Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata)

Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata)

Marine Pollution Bulletin 51 (2005) 784–793 www.elsevier.com/locate/marpolbul Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in ...

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Marine Pollution Bulletin 51 (2005) 784–793 www.elsevier.com/locate/marpolbul

Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata) Satoko Niimi a, Michio X. Watanabe a, Eun-Young Kim a,b, Hisato Iwata Genta Yasunaga c, Yoshihiro Fujise c, Shinsuke Tanabe a b

a,*

,

a Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan Ehime Prefectural Institute of Public Health and Environmental Science, Sanban-cho 8-234, Matsuyama 790-0003, Japan c The Institute of Cetacean Research, Toyomi-cho 4-5, Chuo-ku, Tokyo 104-0055, Japan

Abstract This study presents full-length cDNA sequences of CYP1A1 and 1A2, in common minke whale (Balaenoptera acutorostrata) from the North Pacific. Both CYP1A1 and CYP1A2 cDNAs had an open reading frame of 516 amino acid residues, and predicted molecular masses were 58.3 kDa and 58.1 kDa, respectively. The deduced full-length amino acid sequence of CYP1A1 revealed higher identities with those of sheep (86%) and pig (87%), and that of CYP1A2 was most closely related to human (82%) and monkey CYP1A2 (82%) among species from which CYP1A2 has been isolated so far. Differences in certain conserved and functional amino acid residues of CYP1A1 and 1A2 between common minke whale and other mammalian species indicate the possibility of their specific metabolic function. Concentrations of organochlorine compounds (OCs) including PCBs and DDTs analyzed in common minke whale liver showed no significant correlation with hepatic mRNA expression levels of CYP1A1 and CYP1A2, indicating no induction of these enzymes by such OCs.  2005 Elsevier Ltd. All rights reserved. Keywords: Common minke whale; Cytochrome P4501A1, Cytochrome P4501A2; mRNA expression; cDNA library; Organochlorine compounds; Biomarker

1. Introduction Organochlorine compounds (OCs) such as polychlorinated biphenyls and DDTs (1,1,1-trichloro-2,2-bis(pchlorophenyl) ethane) have generated serious concern because of their ubiquitous distribution, toxicity, and bioaccumulation potential (Tanabe et al., 1994). The common minke whale (Balaenoptera acutorostrata) is chronically exposed to these toxic contaminants as a result of global pollution (Aono et al., 1997). No study has

*

Corresponding author. Tel./fax: +81 89 927 8172. E-mail address: [email protected] (H. Iwata).

0025-326X/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2005.07.012

so far addressed biological, physiological and clinical endpoints to evaluate properly the exposure and effects of these toxic contaminants in this species. Cytochrome P450 (CYP) enzyme participates in classical phase I metabolic reaction in which the substrate is oxidized. The CYP enzyme is primarily responsible for bioactivation and detoxification of a variety of endogenous and xenobiotic compounds. Of the numerous CYP families identified, CYP1-4 families are induced by xenobiotic chemicals, and the induction is mediated by specific or multiple receptors such as aryl hydrocarbon receptor (AhR), constitutive androstane receptor, pregnane X receptor, and peroxisome proliferator-activated receptor (Schmidt and Bradfield, 1996; Waxman,

