ToxSci Advance Access originally published online on March 22, 2007
Toxicological Sciences 2007 97(2):318-335; doi:10.1093/toxsci/kfm066
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Molecular Characterization of Cytochrome P450 1A1, 1A2, and 1B1, and Effects of Polychlorinated Dibenzo-p-dioxin, Dibenzofuran, and Biphenyl Congeners on Their Hepatic Expression in Baikal Seal (Pusa sibirica)
* Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan
Department of Animal Sciences, Teikyo University of Science and Technology, 2525 Yatsusawa, Uenohara 409-0193, Japan
Center for International Cooperation, Ocean Research Institute, The University of Tokyo, Minamidai 1-15-1, Nakano-ku Tokyo 164-8639, Japan
The Eastern-Siberian Scientific and Production Fisheries Center, Hakhalov st. 4, Ulan-Ude, Buryatia, 670034, Russia
1 To whom correspondence should be addressed. Fax: +81-89-927-8172. E-mail: iwatah{at}agr.ehime-u.ac.jp.
Received January 14, 2007; accepted March 16, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study attempts to relate the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent (TEQ) level with certain responses including the catalytic activities and expression of hepatic cytochrome P450 (CYP) 1A and CYP1B in wild population of Baikal seal (Pusa sibirica). We isolated full-length CYP1A1, 1A2, and 1B1 cDNAs, which encode proteins of 516, 512, and 543 amino acids, respectively. Immunochemical analysis demonstrated that a cross-reactive protein with polyclonal antibody against rat CYP1A1 or CYP1B1 was detected in the seal liver. Total TEQ levels showed significant positive correlations with expression levels of CYP1A1, 1A2, and 1B1 mRNAs, and further with both CYP1A- and CYP1B-like proteins, indicating chronic induction of these CYP isozymes by TEQs. The 50% effective concentration for CYP1A-like protein induction was estimated to be 65 pg TEQ/g wet weight. To evaluate the potential of congener-specific metabolism, profiles of negative correlations between the concentrations of eachcongener normalized to a relatively recalcitrant congener, PCB169, and CYP1A-like protein levels were also estimated. Significant negative correlations of 2,3,7,8-tetrachlorodibenzofuran and PCB77 to CYP1A-like protein expression may possibly be due to the preferential metabolism of these congeners. Anti-rat CYP1A1 and CYP1B1 antisera equivalently inhibited ethoxyresorufin O-deethylase (EROD) activity in the seal microsomes, suggesting that both CYPs are involved in EROD activity. Hepatic EROD revealed an increasing trend at lower TEQs, but a declining trend at higher levels, implying a catalytic inhibition of CYP1A and CYP1B. Furthermore, ratios of CYP1B1/CYP1A1 mRNA expression levels increased with TEQs, indicating the enhanced risk of carcinogenicity by preferential induction of CYP1B1 by TEQs in the liver.
Key Words: Baikal seal; toxic equivalent; cytochrome P450 1A; cytochrome P450 1B; catalytic inhibition.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Planar halogenated aromatic hydrocarbons (PHAHs) such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs) are ubiquitous contaminants. These compounds are biomagnified in the food web due to their lipophilic and persistent properties. PHAHs are notably accumulated in a variety of aquatic species (Tanabe et al., 1994
).
Aquatic mammals may be at high risk to PHAHs, as suggested by high incidence of mass mortalities since the 1970s. The accumulation of PHAHs in aquatic mammals has been considered as a contributing factor in the epizootic, although the direct cause for this outbreak was infectious diseases (de Swart et al., 1995). However, the magnitude of effects that these chemicals pose to aquatic mammals remains uncertain because of the lack of direct investigation linking between PHAH contamination levels and certain biochemical/toxicological evidences.
The mechanisms of toxic effects by PHAHs in aquatic mammals still remain unclear, but are likely to involve the aryl hydrocarbon receptor (AHR)-signaling pathway in rodents and human (Okino and Whitlock, 2000). The AHR, a ligand-activated intracellular protein, plays a central role in various biochemical and toxic effects of PHAHs in experimental mammals (reviewed by Poland and Knutson, 1982). Induction of cytochrome P450 (CYP) 1A and CYP1B subfamilies in rodent species is a response to exposure of PHAHs, mediated by AHR, as well as the transcriptional regulation of other AHR target genes. Varieties of endogenous and exogenous compounds, that may be potentially signaling molecules, are oxidized by CYP1A/CYP1B-mediated reactions into more hydrophilic and often less active or more harmful metabolites. Through these processes modulated by AHR-mediated signaling and/or by altered CYP1A/CYP1B-mediated metabolism of signaling molecules, PHAH exposure poses a broad range of effects including reactive oxygen production, hepatocyte hypertrophy, uroporphyria, developmental failure, immune dysfunction, teratogenesis, and modulation of estrogen signaling (Fernandez-Salguero et al., 1996; Ohtake et al., 2003; Schlezinger et al., 1999; Smith et al., 2001; Teraoka et al., 2003;Uno et al., 2004). Therefore, the expression level of CYP1A/CYP1B is considered as a biomarker not only of PHAHs exposure but also of their toxic effects.
In our previous study, a full-length AHR cDNA has been isolated and sequenced from the Baikal seal (Pusa sibirica) (Kim et al., 2002). The high degree of conservation of AHR cDNA sequences in Baikal seal and other aquatic mammalian species including beluga whale (Delphinapterus leucas) and harbor seal (Phoca vitulina) (Jensen and Hahn, 2002; Kim et al., 2002) led to a speculation that the 2,3,7,8-tetrachlorodibenzo-p-dioxin (T4CDD)-binding affinity of these AHRs may be as high as that of the AHR from a dioxin-sensitive (C57BL) strain of mice and that this species may be sensitive to PHAH effects. Apart from the AHR study, we succeeded in isolating a full-length cDNA of aryl hydrocarbon receptor nuclear translocator, a heterodimeric partner of AHR, from Baikal seal liver (Kim et al., 2005). In addition, Baikal seal exhibits high levels of PHAHs in the liver and blubber (Iwata et al., 2004). Total 2,3,7,8-T4CDD toxic equivalents (TEQs) were in the range of 210–920 pg TEQ/g fat weight (wt) (180–800 pg TEQ/g wet wt) in the blubber and 290–7800 pg TEQ/g fat wt (10–570 pg TEQ/g wet wt) in the liver. Comparison of hepatic total TEQs in Baikal seals with 50% effective concentration (EC50) for the induction of mammalian hepatic ethoxyresorufin O-deethylase (EROD) (Safe, 1990) and the T4CDD-binding affinity (Kd = 0.93 ± 0.19nM) of harbor seal AHR (Kim and Hahn, 2002) indicated that most livers of wild Baikal seal population were contaminated with TEQs that may elicit AHR activation and CYP1A induction.
