Hepatic CYP1A Induction by Dioxin-like Compounds, and Congener

The toxic and biochemical mechanism of PHAHs is not entirely known in wild ..... normalized to PCB169 (congener/PCB169 ratio on wet weight basis) in t...
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Environ. Sci. Technol. 2005, 39, 3611-3619

Hepatic CYP1A Induction by Dioxin-like Compounds, and Congener-Specific Metabolism and Sequestration in Wild Common Cormorants from Lake Biwa, Japan A K I R A K U B O T A , † H I S A T O I W A T A , * ,† SHINSUKE TANABE,† KUMIKO YONEDA,‡ AND SACHIKO TOBATA‡ Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, and Japan Wildlife Research Center, Shitaya 3-10-10, Taito-ku, Tokyo 110-8676, Japan

The present study examines the effects of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (CoPCBs) on hepatic cytochromes P450 (CYP) in the wild population of common cormorants from Lake Biwa, Japan, and discusses functional roles of CYP1A in terms of correlation analysis between tissue concentrations of individual congeners and expression levels of CYP1A. Levels of alkoxyresorufin (methoxy-, ethoxy-, pentoxy-, and benzyloxyresorufin) O-dealkylase activities and a protein cross-reacted with anti-rat CYP1A1 polyclonal antibodies showed significant positive correlations with total 2,3,7,8tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) or TEQs for most individual congeners in the liver of cormorants, suggesting induction of CYP1A-like protein by these chemicals. In contrast, TEQs for lower chlorinated congeners, 2,3,7,8-T4CDF and PCB77, showed relatively low correlations with the expression level of CYP1A-like protein. Concentrations of 2,3,7,8-T4CDF and PCB77 normalized to a relatively recalcitrant congener, PCB169, were negatively correlated with the CYP1A-like protein level. These results indicate preferential metabolism of those congeners by CYP1A-like protein that was induced by TEQs. Concentration ratios of liver to pectoral muscle for certain congeners significantly increased with an elevation of the CYP1A-like protein level. Comparing the results in the present study with those of previous studies using rodents treated with certain dioxinlike congeners, these congeners in the liver may be sequestered by CYP1A. Levels of cross-reactive proteins with anti-rat CYP2B1, CYP2C6, and CYP3A2 polyclonal antibodies correlated with neither TEQs nor liver/muscle concentration ratios of congeners. We conclude that the potential for CYP1A induction, and metabolism and sequestration of dioxin-like compounds by CYP1A, may be a critical factor for assessing the ecological risk in wild avian species.

* Corresponding author and address phone/fax: +81-89-927-8172; e-mail: [email protected]. † Ehime University. ‡ Japan Wildlife Research Center. 10.1021/es048771g CCC: $30.25 Published on Web 04/12/2005