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1999; Nebert and Russell, 2002). Therefore, CYP1-4 families are of particular importance as biomarkers of chemical contamination (Nebert and Russell, 2002). The CYP1 family consists of at least three subfamilies, CYP1A, CYP1B and CYP1C. In the CYP1A subfamily, genes of two members, CYP1A1 and CYP1A2 have been identified in mammals. Expression of the CYP1 gene family is mediated by AhR signaling pathway which is activated by planar aromatic hydrocarbons (PAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar PCBs and benzo(a)pyrene (Okino and Whitlock, 1995; Schmidt and Bradfield, 1996; Mimura and Fujii-Kuriyama, 2003). CYP1A1 is expressed in a variety of tissues, whereas the expression of CYP1A2 is mainly confined to the liver (Lewis, 2001). The CYP1A1/2 expression levels have been proposed as a potential early warning biomaker, capable of monitoring exposure to PAHs. However, there is little information on the presence (Teramitsu et al., 2000; Tilley et al., 2002; Kim et al., 2004) and expression (White et al., 1994; Chiba et al., 2002; Iwata et al., 2003) of CYP1A in marine mammals. In cetaceans, only partial sequences of CYP1A1 have been reported in limited number of species, minke whale, DallÕs porpoise and striped dolphin (Teramitsu et al., 2000; Godard et al., 2000; Kim et al., 2004). The aim of this study is to verify whether expression of common minke whale CYP1A mRNA can be a useful biomarker of chemical exposure. Present study initially attempted to isolate full-length cDNA clones of CYP1A subfamilies from common minke whale liver. Also, this study analyzed expression levels of CYP1A mRNAs and residue levels of OCs in the liver of common minke whale, and examined the relationships between CYP1A and OCs levels.

2. Materials and methods 2.1. Samples Nineteen male of common minke whales were collected from the western North Pacific in the 2001 by JARPN II (Japanese Whale Research Program under Special Permit in the Western North Pacific-Phase II). Livers of common minke whales were immediately removed after collection. Subsamples of livers were frozen in liquid nitrogen and stored at 80 C until preparation for total RNA isolation to clone CYP1A cDNAs and quantify their mRNA expression levels. The liver samples for OC analysis were stored at 20 C until analysis. 2.2. cDNA cloning of CYP1A1 and 1A2 Total RNA was isolated from the liver using RNAgent Total RNA Isolation System (Promega). We con-

785

structed a cDNA library from a minke whale liver, using an oligo-capping method (Maruyama and Sugano, 1994; Suzuki et al., 1997). A total of 6930 clones randomly selected in the library were screened for the isolation of cDNA clones encoding CYP1A1 or 1A2. The one pass sequencing of 5 0 -end of inserted cDNA fragments and subsequent annotation by BLAST search in DDBJ resulted in the isolation of cDNA clones that were classified into CYP1A. Plasmids inserted the CYP1A genes were transformed to Escherichia coli and cultured for preparation of sequencing. The CYP1A cDNAs were sequenced using ABI PRISM 310 genetic analyzer. The deduced amino acid sequences of the novel CYP members were aligned using CLUSTAL W ver. 1.7 analysis. A phylogenetic tree of full-length CYP1A1 and 1A2 amino acid sequences were constructed by the neighbor-joining method using MacVector 7.1 program. TM

2.3. Quantification of CYP1A1 and CYP1A2 mRNAs For quantification of CYP1A1 and CYP1A2 mRNA levels, total RNA from 19 liver samples were extracted using TRIzol reagent (Invitrogen). After DNase treatment using RNeasy Mini Kit and RNase-Free DNase set (Qiagen), mRNA expression levels of CYP1A1 were measured by quantitative real-time RT-PCR using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). A set of specific primers and probes for common minke whale CYP1A1 and CYP1A2 were designed by the Primer ExpressTM ver. 1.0 software program (Applied Biosystems): CYP1A1 forward primer, 5 0 -CCGTATCCGGGACATCACAG-3 0 ; CYP1A1 reverse primer, 5 0 -CGACATTAACGATCTTCTCATCTGA-3 0 ; CYP1A1 TaqMan probe, 5 0 -CTGTCAGGGCAAGAGACTGGACGAGAA-3 0 ; CYP1A2 forward primer, 5 0 -GGTGCCCTGTTCAAGCACTACGA-3 0 ; CYP1A2 reverse primer, 5 0 -AGATGGCTGTTGTGATTGGATCAAAC-3 0 ; CYP1A2 TaqMan probe, 5 0 CAACCTTGTCAACGACATCTTCGCAGCC-3 0 . FAM was used as a reporter dye at 5 0 -end of probe and TAMRA as a quencher dye at 3 0 -end. A reaction mixture used for TaqMan One-Step RT-PCR Master Mix Reagent Kit (Applied Biosystems), contained 50 ng or 100 ng of total RNA as template. The primers and probe for CYP1A1 were used at a final concentration of 300 nM and 25 nM, respectively. As for CYP1A2, the reaction solution contained 900 nM of primers and 25 nM of probe. Conditions for quantitative real-time RT-PCR were as follows: 30 min at 48 C, 10 min at 95 C, and 40 cycles of 15 s at 95 C and 1 min at 53 C for CYP1A1, and 30 min at 48 C, 10 min at 95 C, and 40 cycles of 15 s at 95 C and 1 min at 57 C for CYP1A2. The relative CYP1A1/2 mRNA expression levels in each sample were normalized to 18S ribosomal RNA content.