However, only a few studies are available, particularly on the modulation of AHR and CYP signaling pathways by PHAH contamination in wild aquatic mammal population (Chiba et al., 2002; Letcher et al., 1996; White et al., 1994). Furthermore, information on the molecular characterization and expression level of each CYP isozyme for which the transcriptional regulation is mediated by PHAH-activated AHR in aquatic mammals is quite limited. Therefore, the present study attempted to isolate the isozyme-specific CYP1A1, 1A2, and 1B1 cDNAs and to quantify the mRNA expression levels by real-time reverse transcription–polymerase chain reaction (RT-PCR) method. In addition, we detected CYP1A and CYP1B proteins by an immunochemical approach. To examine whether or not PHAH levels modulate AHR and CYP signaling pathways in Baikal seal livers, relationships between hepatic PHAH residue levels and the expression levels of AHR target genes, CYP1A, and CYP1B isoenzymes in the wild population were also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sample collection.
The details of Baikal seal samples were reported previously (Kim et al., 2005
; Nakata et al., 1995). Seals were collected from Lake Baikal in May–June in 1992 and immediately dissected on board after measurement of biometry (body length, body weight, etc.). Liver samples were removed and total liver weight was measured in most of the specimens. The subsamples were frozen in liquid nitrogen and stored at – 80°C until microsome preparation and mRNA isolation. In order to avoid bias due to deterioration of CYP, enzymatic activities, and protein expression during the time spent for postmortem, liver samples (9 males and 19 females) collected within 2 h during postmortem, among all the samples examined for chemical analysis of PCDDs/DFs and Co-PCBs (Iwata et al., 2004), were further used for CYP enzymatic activities and protein expression measurement. Furthermore, to examine the quality of mRNA isolated, we performed electrophoresis of gels loaded with seal mRNAs and checked 28S and 18S ribosomal RNA bands. The results showed that RNA samples from 6 males and 17 females were suitable for quantification of CYP mRNA expression. Ages of animals determined from dentinal and cemental growth layers in a canine tooth have already been reported elsewhere (Kim et al., 2005; Nakata et al., 1995).
RNA isolation and cloning of CYP1A1, 1A2, and 1B1 cDNAs.
For CYP cloning, total RNA was isolated from about 1 g of a liver subsample using RNAgents Total RNA Isolation System (Promega, Madison, WI). Messenger RNA was purified by PolyATtract mRNA Isolation System (Promega) and stored at – 80°C until RT-PCR.
The partial fragments of CYP1A1, 1A2, and 1B1 cDNAs of Baikal seal were isolated by RT-PCR. One microgram of mRNA was reverse transcribed with random hexamers, and cDNAs were amplified using the GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA). PCR primers of CYP1A1, 1A2, and 1B1 were designed from conserved regions of the corresponding genes from mammalian species so far identified. Forward (F) and reverse (R) primer sequences were as follows: BS-1A1-F, 5'-AGAAGGGMCACATTCGGGAY-3'; BS-1A1-R, 5'-ATGCTTCATGGTCAGCCCGTA-3'; BS-1A2-F, 5'-ATTGGTGCCATGTGCTTTGGGCA-3'; BS-1A2-R, 5'-TGTCGTGTCCCTTGTCGTGCTGT-3'; BS-1B1-F, 5'-GCTCAAYCGCAACTTCAGCA-3'; BS-1B1-R, 5'-GAGCTCCATGGACTCTCTGA-3'. PCR amplifications of CYP1A1, 1A2, and 1B1 isozymes were performed under the following conditions: 105 s at 95°C, 30 cycles of 15 s at 95°C, 45 s at 50°C and 1 min at 72°C. A final extension was carried out for 7 min at 72°C following the last cycle.
For 5'- and 3'- rapid amplification of cDNA ends (RACE), double-stranded liver cDNA was synthesized using a Marathon cDNA Amplification Kit (BD Biosciences, San Jose, CA) with DNA polymerase of Advantage 2 PCR Enzyme System (BD Biosciences). The RACE primers were designed based on partial sequences of CYP cDNAs. The following are the primer sequences of CYP1A1, 1A2, and 1B1—BS-1A1-5', 5'-GGCTCCAGGAGATGGCAGTTGTCACGG-3'; BS-1A1-3', 5'-TCCTGGCCATCCTGCTGCAGCAGGT-3'; BS-1A2-5', 5'-GCAAGGCGGGGTTGGGCATATACTGC-3'; BS-1A2-3', 5'-TCACCATCCCGCACAGCACGACAAGG-3'; BS-1B1-5', 5'-CGCGGTGCCTCAGGAACTTGTCGAGG-3'; BS-1B1-3', 5'-GGGCAAACGGCGGTGCATTGGGA-3'. These specific primers were coupled with adaptor primers for PCR. Amplifications of cDNA ends were performed under the following conditions: 30 s at 94°C, 5 cycles of 5 s at 94°C, 4 min at 72°C, 5 cycles of 5 s at 94°C, 4 min at 70°C, 25 cycles of 5 s at 94°C, 4 min at 68°C.
The amplified products were cloned into pGEM-T Easy vector (Promega) and sequenced by ABI PRISM 310 genetic analyzer (Applied Biosystems). The resulting full-length cDNA sequences were submitted to the P450 nomenclature committee (D. R. Nelson, University of Tennessee, Health Science Center, Memphis, TN). The deduced full-length amino acid sequences of Baikal seal CYP1A1, 1A2, and 1B1 were aligned using Mac Vector v.7.1 program (Accelrys, Inc., Madison, WI).