 2005 American Chemical Society

Introduction Highly toxic planar halogenated aromatic hydrocarbons (PHAHs), including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs), have been of great concern as environmental contaminants because of their worldwide distribution, bioaccumulative nature, and possible adverse effects on wildlife and humans. PHAHs produce a broad spectrum of toxic and biochemical effects such as body weight loss, immune dysfunction, teratogenicity, reproductive toxicity, carcinogenicity, and induction of cytochrome P450 (CYP) 1A in experimental animals (1-3). In addition to experimental studies, there are several reports that indicate the impairments of reproductive performance and developmental deformities in wild avian species are possibly caused by high accumulation of dioxin-like compounds (4, 5). The toxic and biochemical mechanism of PHAHs is not entirely known in wild birds but is likely to involve an activation of the aryl hydrocarbon receptor (AHR) (6-8). A well-characterized response to PHAHs through the AHR is the induction of CYP1A (9). Studies using transgenic mice lacking the functional Cyp1a1 (10) or Cyp1a2 gene (11) demonstrated that CYP1A1 and/or CYP1A2 were, at least partially, involved in 2,3,7,8-T4CDD toxicity, including lethality, wasting syndrome, hepatocyte hypertrophy, and uroporphyria. Furthermore, translational inhibition of CYP1A using morpholino antisense oligos reduced PHAH-induced developmental toxicity in zebrafish embryos (12). Therefore, measurement of CYP1A levels both in catalytic activities, including ethoxyresorufin O-deethylase (EROD) activity, and in protein expression is considered as a useful approach to assess not only the environmental exposure to PHAHs, but also their effects. There are a number of field studies examining association of CYP with PHAH mixtures in avian species. For instance, total 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) for AHR-active PCB congeners were associated with hepatic CYP1A levels in pipping black-crowned night-heron (Nycticorax nycticorax) embryos collected from the polluted sites in the Great Lakes and San Francisco Bay, which imply environmental induction of CYP1A by these contaminants (13). Sanderson et al. (14) also found a significant positive correlation between total TEQs for almost all 2,3,7,8-chlorinesubstituted PCDD/DFs and Co-PCBs and EROD activity in the liver of double-crested cormorant (Phalacrocorax auritus) hatchlings collected from five colonies across Canada, with differing levels of contamination. Nevertheless, considering the results of in vitro studies using hepatocyte cultures of several avian species, large differences in responsiveness of CYP1A induction upon exposure to PHAHs were observed (7, 8). Thus, field investigations addressing a wide variety of avian species are necessary to assess the impact of PHAH exposures on CYP expressions. Moreover, most of the field studies have focused only on the potential for CYP induction by these contaminants. The subsequent toxicokinetic effects of individual congeners in which CYP expressions are involved remain to be understood in wild birds. As most of the toxicokinetic data examining the association of PHAH exposures with CYP expressions have been established depending upon acute or subchronic treatment with single or repeated doses of defined congener(s), the appropriateness of extrapolation of the results from such experimental animals to the wild population, which are chronically exposed to a complex mixture of PHAHs, is questionable. VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Our previous study (15) showed significant positive correlations between total TEQs for six Co-PCB congeners and EROD and pentoxyresorufin O-depenthylase (PROD) activities in the liver of common cormorants (Phalacrocorax carbo) collected from Lake Biwa. However, no chemical congener- and CYP isoform-specific correlation analyses were conducted to assess the effects of Co-PCBs on CYP expressions in the cormorants. To date, our group presented the residue levels and toxicokinetic behaviors of all PCDD, PCDF, and Co-PCB congeners, for which toxic equivalency factors (TEFs) were assigned by the World Health Organization (16), for the Lake Biwa cormorant population (17). Total TEQs in the liver of cormorants ranged from 360 to 50000 pg/g lipid weight (from 12 to 1900 pg/g wet weight), and PCB126, 2,3,4,7,8-P5CDF, and 1,2,3,7,8-P5CDD were major total TEQ contributors. Analysis of life-stage-related accumulation and liver-muscle distribution of PCDD/DFs and Co-PCBs revealed that certain congeners could be subjected to hepatic metabolism or sequestration in a TEQ-dependent manner. On the basis of these results, we hypothesized that there was a TEQ-inducible protein(s) in cormorant liver (17) which was involved in hepatic metabolism or sequestration of PHAH congeners, such as CYP1A1 and/or CYP1A2 in rodent (10, 18, 19). Recently, cDNAs of two distinct AHRs were isolated in common cormorant (20), which might play a key role in the AHR-CYP signaling pathway and congener-specific profiles of the residue level in this species. However, no comprehensive data are available for cormorant on CYP induction and on hepatic metabolism and sequestration of PHAH congeners associated with CYP expression. This study therefore investigates the effects of PCDD/DFs and Co-PCBs on CYP protein expressions in the Lake Biwa population of common cormorants. Particular focus is placed on whether environmental induction of CYP enzyme(s) occurs at ambient levels of these contaminants, and whether CYP protein expressions are linked to the toxicokinetics of these chemicals, through a range of correlation analyses between tissue concentrations of individual congeners and expression levels of cross-reactive proteins with anti-rat CYP polyclonal antibodies.