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2.4. Chemical analysis of OCs

2.5. Statistical analysis

Chemicals analysis of OCs was carried out following the method previously described with a slight modification (Watanabe et al., 1999). Briefly, 10-g (approximately) liver samples were homogenized with anhydrous Na2SO4, and OCs were extracted in a Soxhlet apparatus with a mixture of hexane and diethyl ether. The fat content was gravimetrically determined from an aliquot of the extract. Lipid in the remaining extract was removed by gel permeation chromatography (GPC) packed with Bio-Bead S-X 3 (Bio-Rad Laboratories, USA). Dichloromethane (50%) in hexane was used as moving phase and flow rate was set at 5 ml/min. The lipid-removed extract was passed through 12 g activated Florisil (Florisil PR; Wako Chemicals USA, Inc.) packed in a glass column. The first fraction eluted with hexane contained PCBs, p,p 0 -DDE and trans-nonachlor; the second fraction eluted with 20% dichloromethane in hexane contained p,p 0 -DDD, p,p 0 -DDT, HCH isomers (a, b and c), and chlordane compounds (CHLs; cis-chlordane, trans-chlordane, cis-nonachlor, and oxychlordane). Each fraction was concentrated and injected into a gas chromatograph (Agilent 6980N) with a microelectron capture detector (micro-ECD) and an auto-injection system (Agilent 7683 Series Injector) for quantification. The GC column used for OCs analysis was a fused silica capillary (DB-1; 30 m · 0.25 mm i.d. · 0.25 lm film thickness, J&W Scientific Inc.). OC concentrations were calculated from the peak area of the sample to the corresponding external standard. The PCB standard used for quantification was a mixture of 62 PCB isomers and congeners (BPMS) obtained from Wellington Laboratories Inc., Ont., Canada. Concentrations of individually resolved peaks of PCB isomers and congeners were summed to obtain total PCB concentrations. Recoveries of target contaminants through this analytical method ranged from 95% to 105%. DDTs represent the sum of p,p 0 -DDT, p,p 0 DDD and p,p 0 -DDE, and CHLs include cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor and oxychlordane. HCHs include a-, b- and c-isomers. For quality assurance and quality control, we participated in the Intercomparison Exercise for Persistent Organochlorine Contaminants in Marine Mammal Blubber organized by the National Institute of Standards and Technology (Gaithersburg, MD) and Marine Mammal Health and Stranding Response Program of the National Oceanic and Atmospheric AdministrationÕs National Marine Fisheries Service (Silver Spring, MD). Standard reference material SRM 1945 was analyzed for selected PCB congeners and persistent OCs. Data from our laboratory were in good agreement with those for reference materials. The averages of percentage deviation from the certified values were 13% (range: 0.5– 20%) for organochlorine pesticides and 28% (range: 1.3– 57%) for PCB congeners.