Quantitative real-time RT-PCR.
CYP1A1, 1A2, and 1B1 mRNA expression levels in the liver of Baikal seals were measured by quantitative real-time RT-PCR. Total RNA was isolated from liver samples from 6 males and 17 females using MagExtractor-RNA-Nucleic Acid Purification Kit (Toyobo Co., Ltd, Osaka, Japan) by MFX2100 (Toyobo Co., Ltd). Extracted total RNA was treated with DNase, and the concentrations and purities of RNA were measured. For checking the quality of total RNA, the bands of 28S and 18S in ribosomal RNAs from all samples were confirmed by gel electrophoresis. Quantitative real-time RT-PCR was performed with Taqman One-step RT-PCR Master Mix Reagents Kit (Applied Biosystems) using ABI PRISM 7700 Sequence Detector (Applied Biosystems). Specific primers and probes for CYP1A1, 1A2, and 1B1 were designed using the full-length cDNA sequences by Primer Express software v. 1.0 (Applied Biosystems). The 5'- and 3'-end nucleotides of the probes were labeled with a reporter (FAM) and a quencher dye (TAMRA), respectively. The sequences of the primers and probes were as follows: BSRT-1A1-F, 5'-AGGCATTCATCCTGGAGACCT-3'; BSRT-1A1-R, 5'-GGATGTAAAAGCCACTCAGACTTG-3'; BSRT-1A1-probe, 5'-TTCGTCCCCTTCACCATCCCTCATAGTAC-3'; BSRT-1A2-F, 5'-GTGCAGTTCCTGCAGAAAATTG-3'; BSRT-1A2-R, 5'-CTCTGGAGCCCTTCTCATTGTG-3'; BSRT-1A2-probe, 5'-CAGGACATCACAGGTGCCCTCTTGAA-3'; BSRT-1B1-F, 5'-TTCCCAAGGACACGGTGGTT-3'; BSRT-1B1-R, 5'-GCCCGGATCAAAGTCCTCTG-3'; BSRT-1B1-probe, 5'-CTGTGAATCATGACCCAGCGAAGTGGC-3'. The following temperature conditions were employed: 30 min at 48°C for reverse transcription, 10 min at 95°C for hot start, 40 cycles of 15 s at 95°C, and 1 min at 50°C for CYP1A1 and CYP1A2 or 1 min at 60°C for CYP1B1. Template was prepared from total RNA of the liver of a certain specimen. Isoform-specific calibration curves were generated using serially diluted template solutions. Negative control reactions were performed in the absence of template. In addition, 18S ribosomal RNA was measured as an endogenous control using TaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems).
Quantitative values were calculated from the threshold PCR cycle number (Ct) at which the increase in signal associated with an exponential growth of PCR products was detected. Expression level of each CYP mRNA in individual animals was expressed as a relative value calculated from an appropriate calibration curve. The relative mRNA level in each sample was normalized to its ribosomal RNA content. The real-time RT-PCR assays were conducted in triplicate for each sample.
Reagents for enzymatic activities and immunoblotting.
NADPH, glycerol, dithiothreitol, KCl, and sodium dodecyl sulfate (SDS) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Methoxyresorufin, ethoxyresorufin, pentoxyresorufin, benzyloxyresorufin, resorufin, and Tris-HCl were purchased from Sigma-Aldrich CO. (St. Louis, MO). EDTA and HCl were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Chemicals of highest grades were commercially available.
Preparation of microsomal fraction.
Hepatic microsomal fractions were prepared following the method of Guengerich (1982). Seal liver tissue (
6 g) was homogenized in five volumes of cold homogenization buffer (50 M Tris-HCl, 0.15M KCl, pH 7.4–7.5) with a teflon-glass homogenizer (10 passes), and centrifuged for 10 min at 750 x g. The supernatant was then centrifuged at 12,000 x g for 10 min. The supernate was further centrifuged at 105,000 x g for 70 min. The supernatant (cytosol) fraction was removed, and microsomal pellets were resuspended in one volume of resuspension buffer [50mM Tris-HCl, 1mM EDTA, 1mM dithiothreitol, 20% (vol/vol) glycerol, pH 7.4–7.5]. All the microsomal fractions were immediately frozen in liquid nitrogen, and stored at – 80°C until further CYP enzyme assays and immunoblotting.
Protein concentrations in microsomal fractions were determined by the bicinchoninic acid method (Smith et al., 1985). BCA Protein Assay Reagent (Pierce, Rockford, IL) was used for the protein assay and bovine serum albumin as a standard. Absorbance at 560 nm was measured using a multiwell plate reader (SpectraFluor Plus, Tecan Austria GmbH, Groedig, Austria).
CYP contents and enzymatic activities.
The content of hepatic microsomal CYP was determined from dithionite difference spectra of CO-treated samples (Omura and Sato, 1964) with a spectrophotometer. Measurements of methoxyresorufin O-demethylase (MROD), EROD, pentoxyresorufin O-depenthylase (PROD) and benzyloxyresorufin O-debenzylase (BROD) in microsomal fractions were performed as described previously with slight modification (Iwata et al., 2002; Kubota et al., 2005, 2006). EROD activity was measured with 0.002mM substrate and 1.33mM NADPH concentrations using a multiwell plate reader (SpectraFluor Plus, Tecan Austria GmbH) at 37°C. Measurements of MROD, PROD, and BROD were perfomed similarly with 0.005mM substrate and 1.33mM NADPH concentrations. Resorufin formed by CYP enzymatic activity was excited at 535 nm wavelength and detected at 595 nm wavelength.
Immunoblotting.