Materials and Methods Reagents for Enzyme Assay. Resorufin, methoxyresorufin, ethoxyresorufin, pentoxyresorufin, and benzyloxyresorufin were purchased from Sigma Chemical (St. Louis, MO). NADPH was obtained from Nacalai Tesque (Kyoto, Japan). Goat anti-rat CYP1A1, CYP2B1, and CYP2C6 antisera and a rabbit anti-rat CYP3A2 antiserum for the immunochemical analysis of the cross-reactive proteins in cormorants were purchased from Daiichi Pure Chemicals (Tokyo, Japan), and rat standard microsomes in which the corresponding CYP proteins were expressed were from BD Gentest (Woburn, MA). Horseradish peroxidase (HRP)-linked anti-goat immunoglobulin G (IgG) and anti-rabbit IgG were purchased from Bethyl Laboratories (Montgomery, TX) and Cell Signaling Technology (Beverly, MA), respectively. The other chemicals and reagents used were of biochemical grades commercially available. Sample Collection. Twenty-six common cormorants captured under license from Shiga Prefecture in May 2001 from the southern part of Lake Biwa were used for this study. Cormorants were immediately dissected on board after measurements of biometry. Subsamples of liver for enzyme assays were flash frozen in liquid nitrogen and stored at -80 °C until microsome preparation. The rest of the livers and pectoral muscles were stored at -20 °C until chemical analysis. The growth stage of the cormorants was determined from the development of the reproductive organs. For nine female specimens, however, the growth stage could not be 3612

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determined due to the involution of the reproductive organ, as the specimens were collected at the end of the reproductive period. The sample sizes of individual life stages were 6 juveniles and 11 adults. Nine specimens were of unknown reproductive stage. Microsomal Preparation and Protein Determination. Approximately 5.0 g of liver sample was minced and homogenized in a 10 mL Teflon-glass homogenizer containing a cold buffer (50 mM Tris-HCl, 0.15 M KCl, adjusted to pH 7.4-7.5 at 25 °C). The sample was then centrifuged at 750g for 10 min at 4 °C. After removal of the nuclear fraction, the supernatant was centrifuged at 12000g for 10 min at 4 °C. The supernatant was further centrifuged at 105000g for 1.5 h at 4 °C. Following centrifugation, the supernatant was removed and the microsomal pellet was resuspended in an equivalent volume of resuspension buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.4-7.5, dissolved in a 20% glycerol solution). Microsomes were placed in cryostorage vials, flash frozen in liquid nitrogen, and stored at -80 °C until alkoxyresorufin O-dealkylase (AROD) activity assay and Western blotting. Total protein concentrations in the hepatic microsomes of cormorants were determined by the method of bicinchoninic acid assay (21) using a BCA protein assay kit (Pierce, Rockford, IL). AROD Activity Assay. AROD activities, including methoxyresorufin O-demethylase (MROD), EROD, PROD, and benzyloxyresorufin O-debenzylase (BROD) activities, were measured in 48-well microplates in duplicate using a spectrofluorometer, SpectraFluor Plus (Tecan, Maennedorf, Switzerland), following the method described by Iwata et al. (22), with some modifications. All substrate solutions were stocked in methanol or dimethyl sulfoxide. Reactions were initiated by adding respective substrates (2.0 µM), microsomes (approximately 1.0 mg/mL), and NADPH (1.33 mM), and then the reaction mixtures were incubated for 5 min at 37 °C. Resorufin formed by each reaction was measured at an excitation wavelength of 535 nm and an emission wavelength of 595 nm. All catalytic activities were determined using a regression line generated from a set of resorufin standard solutions (2.0-100 pmol/well), and were expressed by the amount of resorufin formed per minute per milligram of microsomal protein (pmol of resorufin/min/ mg of protein). Western Blotting. Liver microsomal proteins from cormorants were resolved by sodium deodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as originally described by Laemmli (23). Samples of 20 µg of microsomal proteins were placed on 5-20% gradient polyacrylamide gels (ATTO, Japan) in duplicate. The proteins were separated by electrophoresis using a powered mini PAGE system, pageRun (ATTO). Rat CYP1A1, CYP2B1, CYP2C6, or CYP3A2 standard microsomes were simultaneously loaded onto the gradient gels as positive controls for the corresponding cross-reactive proteins in cormorant microsomes. Gel electrophoresis was performed at 125 V for 2 h. Western blotting analysis was conducted according to Kyhse-Andersen (24). The resolved proteins were electrophoretically transferred to polyvinylidene fluoride membranes at 40-45 mA for a period of 1 h using a SemiPhor semidry transfer unit (Amersham Biosciences, Piscataway, NJ). The membranes were probed with the polyclonal antibodies against rat CYP1A1, CYP2B1, and CYP2C6 (goat antiserum) and CYP3A2 (rabbit antiserum). The secondary antibody was rabbit anti-goat IgG linked to HRP for CYP1A, CYP2B, and CYP2C detection, and goat antirabbit IgG linked to HRP for CYP3A detection. Detection of the antibody cross-reactive proteins was performed using a highly sensitive ECL Western blotting system (Amersham Biosciences). Protein bands were visualized by an imaging analyzer, ChemiDoc, and quantified using