The Mann–Whitney U test was employed to detect the differences in OC concentrations, CYP1A1 and CYP1A2 mRNA expression levels between immature and mature whales. SpearmanÕs rank correlation coefficient was used to detect the relationships between CYP1A mRNA expression levels and OCs concentrations. All statistical analysis was executed by StatView for Windows (Version 5.0, SAS Institute Inc., USA).

3. Results and discussion 3.1. Characteristics of CYP1A1 and CYP1A2 amino acid sequences As a result of screening of 6930 clones randomly selected in a liver cDNA library, we obtained full-length cDNA sequences of two CYP1A subfamilies, CYP1A1 and 1A2. Both CYP1A1 and CYP1A2 cDNAs from common minke whale had an open reading frame of 516 amino acid residues. The predicted molecular masses of CYP1A1 and 1A2 were 58.3 kDa and 58.1 kDa, respectively (Figs. 1 and 2). In CYP1A1 and 1A2 sequences, the 5 0 -UTRs were 135 bp and 62 bp, and the 3 0 -UTRs included 1321 bp and 367 bp with a poly(A) + tail, respectively. A polyadenylation signal sequence (AATAAA) was not found in CYP1A2, but in CYP1A1. The highly conserved amino acid motif (FXXGXXXCXG) with the heme-binding cysteine was maintained in common minke whale (Figs. 3 and 4). The deduced amino acid sequence of common minke whale CYP1A1 revealed higher identities with those of pig (87%) and sheep (86%), followed by those of pinnipeds such as grey seal, harp seal and Baikal seal (84%) (Table 1). An amino acid replacement from Val to Leu at position 386 in common minke whale CYP1A1 implies low metabolic potential to oxidize substrates (Fig. 3), as suggested by modelling predictions of CYP1A1 and experimental results using human CYP1A1 expressed in E. coli (Liu et al., 2003). The amino acid sequence of common minke whale CYP1A2, this being the first report in cetaceans, was most closely related to human (82%) and monkey CYP1A2 (82%), but shared lower identities with grey seal (77%) and harp seal CYP1A2 (77%). The common minke whale CYP1A2 displayed 73% amino acid identity with CYP1A1, as has been observed in other species (67–75%). Thr-321, which is an amino acid residue conserved over the species and CYP family, is known to play an important role in binding oxygen molecule in the process of substrate metabolism. The corresponding amino acid in common minke whale CYP1A2 was Pro (Fig. 4). This implies a low metabolic potential of CYP1A2 in this species (Lewis, 2001).

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Fig. 1. The nucleotide sequence of a full-length CYP1A1 cDNA of common minke whale and its deduced amino acid sequence. The stop codon is indicated by an asterisk. GenBank accession no.: AB231891.

3.2. Phylogenetic analysis Phylogenetic analysis from multiple alignments of the full-length amino acid sequences of vertebrates CYP1A subfamily revealed that common minke whale CYP1A1

and CYP1A2 were positioned into separate CYP clade (Fig. 5). Common minke whale CYP1A1 belonged to the same group as pig and sheep. This result agrees with the evolutionary process of cetacean. CYP1A2 of common minke whale belonged to the same group as human

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Fig. 2. The nucleotide sequence of a full-length CYP1A2 cDNA of common minke whale and its deduced amino acid sequence. The stop codon is indicated by an asterisk. GenBank accession no.: AB231892.

and monkey. Because CYP1A2 genes in pig and sheep were not isolated, the common minke whale CYP1A2 amino acid sequence was not able to compare with those artiodactyls CYP1A2. 3.3. Expression levels of CYP1A1 and CYP1A2 mRNAs in the liver Several studies have shown that notable CYP1A induction was observed in the liver, not in the blubber.