Immunoblotting of microsomal fraction was performed as described previously (Iwata et al., 2002; Kloepper-Sams et al., 1987). Proteins (CYP1A: 8 µg/lane, CYP1B: 40 µg/lane) in microsomal fractions were resolved by electrophoresis on 5–20% gradient polyacrylamide gel (ATTO, Tokyo, Japan) in a SDS-containing buffer and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. Microsome containing rat CYP1A1 or CYP1B1 heterologously expressed in a baculovirus system (BD Biosciences) was simultaneously subjected to electrophoresis as a positive control. Goat polyclonal antibody raised against rat CYP1A1 (Daiichi Pure Chemicals, Tokyo, Japan) and a whole serum prepared from rabbit immunized with a peptide specific for rat CYP1B1 (BD Gentest, Woburn, MA) were used for detecting CYP1A and CYP1B homologues in seal liver, respectively. For CYP1A, the secondary antibody was anti-goat immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugate (Bethyl Laboratories, Inc., Montgomery, TX). As for CYP1B antibody, anti-rabbit IgG-HRP (BD Bioscience) was used as a secondary antibody. Detection of the antibody cross-reactive proteins was performed using a highly sensitive ECL Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ). As the Western blot analyses demonstrated that single band emerges at the expected molecular weight region by using the rat CYP1A1 antibody, revealing a specific cross-reaction, quantification of seal CYP1A protein was performed by dot blot analysis. As for the anti-rat CYP1B1 antiserum, multiple bands at unexpected molecular weight regions were obtained in seal microsomes. Thus, the seal CYP1B protein was quantified from the band emerged at the expected molecular weight region by Western blot analysis.
For the quantification of seal CYP1A by dot blot analysis, protein (8 µg per well) in microsomal fraction was placed on a PVDF membrane and allowed to air dry for 1 h in Bio-Dot Microfiltration Apparatus (Bio-Rad Laboratories, Hercules, CA). After blocking the membrane using 5% skim milk in Tris-buffered saline, diluted rat CYP1A1 polyclonal antibody was incubated with coated microsomal fraction on the membrane at 4°C for 12 h. After washing the membrane, first antibody bound on the membrane was assayed with anti-goat IgG-HRP and was detected by ECL Plus Western Blotting Detection System (Amersham Biosciences).
The bands or spots emerged on the PVDF membrane were visualized by an imaging analyzer, ChemiDoc (Bio-Rad Laboratories), and the luminescent intensities were then measured using Quantity One (Bio-Rad Laboratories). The relative expression levels of individual CYP proteins in hepatic microsomes were expressed as the optical densities of the antibody cross-reactive proteins relative to those in a certain hepatic microsomes that showed highest EROD activities.
Antibody inhibition of catalytic activities.
One liver microsome sample with the highest EROD activity was selected for the antibody inhibition test. Seal hepatic microsomes were preincubated with 0, 1, 2, 5, 10, 20, or 25 µl of anti-rat CYP1A1 polyclonal antibody (Daiichi Pure Chemicals), anti-rat CYP1B1 antiserum (BD Gentest), anti-dog CYP2B11 polyclonal antibody (Daiichi Pure Chemicals), or control goat sera for 30 min at room temperature prior to initiation of EROD reaction by adding NADPH. The EROD activity was measured as described above.
2,3,7,8-T4CDD toxic equivalents.
The concentrations of individual PCDDs/DFs and Co-PCBs congeners in Baikal seal livers were quantified by a gas chromatograph equipped with a high-resolution mass selective detector and have already been reported elsewhere (Iwata et al., 2004). TEQs were calculated from World Health Organization mammalian toxic equivalency factors of individual congeners proposed by Van den Berg et al. (1998).
Statistical analysis.
Statistical analysis was performed using StatView v.5.0 (SAS Institute, Cary, NC) and SPSS Advanced Models 12.0J (SPSS Japan, Inc., Tokyo, Japan). Data are reported as means ± SD (Table 1). Prior to the comparison of data among groups, Levene's test was performed to check homogeneity of variance of data. When the equality of variance was assumed, data were then subjected to ANOVA and Scheffe's F-test. When the equality of variance was not assumed, a nonparametric statistical comparison, the Kruskal-Wallis test was employed, and data difference between groups was assessed using individual Mann-Whitney U-test with Bonferroni's adjustment. Correlations among quantified values were examined by Spearman's rank correlation analysis. p Values below 0.05 were regarded as statistically significant. When more than 50% of the observations were below the quantification limit, statistical analysis was not conducted, and the result was shown as no analysis (NA). The EC50 of TEQ was calculated using GraphPad PRISM v.4 (GraphPad Software, Inc., San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of Baikal Seal CYP1A1, 1A2, and 1B1 cDNAs
By employing RT-PCR and RACE methods, full-length cDNAs of CYP1A1, 1A2, and 1B1 were isolated from the liver of Baikal seal. The Baikal seal CYP1A1 cDNA has a 1551-bp open reading frame that encodes 516 amino acid residues with a predicted molecular mass of 58.2 kDa, while the Baikal seal CYP1A2 cDNA has a 1539-bp open reading frame encoding 512 amino acid residues with a predicted molecular mass of 57.8 kDa (Figs. 1A and 1B). The Baikal seal CYP1B1 cDNA consists of 1632-bp open reading frame encoding 543 amino acid residues with a predicted molecular mass of 60.2 kDa (Fig. 1C). The C-terminal sequences in 3'-untranslated region of CYP1A1 and CYP1A2 included poly (A)+ tail preceded by a polyadenylation signal sequence (AATAAA). The start codon was recognized by comparing Kozak sequences of Baikal seal CYP1A1, 1A2, and 1B1 with corresponding homologue genes from other species. The nomenclature of CYP1A1, 1A2, and 1B1 assigned to each Baikal seal CYP sequence was confirmed by D. R. Nelson (University of Tennessee, Memphis, TN).
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Phylogenetic Analysis of Baikal Seal CYP1 Genes
A phylogenetic tree was constructed from multiple alignments of the full-length amino acid sequences of CYP1A1, 1A2, and 1B1 from Baikal seal and other vertebrate species, using the neighbor-joining method (Fig. 2). The amino acid sequences of CYP1 family isozymes were aligned using ClustalW analysis. The phylogenetic analysis showed that Baikal seal CYP1A1, 1A2, and 1B1 belonged to mammalian CYP1A1, 1A2, and 1B1 clades, respectively. Each sequence of Baikal seal CYP1A1 and CYP1A2 clustered together with the corresponding CYP1As in other seal species, such as harp seal (Pagophilus groenlandicus), gray seal (Halichoerus grypus) and ribbon seal (Histriophoca fasciata). Since no other full-length CYP1B1 sequence from aquatic mammals was reported, Baikal seal CYP1B1 was in the same cluster as human CYP1B1.