TABLE 1. CYP-Dependent Enzymatic Activities (pmol/min/mg protein) in Liver Microsomes of Common Cormorantsa juvenile

adult

TABLE 2. Spearman’s Rank Correlations among CYP Protein Expression Levels in Liver Microsomes of Common Cormorantsa

male (n ) 3) female (n ) 3) male (n ) 8) female (n ) 3) MROD EROD PROD BROD

29 ( 12 (20-42) 56 ( 25 (36-85) 1.7 ( 0.30 (1.4-2.0) 4.5 ( 1.6 (3.0-6.2)

67 ( 31 (35-97) 160 ( 86 (68-240) 3.3 ( 0.92b (2.2-4.0) 24 ( 24 (6.2-51)

70 ( 24b,c (48-120) 190 ( 120c (110-470) 3.5 ( 1.1c (2.5-6.0) 23 ( 18c (11-58)

44 ( 11 (32-55) 130 ( 31 (100-160) 2.9 ( 0.87 (2.0-3.7) 15 ( 8.3 (5.9-21)

a Numbers represent the mean ( SD (minimum-maximum). b Comparison with the other sex (p < 0.05). c Comparison with the other life stage (p < 0.05).

Quantity One (Bio-Rad Laboratories, Hercules, CA). Expression levels of the cross-reactive proteins in individual animals were expressed as a relative value to the staining intensity of the antibody cross-reactive protein in a specimen. PCDD, PCDF, and Co-PCB Concentrations. Data on the concentrations and TEQs of PCDD/DF and Co-PCB congeners in all 26 livers and in 20 pectoral muscles of common cormorants from Lake Biwa used in this study have already been reported elsewhere (17). TEQs were calculated using TEFs for birds reported by Van den Berg et al. (16). Statistical Analyses. All the statistical analyses were performed using StatView v. 5.0 (SAS Institute, Cary, NC). The Mann-Whitney U test was employed to detect differences in AROD activities by sex and life stage, using the data obtained from the specimens in which the life stage was determined. Spearman’s rank correlation test was performed to evaluate the relationships among TEQs for individual dioxin-like congeners, AROD activities, expression levels of antibody cross-reactive proteins, and liver/muscle concentration ratios. For values below the quantification limit, half of the values were substituted. When more than 50% of the observations were below the quantification limit, statistical analyses were not conducted for the congener, and those results are shown as “no data available (NA)”. A p value of < 0.05 was regarded as statistically significant.