Most of the OCs body burden is present in the blubber, but such lipophilic chemicals are dissolved in lipid in the body and their tissue distribution is dependent upon lipid contents. Due to such a kinetic property of OCs, their tissue concentrations expressed on lipid weight basis are expected to be almost equal in blubber and liver (Tanabe et al., 1981). Therefore, we analyzed OCs concentrations in the liver, and discussed relationships between hepatic CYP mRNA expressions and OCs concentrations. OCs including PCBs, DDTs, HCHs, CHLs

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Fig. 3. Alignment of CYP1A1 amino acid sequences of common minke whale and other vertebrates. Accession numbers used are as follows: pig 1A1 (AB052254), harp seal 1A1 (AJ621380) and human 1A1 (P04798). The predicted regions of a-helix is shown square (Edwards et al., 1989). The conserved amino acid motif with the heme-binding cycteine is underlined. A mark (*), L386 is a specific amino acid residue in common minke whale, which may be involved in substrate oxidization.

and HCB were detected in all samples of common minke whale liver analyzed in this study. Residue levels of these OCs were 5.5–93 ng/g wet weight for PCBs, 4.1–210 ng/g wet weight for DDTs, 14–48 ng/g wet weight for HCHs, 2.7–67 wet weight for CHLs and 3.5–57 ng/g wet weight for HCB (Table 2), which were higher than those in fishes from North Pacific (Ueno et al., 2003). Furthermore, the concentrations in common minke whale were lower than those in pilot whales from the coast of Massachusetts that is known to be a highly polluted region (Tilbury et al., 1999), but higher than those in bowhead whales from Alaska (Hoekstra et al., 2002). Residue levels of PCBs and DDTs in mature male were significantly higher than those in immature male (p < 0.05). To investigate whether CYP expression levels are altered by exposure to OCs, this study examined the relationships between hepatic OC residue levels and CYP1A1 or CYP1A2 mRNA expression levels. Neither of mRNA expression levels of CYP1A1 nor CYP1A2 showed significant difference between immature and

mature specimens. Furthermore, no relationship between CYP1A1 or CYP1A2 mRNA expression and OCs residue levels was found, even when data from all the specimens were used for the statistical analysis, or data from immature and mature animals were treated separately (Figs. 6 and 7). According to Mattson et al. (1998), hepatic CYP1A activities and the apoprotein expression levels in ringed seals exposed to high levels of PCBs (66 lg/g lipid weight) and DDTs (38 lg/g lipid weight) in the Baltic Sea were much higher than those in seals from a relatively unpolluted area, Svalbard Region (PCBs; 1.1 lg/g lipid weight, DDTs; 0.4 lg/g lipid weight). Concentrations of PCBs and DDTs in the liver of common minke whales were lower than those in ringed seals from Baltic Sea, and comparable to those in seals from Svalbard (Table 2). Therefore, results of this study imply that these chemicals may not be directly responsible for induction of CYP1A1 and CYP1A2 in common minke whale liver. Expression of the CYP1A genes is known to be related to planar compounds

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Fig. 4. Alignment of CYP1A2 amino acid sequences of common minke whale and other vertebrates. Accession numbers used are as follows: marmoset 1A2 (D86475), human 1A2 (P05177) and harp seal 1A2 (AJ621381). The predicted regions of a-helix is shown square (Edwards et al., 1989). The conserved amino acid motif with the heme-binding cycteine is underlined. A mark (*), P321 is a specific amino acid residue in marine mammal, which may be involved in low substrate oxidization.

Table 1 Identity percentages of deduced amino acid sequences of common minke whale CYP1A1 and CYP1A2 with those of other vertebrate CYP1As Common minke whale