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Sequence Comparison
Deduced amino acid sequences of Baikal seal CYP1A1, 1A2, and 1B1 were compared with CYP1 family amino acid sequences of other vertebrates (Table 2). The Baikal seal CYP1A1 amino acid sequence was most closely related to harp seal, gray seal, and ribbon seal CYP1A1 (99%) sequences, and CYP1A2 was highly similar to harp seal CYP1A2 (96%) and to gray seal and ribbon seal CYP1A2s (97%) in overall amino acid identities (Tilley et al., 2002
). There was no comparable sequence data on CYP1B1 from other seal species. The Baikal seal CYP1B1 amino acid sequence showed the highest identity with a partial sequence of CYP1B1 from striped dolphin (Stenella coeruleoalba) (91%), when the corresponding partial sequence of Baikal seal CYP1B1 (203–543 amino acid residues) was employed for the comparison.
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The Baikal seal CYP1A1, 1A2, and 1B1 amino acid sequences were aligned with amino acid sequences of other vertebrate CYP1 families (Fig. 3). The heme-binding motif (FxxGxxxCxG), which is known to be a highly conserved segment (Porter and Coon, 1991
; Tudzynski and Hölter, 1998), and the proline-rich region (PPGPxxxP), important for directing the folding pathway leading to the functional CYP (Kusano et al., 2001), are preserved in deduced amino acid sequences of all Baikal seal CYPs isolated in this study. The threonine-321 in human CYP1A1, which participates in a proton delivery network in the enzymatic active site and involved in oxygen activation (Blobaum et al., 2004; Hiroya et al., 1994; Vaz et al., 1996, 1998), was conserved in CYP1A1, but not in CYP1A2, where the amino acid at the corresponding site was proline in CYP1A2. In contrast, the amino acid residue corresponding to Val-382 in human CYP1A1, which is a critical residue of alkoxyresorufin metabolism (Liu et al., 2003, 2004), was conserved in all Baikal seal CYP1 sequences.
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Total CYP Content in Hepatic Microsomes
Average values of total CYP content in hepatic microsomes from immature and mature males of Baikal seals were 0.43 ± 0.22 nmol/mg and 0.68 ± 0.04 nmol/mg, respectively (Table 1). Female CYP contents were 0.32 ± 0.05 nmol/mg in immature and 0.43 ± 0.11 nmol/mg in mature animals. Statistical analysis showed that total CYP contents in mature males were significantly higher than those in mature females.
Immunoblot Analysis of CYP1A- and CYP1B-like Proteins
To determine whether the seals express CYP1 family proteins in the livers, Western blot analyses were conducted (Fig. 4). All seal hepatic microsomal samples cross-reacted notably with both anti-rat CYP1A1 polyclonal antibody and anti-rat CYP1B1 antiserum. Each band with the anticipated molecular weight was conveniently noted as seal CYP1A- and CYP1B-like proteins. Statistical analysis showed that expression levels of CYP1A-like proteins in mature males were significantly higher than those in immature and mature females, and CYP1B-like proteins in mature males were significantly higher than those in immature males and immature and mature females (Table 1).
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CYP Enzymatic Activities
Activities of MROD, EROD, PROD, and BROD in Baikal seal liver microsomes were characterized by higher activity of EROD, followed by MROD (Table 1). In contrast, BROD and PROD activities were apparently low. Neither significant sex difference nor growth stage difference was detected in these enzymatic activities. Spearman's rank correlation analysis revealed that each enzyme activity had significant positive correlation (p < 0.01) with others (Table 3). Regarding the relationships of the enzymatic activities, significant positive correlations were found between MROD and EROD (r = 0.91, p < 0.001) and between PROD and BROD (r = 0.86, p < 0.001).
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To investigate which CYP subfamily is responsible for alkoxyresorufin-O-dealkylation (AROD) activities, correlation analyses were conducted between CYP1A- or CYP1B-like protein levels and each AROD activity (Table 3). Significant positive correlations were detected between activities of MROD or EROD and expression levels of CYP1A-like protein. In addition, the expression levels of CYP1B-like protein were also positively correlated with MROD and EROD activities. No correlation was observed between PROD or BROD activities and CYP1A- or CYP1B-like protein levels.
Antibody Inhibition
To further characterize whether the seal CYP1A and CYP1B subfamilies are involved in the EROD activity, the inhibition tests were conducted by incubating each CYP antibody with seal microsomes. Both anti-rat CYP1A1 polyclonal antibody and anti-rat CYP1B1 antisera suppressed microsomal EROD activity in a dose-dependent manner, while no inhibition was shown by anti-dog CYP2B11 (Fig. 5). Taking together with the results of correlation analysis between AROD activities and CYP protein expression levels, these results suggest that EROD activity, and perhaps MROD activity also, may be catalyzed by both of CYP1A and CYP1B.
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Relationships between CYP mRNA and Protein Expression Levels
The CYP1A1, 1A2, and 1B1 mRNA expression levels in the liver of Baikal seals were measured by quantitative real-time RT-PCR. Statistical analysis showed that CYP1A2 mRNA expression levels in mature males were significantly higher than those in immature females, whereas the expression levels of CYP1A1 and CYP1B1 mRNAs showed no significant sex difference and growth stage differences (Table 1). All combinations of expression levels of CYP1A1, 1A2, and 1B1 mRNAs were strongly correlated (Table 3). The mRNA levels of both CYP1A1 and CYP1A2 revealed significant positive correlations with expression levels of CYP1A-like protein (Figs. 6A and 6B). In addition, CYP1B1 mRNA levels also showed a positive correlation with CYP1A-like protein levels. Correlation between CYP1B1 mRNA and CYP1B-like protein levels was less significant (p = 0.062), but the mRNA levels represented an increasing trend with protein levels (Fig. 6C).
