaagtttttgaatctattttat 1924 ggaaagacgcgcaaaaacttcaaaattttaaataaagccAATAAA 1969 gctatgatt 1978

Fig. 1 – Nucleotide and conceptual amino acid sequences of Chironomus riparius full length cDNA of CYP9AT2. The nucleotides are numbered on the left. Both start codon (atg) and stop codon (taa) are boxed. The polyadenylation signal (AATAAA) is in bold italics. The typical p450 signature sequence of FGIGPRNCIG is dark shaded.

In this study, we investigated the differential transcript expression levels of CrCYP9AT2 gene in different developmental stages such as egg, fourth instar larvae, pupae, adult males and adult females. Our results showed that the expression of CrCYP9AT2 was highest in fourth instar larvae. Similarly it was reported that in A. aegypti, the expression levels of a CYP9 gene, i.e. CYP9M9 was highest in larvae compared to pupae (Poupardin et al., 2010). It was speculated that the reason for the highest expression levels of CYP9M9 gene in larval stage as compared to other stages might be due to the feeding behavior since the larvae are more exposed to dietary xenobiotics (Poupardin et al., 2010). Similar reason might have resulted in the highest up-regulation of CYP9AT2 gene in fourth instar larvae of C. riparius as observed in this study. It is known that, in insects, CYP9 families are involved in detoxification of xenobiotics and metabolic resistance to insecticides (Poupardin et al., 2010). The mRNA modulation CrCYP9AT2 gene was analyzed in fourth instar larvae of C. riparius after exposure to different environmental pollutants that are reported to be present in fresh water systems. The induction of different isoforms of CYPs on exposure to

environmental xenobiotics has been reported from several aquatic organisms. The presence of an inducible cytochrome p450 has been reported from C. tentans (Miota et al., 2000; ˜ et al., 2004, 2007). Among the different chemicals Londono used, the highest expression of CrCYP9AT2 was observed after exposure to B(a)P which was about 4–6 fold higher than the control sets. Uppstad et al. (2010) reported that exposure of human lung cell lines to B(a)P for 24 h has resulted in an approximately 100 fold increase in P450 gene expression levels. In this study, it was observed that, exposure to CP also made significant up-regulation in the expression of CrCYP9AT2. Similarly, induction of CYP450 gene after exposure to atrazine and CP has been reported from C. tentans ˜ et al., 2004; Rakotondravelo et al., 2006; Jin-Clark (Londono et al., 2008). In the present study, we also found that heavy metal Cd increased the expression of CrCYP9AT2 mRNA in C. riparius. It has been reported that in Hepa 1c1c7 cells, exposure to Cd increased the mRNA expression level of CYP1A1 (Elbekai and El-Kadi, 2007). It was proposed by earlier researchers that one of the reasons for the induction of CYP genes following xenobiotic exposure could be due to the oxidative stress (Ding et al., 2005; Poupardin et al., 2010). For

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Fig. 2 – Alignment of CYP9 family amino acid sequences with Chironomus riparius CYP9AT2 gene. The deduced amino acid sequence for CrCYP9AT2 was aligned with CYP9 genes from different species using ClustalW. Center of ␣-helix I which contains conserved threonine residue (marked with asterisk symbol), and heme-binding motif are boxed. GenBank accession numbers for sequences used are given in brackets. Drosophila melanogaster CYP9f2 (NP 650189.1), D. melanogaster CYP9h1 (NP 610820.1), Culex quinquefasciatus CYP9b2 (XP 001862746.1), C. quinquefasciatus CYP9b1 (XP 001855241.1), C. quinquefasciatus CYP9c1 (XP 001855254.1), C. quinquefasciatus CYP9J40 (AEN19673.1), Aedes aegypti CYP9J10, partial (AEN19673.1), Zygaena filipendulae CYP9A36 (ACZ97417.2), Tribolium castaneum CYP9Z4 (NP 001164248.1), Bombyx mori CYP9A19 (ABQ08709.1). Identical amino acids are shaded with same color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