Aquatic mammal

Terrestrial mammal

Bird

1A1 n=3

1A2 n=2

1A1 n = 10

1A2 n=8

1A4 n=1

1A5 n=1

1A n=9

1A1 1A2

84 70

68 77

76–87 65–69

62–70 69–82

58 51

62 65

55–57 47–50

including 2,3,7,8-TCDD and coplanar PCBs. Specific analysis of such chemicals was not conducted in the present study. On the other hand, our previous study demonstrated that PCBs residue levels were positively correlated (p < 0.0001, SpearmanÕs rank correlation test) with 2,3,7,8-TCDD toxic equivalents (TEQs) of coplanar PCBs, including non-ortho (IUPAC Nos. 77, 126 and 169) and mono-ortho (IUPAC Nos. 105, 118 and 156) PCB congeners, in the blubber of common minke whales from the North Pacific (unpublished data). In addition, larger contribution from coplanar PCBs to total TEQs, and low contributions from PCDDs and

Fish

PCDFs in cetacean tissues have been reported (Tanabe et al., 1989; Ross et al., 2000). Therefore, PCBs concentration may reveal a positive correlation with total TEQs in common minke whale. TEQs (22 pg TEQ/g wet weight on average) in the liver of common minke whales in this study was estimated using a liner regression formula of PCBs concentrations (ng/g wet weight) and TEQs (pg TEQ/g wet weight) in the blubber (TEQs = 0.0358 PCBs + 20.8, n = 58, r2 = 0.67). Several in vitro studies showed that the EC50 values for EROD induction by TCDD in the human hepatoblastoma cell line HepG2, rat hepatoma cell line H4IIE

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791

human 3A4 guinea pig 1A2

94 100 100

rat 1A2 mouse 1A2 rabbit 1A2

minke whale 1A2

80

marmoset 1A22 monkey 1A2 human 1A2

100 89

100 100

100 100

100 100 88

96

88 100

mammal 1A2

dog 1A2 grey seal 1A2 harp seal 1A2 monkey 1A1 human 1A1 dog 1A1 grey seal 1A1 harp seal 1A1 Baikal seal 1A1 sheep 1A1 pig 1A1

mammal 1A1

minke whale 1A1 guinea pig 1A1 100

100 100

98

100

hamster 1A1 rat 1A1 mouse 1A1 rabbit 1A1 chicken 1A5 chicken 1A4 killifish 1A

100

100 100

100 0

rainbow trout 1A33 rainbow trout 1A2 rainbow trout 1A1 toadfish 1A European sea bass 1A dab 1A scup 1A

bird 1A4/1A5

fish 1A

gilthead sea bream 1A

Fig. 5. Phylogenetic analysis of amino acid sequences from vertebrate CYP1As. Full-length CYP1A sequences were aligned using CLUSTAL W ver. 1.7 and a neighbor-joining tree was constructed using MacVector 7.1 program. Bootstrap values based on 1000 sampling are shown above each branch. Positions with gaps are excluded and corrections were made for multiple substitutions. Accession numbers used are as follows: CYP1A1 (Q06367) and CYP1A2 (Q64391) of guinea pig, rat (P00185, P04799), mouse (P00184, P00186), rabbit (P05176, P00187), marmoset (D86475/ CYP1A2), monkey (P33616, D86474), human (P04798, P05177), dog (P56590, P56592), grey seal (AJ621378, AJ621379), harp seal (AJ621380, AJ621381), sheep (P56591/CYP1A1), pig (AB052254/CYP1A1), hamster (Q00557/CYP1A1), chicken CYP1A4 (P79760), chicken CYP1A5 (P79761), killifish (AF026800), rainbow trout (Q92110/CYP1A1, S69278-1/CYP1A2, Q92109/CYP1A3), toadfish (Q92095), European sea bass (P79716), dab (O42430), scup (Q92116), gilthead sea bream (O424579) and Baikal seal (unpublished). Human CYP3A4 (AF109068) was used as an outgroup.