Results and Discussion Enzymatic Activities and Immunochemical Detection of CYP Proteins. Hepatic microsomal AROD activities, including MROD, EROD, PROD, and BROD activities in common cormorants, were characterized by the highest activity of

CYP1A CYP2B CYP2C a

CYP2B

CYP2C

CYP3A

-0.34

-0.26 -0.048

0.12 -0.23 0.38

Numbers represent F values.

EROD, followed by MROD, BROD, and PROD (Table 1). No clear sex difference in AROD activities was observed except for MROD in the adult (p ) 0.041), which was higher in the male than the female, and for PROD in the juvenile (p ) 0.049), which was higher in the female than the male. There is no comprehensive study available that has examined a gender difference in AROD activities of wild birds. A study using wild ribbon seals (Phoca fasciata) collected from Hokkaido, Japan, showed significantly higher MROD and EROD activities in the male (25), which is consistent with our results. AROD activities in adult male specimens were significantly higher than those in juveniles; it is unclear whether the life stage is an influencing factor, because adult birds contained significantly higher TEQs than juveniles (17). To determine whether cormorants expressed hepatic CYP1A-, CYP2B-, CYP2C-, and CYP3A-like proteins and to measure their expression levels, Western blotting was performed using polyclonal antibodies against rat CYP1A1, CYP2B1, CYP2C6, and CYP3A2, respectively. Proteins crossreacted with these antibodies in cormorant hepatic microsomes showed molecular weights similar to those of the corresponding rat CYP standards (Figure 1). Spearman’s rank correlation test revealed that none of the expression levels of CYP1A-, CYP2B-, CYP2C-, and CYP3A-like proteins showed significant correlation with each other (Table 2). These results imply that all the anti-rat CYP polyclonal antibodies used in this study recognize different CYP isoforms. Our results might be supported by the fact that an avian paradigm species, chicken, has multiple CYP isoforms belonging to subfamilies of CYP1A (26), CYP2C (27), and CYP3A (28). It is likely that there are two CYP1A isoforms in cormorant as well as chicken, but both were not resolved on SDS gels, which eventually resulted in a single broad band. A similar single protein band has been detected in hepatic microsomes of wild and chemically treated birds using various polyclonal antibodies against rat CYP1A1 (14, 29-31). Alternatively, only one of the CYP1As might show cross-reactivity with anti-rat CYP1A1 due to the epitopes that were involved. As for CYP2B, our

FIGURE 1. Cross-reactive proteins with polyclonal antibodies against rat CYP1A1, CYP2B1, CYP2C6, and CYP3A2 in hepatic microsomes of common cormorants. Rat standard microsomes for each CYP isoform are included as positive controls for the corresponding CYP proteins in cormorant hepatic microsomes. VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Relationships among AROD activities in hepatic microsomes of common cormorants. results are different from those of previous studies that showed weak or no cross-reactivity with anti-rat CYP2B1 in hepatic microsomes of birds (14, 29-32), which may also be explained, in part, by differences in the epitopes with which the antibodies can react. Expression of cross-reactive protein with anti-rat CYP2B1 in cormorant livers may be a reflection of CYP2H or CYP2C, as the rat CYP2B1 amino acid sequences showed 48% identity both with chicken CYP2H1/2H2 and with chicken CYP2C45. In the liver microsomes of common cormorants, there were strong positive correlations (p < 0.001) among M-, E-, P-, and BROD activities: one activity was in parallel with the other activities (Figure 2). Similar relationships have been reported for multiple avian species, including great blue herons (Ardea herodias) (33), common terns (Sterna hirundo) (32), and herring gulls (Larus argentatus) (34, 35). To further investigate which CYP subfamily was responsible for AROD activities in the liver of cormorants, correlation analyses were also conducted between expression levels of immunochemically detected CYP1A-, CYP2B-, CYP2C-, and CYP3A-like proteins and AROD activities (Table 3). Significant positive correlations were found between the CYP1A-like protein level and the EROD activity (Figure 3) and the other three catalytic activities, whereas none of the expression levels of CYP2B-, CYP2C-, and CYP3A-like proteins were correlated with AROD activities. These findings suggest that AROD activities in the wild population of cormorants may be mainly catalyzed by CYP1A, and avian CYP1A may have catalytic properties different from those of rodent CYP1A, which is less preferable for PROD and BROD activities to CYP2B (36, 37). 3614