example, the induction of CYP genes mediated through oxidative stress after exposure to B(a)P has been reported (Tsuji et al., 2011). In the present study, it was observed that, exposure to estrogenic compounds such as BPA, NP and EE significantly decreased the expression of CrCYP9AT2. Recently it was reported that the expression of CrCYP4G gene was down regulated after exposure to different concentrations of NP and BPA (Martínez-Paz et al., 2012). Hanioka et al. (2000) reported that exposure to BPA has resulted in the inhibition of CYP2 and CYP3. In a previous report, in vivo and in vitro experiments showed that NP significantly suppressed hepatic p450 gene expression levels, EROD activity and p450 protein in Atlantic salmon (Arukwe et al., 2000). Similarly in Salmo salar, exposure to NP significantly suppressed hepatic CYP gene expression level which was correlated with decrease in EROD activity and protein levels (Arukwe et al., 2000). In trout hepatocytes, NP exposure resulted in the reduction of CYP gene expression (Navas and Segner, 2000). In marine fish, Gobius niger, exposure to NP produced a decrease of CYP mRNA levels

Fig. 3 – Real-time PCR analysis of Chironomus riparius CYP9TA2 mRNA transcripts at different developmental stages. The mRNA expression of CrCYP9TA2 gene was quantified using real time PCR and normalized using Chironomus actin gene.

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Fig. 4 – Analysis of the expression of Chironomus riparius CYP9TA2 mRNA after 24 h exposure to various environmental xenobiotics in fourth instars Chironomus riparius larvae. Expression of CrCYP9TA2 mRNA after exposure to (A) cadmium chloride; (B) nonylphenol; (C) benzo(a)pyrene; (D) bisphenol A; (E) chlorpyrifos, and (F) ethinylestradiol. Real-time PCR experiments were performed using gene-specific primer and Chironomus actin gene was used as an internal control to normalize gene expression and to calculate relative mRNA expression levels. The expression level under control conditions was set to 1. The average and standard errors of measurements taken in three independent experiments with three sample replicates for each exposure condition are shown. Significant differences (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001).

(Maradonna et al., 2004). In Atlantic cod (Gadus morhua), NP exposure resulted in significant inhibition of five CYP genes (Olsvik et al., 2009). Similar to the results obtained in our study, exposure to EE decreased the expression of CYP gene in Zebra fish (Danio reiro) (Notch et al., 2007). Chang et al. (2009) also reported that exposure to EE inhibited eleven human CYP450s. Down-regulation of murine p450 in mouse hepatoma

Hepa-1c1c7 cells by BPA has been reported (Jeong et al., 2000). Although we observed the modulation of CrCYP9AT2 mRNA expression by different environmental pollutants in this study, the molecular mechanisms through which such up or down regulation occurs is not clear. Therefore, further studies are required to understand the involvement of CrCYP9AT2 in xenobiotic metabolism in C. riparius.

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5.

Conclusion

In conclusion, determination of the effects of environmental pollutants belonging to several classes at gene level, apart from studying at the organism level, will give an immediate idea about each chemicals effect at molecular level. Moreover, since C. riparius is used as an important species in eco-toxicological studies, identification and characterization of CYP9 (CrCYP9AT2) could be used as molecular biomarkers. In addition, studying the modulation of CrCYP9AT2 will give an idea about the involvement of cytochrome p450 family mediated detoxification of environmental pollutants in C. riparius, an eco-toxicologically important organism.

Conflict of interest statement The authors have declared that no conflict of interest exists.

Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2041679).

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Characterization and expression of cytochrome p450 cDNA (CYP9AT2) in Chironomus riparius fourth instar larvae exposed to multiple xenobiotics.

We identified and characterized a CYP9 family gene, CrCYP9AT2, from Chironomus riparius, an eco-toxicologically important model organism. The 1978 bas...
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