Table 2 Mean and range of organochlorine concentrations in the liver of male common minke whales collected from the North Pacific in 2001 Species

Common minke whale

Growth stage

Immature

Mature

n

9

10

Body length (m)

Lipid (%)

Mean

5.81

3.2

Range

4.44–6.97

2.5–3.9

Mean

7.30

3.5

Range

6.87–7.67

2.6–4.8

Concentration PCBs

DDTs

HCHs

CHLs

HCB

13 (400) 5.5–22 (210–660)

14 (450) 4.1–31 (160–960)

24 (750) 16–30 (560–930)

7.4 (240) 3.2–13 (120–380)

11 (330) 6.3–15 (190–460)

35 (950) 5.6–93 (190–1900)

62 (1600) 5.7–210 (190–4300)

28 (790) 14–48 (480–1300)

20 (540) 2.7–67 (92–1400)

16 (430) 3.5–57 (120–1200)

Concentrations are expressed as ng/g on wet weight basis. Numbers in parenthesis are concentrations expressed as ng/g on lipid basis. n: number of samples. DDTs: p,p 0 -DDT + p,p 0 -DDE + p,p 0 -DDD; HCHs: a-HCH + b-HCH + c-HCH; CHLs: trans-chlordane + cis-chlordane + trans-nonachlor + cis-nonachlor + oxychlordane.

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S. Niimi et al. / Marine Pollution Bulletin 51 (2005) 784–793 1.2

1.0

p=0.96 0.8 0.6 0.4 0.2 0

0

20

40

60

80

1.2

DDTs

1.0

p=0.89 0.8 0.6 0.4 0.2 0

100

0

1.2

100

150

200

p=0.79 0.8 0.6 0.4 0.2 0

250

0

20

40

60

Concentration (ng/g wet wt.)

1.2

CHLs

1.0

Relative CYP1A1 mRNA level

Relative CYP1A1 mRNA level

50

HCHs

1.0

Concentration (ng/g wet wt.)

Concentration (ng/g wet wt.)

p=0.82 0.8 0.6 0.4 0.2 0

Relative CYP1A1 mRNA level

PCBs

Relative CYP1A1 mRNA level

Relative CYP1A1 mRNA level

1.2

0

20 40 60 Concentration (ng/g wet wt.)

HCB 1.0

p=0.99

0.8 0.6 0.4 0.2 0

80

0

20 40 Concentration (ng/g wet wt.)

60

Fig. 6. Relationships between CYP1A1 mRNA expression and OC concentrations in the liver of common minke whales.

PCBs

1.0

p=0.20 0.8 0.6 0.4 0.2 0

DDTs

1.0

p=0.12 0.8 0.6 0.4 0.2

20 40 60 80 Concentration (ng/g wet wt.)

p=0.72 0.8 0.6 0.4 0.2 0

0

100

HCHs

1.0

0 0

50 100 150 200 Concentration (ng/g wet wt.)

250

0

20 40 Concentration (ng/g wet wt.)

60

1.2 Relative CYP1A2 mRNA level

1.2 Relative CYP1A2 mRNA level

1.2 Relative CYP1A2 mRNA level

1.2 Relative CYP1A2 mRNA level

Relative CYP1A2 mRNA level

1.2

CHLs

1.0

p=0.32 0.8 0.6 0.4 0.2

HCB

1.0

p=0.92 0.8 0.6 0.4 0.2 0

0 0

20 40 60 Concentration (ng/g wet wt.)

80

0

20 40 Concentration (ng/g wet wt.)

60

Fig. 7. Relationships between CYP1A2 mRNA expression and OC concentrations in the liver of common minke whales.

and rat primary hepatocytes were 220, 16 and 7 pg TEQ/g wet weight, respectively (Zeiger et al., 2001). In this regard, residue levels of TEQs in common minke whale may be low for inducing hepatic CYP1A1 induction. Further study is necessary to verify whether other CYP expression can be potential biomarkers of the OC exposure.

Acknowledgements The authors thank Prof. A. Subramanian (CMES, Ehime University) for critical reading of the manuscript. This study was supported by Grants-in-Aid for Scientific Research (A) (Nos. 17208030 and 16202014) and (B) (No. 13480170) from Japan Society for the

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