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Relationships between the Hepatic TEQ and CYP mRNA, Protein, or AROD Activities
The CYP1A1, 1A2, and 1B1 mRNA expression levels showed significant positive correlations with total TEQ in the liver of Baikal seals (Table 4, Fig. 7). Congener-specific analyses in most cases revealed significant positive correlations between mRNA levels of CYP1A1, 1A2, or 1B1 and concentrations of each congener. On the other hand, no significant positive correlation was detected in some cases; between CYP1A1 and octachlorodibenzo-p-dioxin (O8CDD) and between CYP1A2 and 2,3,7,8-T4CDD, 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-T4CDF), PCB77, PCB169, PCB123, PCB157, or PCB189.
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In addition, expression levels of seal microsomal CYP1A- and CYP1B-like proteins, which were quantified by immunoblot analyses, also had positive correlations with total TEQ (Fig. 8). Since the relationship between total TEQ and CYP1A-like protein showed a sigmoidal curve, the EC50 was estimated to be 65 pg TEQ/g wet wt. Although concentrations of most congeners exhibited significant positive correlations with CYP1A-like protein levels, concentrations of highly chlorinated O8CDD showed no correlation (Table 4). Among the congeners whose concentrations were positively correlated with CYP1A-like protein level, 1,2,3,4,6,7,8-H7CDD, 2,3,7,8-T4CDF, PCB77, PCB169, and all mono-ortho Co-PCB congeners had relatively low correlations. CYP1B-like protein also showed significant positive correlations with PCDD congeners except O8CDD and 2,3,7,8-T4CDF, 1,2,3,7,8-P5CDF, 2,3,4,7,8-P5CDF, PCB81, and PCB126. However, correlation of each congener concentration with CYP1B-like protein expression levels was less prominent than those with CYP1B1 mRNA expression levels. Considering no significant correlation between CYP1B1 mRNA and CYP1B-like protein expression levels, it can be assumed that the quantitative accuracy of CYP1B-like protein may be low due to the nonspecific cross-reaction with anti-rat CYP1B1 antiserum.
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Regarding the lower levels of correlations of 2,3,7,8-T4CDF, PCB77, and mono-ortho Co-PCB congeners with CYP1A or CYP1B expression levels, preferential metabolism of these congeners by these CYP induction was hypothesized. To examine this hypothesis, concentrations of individual congeners were normalized to a relatively recalcitrant congener, PCB169, and the relationships between the congener ratios and the CYP expression levels were further assessed. The statistical analyses showed that CYP1A protein levels were negatively correlated with 1,2,3,4,6,7,8-H7CDD/PCB169, 2,3,7,8-T4CDF/PCB169, PCB77/PCB169, PCB105/PCB169, PCB114/PCB169, PCB118/PCB169, and PCB123/PCB169 ratios, while 2,3,4,7,8-P5CDF/PCB169 and PCB126/PCB169 ratios showed no correlation (Table 5). Regarding the relationships between CYP1B-like protein expression levels and the ratios of these congeners, similar correlation profiles as that of CYP1A-like protein expression levels were observed.
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Microsomal EROD activities were not correlated with total TEQ in seal livers. Relationship between hepatic total TEQ and microsomal EROD revealed an increasing trend of EROD with an elevation of total TEQ up to 200 pg TEQ/g wet wt and a declining trend at higher TEQ (Table 4, Fig. 9).
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Relationships between Hepatic TEQ Levels and CYP1B1/CYP1A1 mRNA Ratios
CYP1A1 is involved in the metabolism of 17ß-estradiol (E2) to noncarcinogenetic 2-hydroxyestradiol (2-OHE2), whereas CYP1B1 preferentially metabolizes E2 to carcinogenetic 4-hydroxyestradiol (4-OHE2) (Yager and Liehr, 1996
). Thus, the ratio of 4-OHE2/2-OHE2 could be a critical parameter of carcinogenicity of E2, and the relative expression of CYP1B1/CYP1A1 could also be crucial in terms of carcinogenesis (Coumoul et al., 2001; Liehr and Ricci, 1996). Considering the induction of these CYP isozymes by PHAHs, this study investigated the relationships between the hepatic accumulation levels of PHAHs and the ratios of CYP1B1/CYP1A1 mRNA expression levels. The results showed that the ratios of CYP1B1/CYP1A1 mRNA expression levels are positively correlated with total TEQ and individual congener concentrations (Table 6, Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Characterization of Baikal Seal CYP1A1, 1A2, and 1B1
In aquatic mammals, full-length CYP1A1 and/or CYP1A2 cDNA sequences have been reported only in gray seal, harp seal, ribbon seal, minke whale, and Atlantic white-sided dolphin. Partial sequences of CYP1A1 from dall's porpoise, steller sea lion, and largha seal and those of CYP1A2 and CYP1B1 from steller sea lion and striped dolphin were reported (Niimi et al., 2005
; Teramitsu et al., 2000; Tilley et al., 2002). In the present study, we attempted to isolate full-length CYP1A1, 1A2, and 1B1 cDNAs from the liver of Baikal seal (Fig. 1). The start site of translation of Baikal seal CYP1A1 cDNA sequence was different from those of harp seal and gray seal CYP1A cDNAs deposited in DNA Data Bank of Japan. In Baikal seal CYP1A1 cDNA, three start codon sequences of methionine (ATG) were found around the deduced start site in the 5'-terminal sequence (Fig. 1). One of them is the site corresponding to the start site of ribbon seal CYP1A1. Other two sites were located in the same sites as in harp seal and gray seal CYP1A1s and in the 3'-flanking neighbor site, respectively. Therefore, we made an interspecies comparison of Kozak sequence, a translation initiation site, and speculated the start codon site of CYP1A1 in seals. The alignment of Kozak sequences of mammalian CYP1A1s showed that deduced start codon in the most 3'-flanking site was highly conserved among mammalian CYP1A1s than other sites; this site may be the translation initiation site of Baikal seal CYP1A1. The deduced start codons of ribbon seal, harp seal, and gray seal CYP1A1s so far reported may be incorrect.