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TABLE 3. Spearman’s Rank Correlations among CYP Protein Expression Levels in Liver Microsomes of Common Cormorantsa CYP1A CYP2B CYP2C CYP3A a

MROD

EROD

PROD

BROD

0.77b

0.83c

0.73b

0.68b -0.045 -0.19 0.027

-0.22 -0.022 0.29

-0.18 -0.12 0.28

Numbers represent F values.

b

-0.11 -0.17 0.11

p < 0.001. c p < 0.0001.

Induction of CYP1A by TEQs. Our previous study reported the residue levels of PCDD/DF and Co-PCB congeners in the same specimens used in this study (17). Total TEQs of PCDD/ DFs and Co-PCBs in the liver of cormorants were in the range of 360-50000 pg/g of lipid weight (12-1900 pg/g wet weight). To examine whether these chemicals that accumulated in the liver of cormorants altered hepatic CYP protein expressions, correlation analyses were performed between hepatic total TEQs and CYP protein levels or AROD activities. The relationships showed that both of CYP1A-like protein and its catalytic activities significantly increased with an increase in total TEQs (Figure 4), suggesting the induction of CYP1Alike protein by PCDD/DFs and Co-PCBs in cormorant livers. In contrast, expression levels of CYP2B-, CYP2C-, and CYP3Alike proteins were correlated neither with total TEQs nor with TEQs of the individual congeners (p > 0.05), indicating no significant induction of these enzymes by dioxin-like compounds.

FIGURE 3. Relationship between the expression level of CYP1Alike protein and the EROD activity in hepatic microsomes of common cormorants.

FIGURE 4. Relationships between total TEQs (wet weight basis) and the EROD activity or expression level of CYP1A-like protein in the liver of common cormorants. These measurements were logarithmically transformed to make the relationships clearly visible. Total TEQs were cited from Kubota et al. (17), derived from the same specimens used in this study. Significant concentration-dependent induction of CYP1A protein expression and/or its catalytic activities has been reported in various wild avian species, including great blue herons (33), black-crowned night-herons (13), double-crested cormorants (14), cormorants (P. carbo) (38), common terns (32, 39), bald eagles (Haliaeetus leucocephalus) (40), and ospreys (Pandion haliaetus) (41). In accordance with our results, Ronis et al. (29, 30) demonstrated no correlation between cross-reactive protein with anti-rat CYP2B1 polyclonal antibodies and concentrations of PCBs in the liver of six fish-eating birds, including cormorants. Comparable results were also reported for a hepatic cross-reactive protein with anti-rat CYP2B1/2 in double-crested cormorants (14) and common terns (32) contaminated by PCDD/DFs and PCBs. In 2,3,7,8-T4CDD-treated chickens, there was no inducible hepatic cross-reactive protein with anti-rat CYP2B1 and CYP3A1 polyclonal antibodies (31). On the other hand, proteins cross-reacted with anti-rat CYP2C11 were modestly