A phylogenetic analysis showed that each amino acid sequence deduced from three CYP cDNA sequences of Baikal seal was separately localized into CYP1A1, 1A2, or 1B1 clade (Fig. 2). The seal CYP1A sequences were most closely related to dog CYP1A isozymes and belonged to clusters that are different from those where cetacean CYP1As are located. This result agrees with the evolutionary history of pinnipeds and cetaceans (Niimi et al., 2005; Teramitsu et al., 2000; Tilley et al., 2002). The seal CYP1B1 sequence was closest to human CYP1B1. In comparison with amino acid sequences of other vertebrate CYP1 family, Baikal seal CYP1A1, 1A2, and 1B1 showed more than 70% identities with other mammalian CYP1A1, 1A2, and 1B1 amino acid sequences, respectively (Table 2). In addition, Baikal seal CYP1A1 and CYP1A2 showed higher identities with fish CYP1As (46–57%) than fish CYP1Bs (34–38%), while Baikal seal CYP1B1 indicated higher identities with fish CYP1Bs (51–52%) than fish CYP1As (37%). To classify the CYP families and subfamilies, a CYP nomenclature committee recommended a criteria stating that amino acid sequence identities should be >40% within the same family and >55% within the same subfamily (Nelson et al., 1996). The amino acid sequences of Baikal seal CYP1A1, 1A2, and 1B1 met this requirement for the nomenclature.
The proline-rich region and heme-binding motif were conserved in Baikal seal CYP1 isozymes, whereas conserved threonine was replaced by proline at the 
-helix I site of CYP1A2 in Baikal seal as well as harp seal (Fig. 3). Since the threonine is involved in oxygen activation, amino acid replacement at this site might affect the oxidization of substrates in seals (Blobaum et al., 2004; Hiroya et al., 1994; Vaz et al., 1996, 1998). Valine at position 382 of human CYP1A1, which is critical for alkoxyresorufin metabolism (Liu et al., 2003), was conserved in both CYP1A isozymes of Baikal seal. In human, the corresponding site of CYP1A2 is leucine, and this difference in amino acid is responsible for the different metabolic profiles of AROD between CYP1A1 and CYP1A2 (Liu et al., 2004). Baikal seal CYP1A1 and CYP1A2 share this residue, but whether this has any bearing on their metabolic function is unclear.
CYP1A and CYP1B Expression and AROD Activities
Total CYP contents in hepatic microsomes from mature males of Baikal seals were significantly higher than those in mature females. In addition, CYP1A-, CYP1B-like proteins and CYP1A2 mRNA expression levels in mature males were also found to be higher than in other groups. Our previous study showed that in the liver of male seals, concentrations of PHAH congeners showed an increasing trend with age (Iwata et al., 2004). Total TEQ in mature males were also significantly higher than those in mature females. Therefore, accumulation levels of PHAHs may account for the differences in CYP expression levels among sex and/or growth stage.
The present study measured the expression levels of CYP1A- and CYP1B-like proteins and CYP1A1, 1A2, and 1B1 mRNAs in the liver of Baikal seals. Considering the result that expression levels of CYP1A1, 1A2, and 1B1 mRNAs had strong correlations with each other, these CYP1 enzymes may be assumed to be induced by a shared mechanism, probably through activation of AHR by PHAHs (Table 3) (Shimada et al., 2002).
Both CYP1A1 and CYP1A2 mRNA expression levels showed significant positive correlations with CYP1A-like protein expression levels, suggesting that expression levels of this protein cross-reacted with the anti-rat CYP1A1 polyclonal antibody reflect both CYP1A1 and CYP1A2 mRNA expression levels (Figs. 6A and 6B). As the difference in anticipated molecular masses of the two CYP1A enzymes was less than 0.5 kDa, it is likely that detection of only a single cross-reactive band by Western blot analysis was due to the lack of resolution in SDS-PAGE. This result indicates that such an immunochemical approach may limit isoform-specific detection of multiple CYP1A isozymes, and thus measurement of CYP1A1 and CYP1A2 mRNA expression levels appear to be a useful approach for investigating the isozyme-specific effect. Intriguingly, CYP1B-like protein levels reacted with anti-rat CYP1B1 antiserum had a higher correlation with expression levels of CYP1A1 and CYP1A2 mRNA and CYP1A-like protein than CYP1B1 mRNA level. Correlation analyses suggest that anti-rat CYP1B1 antiserum used in the present study may be nonspecific to seal CYP1B1 protein and, probably, recognize CYP1A proteins.
With regard to AROD activities in Baikal seal liver microsomes, MROD and EROD activities showed significant positive correlations with both CYP1A- and CYP1B-like proteins, whereas PROD and BROD activities exhibited no correlation. Correlations between CYP1 mRNA expression levels and AROD activities were not statistically significant. Results from the correlation analyses, together with those from antibody inhibition test, suggest that at least EROD, and probably MROD also, might be dependent on CYP1 proteins recognized by anti-rat CYP1A1 polyclonal antibody and/or anti-rat CYP1B1 antiserum and not on CYP2B-like protein in the hepatic microsomes of Baikal seals (Table 3, Fig. 5). Previous studies have shown that in human and rodent species, EROD activity is preferentially catalyzed by CYP1A1 and MROD activity by CYP1A2 (Burke et al., 1994; Shimada et al., 1997). When compared with other species previously examined in our laboratory, the hepatic expression levels of CYP1A-like proteins in the wild population of common cormorants (Phalacrocorax carbo) revealed highly significant positive correlations with MROD, EROD, PROD, and BROD (Kubota et al., 2005). The hepatic CYP1A-like protein levels in jungle crows (Corvus macrorhynchos) were positively correlated only with PROD (Watanabe et al., 2005); cormorant CYP1A may be responsible for all the four activities and crow CYP1A may be active only for PROD. These results suggest that the substrate specificity for AROD activities in which CYP1 isozymes are involved is different among species (Burke et al., 1994).
PHAHs Accumulation and CYP1 Expression or AROD Activities
Baikal seal accumulates high levels of PHAHs (Iwata et al., 2004). Significant positive correlations between total TEQ and CYP1A1, 1A2, or 1B1 mRNA, CYP1A-like, or CYP1B-like protein expression levels indicate the transcriptional and translational induction of these CYP isozymes by PHAHs in Baikal seal livers (Figs. 7 and 8). The EC50 of TEQ for CYP1A-like protein induction was estimated to be 65 pg TEQ/g wet wt.
The correlation analyses revealed that the levels of significance are congener specific (Table 4). Lower levels of correlations between individual congener TEQs and CYP mRNA or protein levels were found for 2,3,7,8-T4CDF, O8CDD, PCB77, PCB169, and mono-ortho Co-PCB congeners. This may be due to the lower residue levels and/or poor CYP induction potencies of these congeners in Baikal seal liver. Concentration ratios of certain PHAH congeners to a stable congener, PCB169, revealed negative correlations with CYP mRNA and CYP protein levels (Table 5). The ratios of 2,3,7,8-T4CDF, PCB77, and some mono-ortho Co-PCBs to PCB169 especially had strong negative correlations with CYP1A-like protein or CYP1B1 mRNA expression levels, suggesting preferential metabolism of these congeners by the induced CYPs in Baikal seal. There are a certain number of investigations that support our observations. Several studies have shown that the metabolism of these two congeners increased when CYP1A is induced, although CYP1 isoform involved in the metabolism is not assigned. Ishida et al. (1991) showed that CYP1A1 play a major role in the metabolism of PCB77 in microsomes of 3-methylcholanthrene-treated rats. In vitro studies have demonstrated that hepatic CYP1A expression level and EROD activity are associated with microsomal metabolism of PCB77 in several vertebrate species including harbor seal (Murk et al., 1994; Schlezinger et al., 2000). Studies have also exhibited that 2,3,7,8-T4CDD pretreatment in rats, which increases hepatic CYP1A1 and CYP1A2 expresssion, enhances metabolism of 2,3,7,8-T4CDF (McKinley et al., 1993; Olson et al., 1994; Tai et al., 1993). Our recent studies have also shown that the ratios of PCB77/PCB169 were negatively correlated with CYP1A protein expression levels in the liver of jungle crow and common cormorant (Kubota et al., 2005; Watanabe et al., 2005).
There is a slight difference between CYP1A and CYP1B in terms of the correlations with TEQs; CYP1B1 mRNA levels showed stronger correlations with PHAH congeners than CYP1A1 and CYP1A2 mRNA levels. This suggests that induction efficiency of CYP1B1 mRNA by PHAHs may be more intensive than those of CYP1A1 and CYP1A2 mRNAs. Using DNA microarray to identify unique hepatic gene expression patterns, Vezina et al. (2004) also showed that the induction potentials of CYP1A1, 1A2, and 1B1 by 2,3,7,8-T4CDD, 2,3,4,7,8-P5CDF, and PCB126 are isozyme specific.
The relationship between total TEQ and EROD activity suggests induction of CYP1A and CYP1B catalytic activities at lower TEQ levels but an inhibition of these CYP function at higher TEQ (Fig. 9). In porcine aorta endothelial cells, a similar trend has been observed; EROD activities in cells treated with 2,3,7,8-T4CDD, PCB77, and benzo[a]pyrene increased with the dosage and then declined at the higher doses (Stegeman et al., 1995). These results support the catalytic inhibition of CYP1A and/or CYP1B isozymes, but not the suppression of CYP expression, by greater TEQ exposure. Therefore, PHAHs accumulation levels may be so high that the EROD activity is inhibited in the liver of Baikal seals.
The CYP1A1, 1A2, and 1B1 are known to be involved in the metabolism of estrogens and to mediate the initiation of cancer that is triggered by the metabolites (Martucci and Fishman, 1993; Yager, 2000). Several studies have shown that CYP1A1 and CYP1B1 are responsible for the different pathways of estrogen metabolism. CYP1A1 displays hydroxylase activity at the C2 position of E2, leading to the formation of noncarcinogenic 2-OHE2, whereas CYP1B1 catalyzes the hydroxylation at C4 position producing carcinogenic 4-OHE2 (Cavalieri et al., 1997; Hayes et al, 1996; Spink et al., 1992; Yager and Liehr, 1996). Therefore, the ratio of 4-OHE2/2-OHE2 and furthermore the ratio of CYP1B1/CYP1A1 are considered to be indicative parameters reflecting the carcinogenic potential of E2 (Coumoul et al., 2001; Liehr and Ricci, 1996). In this study, the ratios of CYP1B1/CYP1A1 mRNA expression levels in Baikal seal livers had positive correlations with total TEQ and concentrations of all individual congeners examined (Table 6, Fig. 10). This result implies that CYP1B1 is preferentially induced by these chemicals, and the potential risk of carcinogenicity in the liver of Baikal seal may be enhanced by an increase in CYP1B1/CYP1A1 ratio. On the other hand, the ratios of CYP1A/CYP1B protein expression levels showed no correlation with total TEQ or individual congener TEQs. The redundant cross-reactivities of anti-rat CYP1A1 polyclonal antibody and anti-rat CYP1B1 antiserum might be the reason for such a poor correlation.
Our study reveals that the basic mechanism of AHR-mediated responses is conserved both in Baikal seal and experimental animals. Considering the relationships between TEQs and EROD or CYP1 isozymes in Baikal seal, we suggest that hepatic CYP1A and CYP1B subfamilies were induced by TEQ, but the catalytic function of CYP1 isozyme may be inhibited by a greater accumulation of PHAHs. Most PHAH congeners contributing to total TEQ were not likely to be metabolized by induced CYP1A and CYP1B, leading to the chronic CYP induction. In contrast, low-chlorinated congeners, 2,3,7,8-T4CDF and PCB77, may be preferentially metabolized by the induced CYPs in Baikal seal liver. Due to the continuous disruption of AHR- and CYP1-mediated signaling pathways by recalcitrant PHAHs, Baikal seal population may experience a serious threat by these contaminants.
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ACKNOWLEDGMENTS |
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The authors wish to thank Professor An. Subramanian, Ehime University, for critical reading of this article. This study was supported by Grants-in-Aid for Scientific Research (A) (Nos. 17208030 and 16201014) and (B) (No. 13480170) from Japan Society for the Promotion of Science and for Scientific Research on Priority Areas (A) (No. 13027101). Financial assistance was also provided by "21st Century Center of Excellence Program" from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by Feasibility Studies for Basic Research in ExTEND 2005 (Enhanced Tack on Endocrine Disruption) from the Ministry of the Environment, Japan.
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