FIGURE 5. Relationships between the expression level of CYP1Alike protein and congener concentrations normalized to PCB169 (congener/PCB169 ratio on wet weight basis) in the liver of common cormorants. These measurements were logarithmically transformed to make the relationships clearly visible. Concentrations of individual congeners were cited from Kubota et al. (17), derived from the same specimens used in this study. induced in 2,3,7,8-T4CDD-treated chicken livers (31). The isoform difference in cross-reactive CYP2C proteins between this study (anti-rat CYP2C6 polyclonal antibodies) and the chicken study (anti-rat CYP2C11 polyclonal antibodies) possibly resulted in a distinct response to dioxin-like compounds. This difference may also be attributed to the differences in the species and uptake route of chemicals: the wild cormorant absorbed the congeners through the gastrointestinal tract, whereas in the chicken 2,3,7,8-T4CDD was injected into the allantoic fluid. Considering these results, CYP1A is regarded as a highly sensitive and specific biomarker for exposure to dioxin-like compounds. Relationships between TEQs for individual congeners and CYP1A-like protein level were also examined (Table 4). TEQs for most individual congeners, including PCB126, 2,3,4,7,8P5CDF, and 1,2,3,7,8-P5CDD, which were the main contributors to the total TEQs in these specimens (17), showed significant positive correlations with CYP1A (p < 0.0001). In VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Relationships between the expression level of CYP1A-like protein and liver/pectoral muscle concentration ratios in common cormorants. These measurements were logarithmically transformed to make the relationships clearly visible. Concentrations of individual congeners were cited from Kubota et al. (17), derived from the same specimens used in this study.

TABLE 4. Spearman’s Rank Correlations between TEQs and the Expression Level of CYP1A-like Protein in the Liver of Common Cormorantsa PCDD/DFs

G value

Co-PCBs

G value

2,3,7,8-T4CDD 1,2,3,7,8-P5CDD 1,2,3,4,7,8-H6CDD 1,2,3,6,7,8-H6CDD 1,2,3,7,8,9-H6CDD 1,2,3,4,6,7,8-H7CDD 1,2,3,4,6,7,8,9-O8CDD 2,3,7,8-T4CDF 1,2,3,7,8-P5CDF 2,3,4,7,8-P5CDF 1,2,3,4,7,8-H6CDF 1,2,3,6,7,8-H6CDF 1,2,3,7,8,9-H6CDF 2,3,4,6,7,8-H6CDF 1,2,3,4,6,7,8-H7CDF 1,2,3,4,7,8,9-H7CDF 1,2,3,4,6,7,8,9-O8CDF

0.78d 0.79e 0.81e 0.77d 0.65c 0.55c 0.45b 0.38 NA 0.81e 0.79e 0.77d NA 0.77d 0.67d NA NA

PCB77 PCB81 PCB126 PCB169

0.48b 0.76d 0.83e 0.81e

PCB105 PCB114 PCB118 PCB123 PCB156 PCB157 PCB167 PCB189

0.79e 0.79e 0.79e 0.81e 0.81e 0.78e 0.80e 0.80e

a NA denotes no data available because more than 50% of the observations were below the quantification limit (17). b p < 0.05. c p < 0.01. d p < 0.001. e p < 0.0001.

contrast, O8CDD (p ) 0.024) and lower chlorinated congeners, 2,3,7,8-T4CDF (p > 0.05) and PCB77 (p ) 0.018), exhibited relatively low correlations. Because O8CDD is poorly absorbed from the gastrointestinal tract, tissue concentration of this congener does not increase with an increase in the total TEQs (17), thus resulting in the weak correlation between O8CDD and CYP1A-like protein. For the lower correlations of 2,3,7,8T4CDF and PCB77, preferential metabolism of these congeners by CYP1A induction may be speculated. Implication of Congener-Specific Metabolism by CYP1A. To provide more evidence of preferential metabolism of lower chlorinated congeners by induced CYP1A, concentrations of 2,3,7,8-T4CDF and PCB77 were normalized to a relatively recalcitrant congener, PCB169 (15), and the relationships between the CYP1A-like protein level and the congener ratios were examined. Spearman’s rank correlation test revealed 3616

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that the CYP1A-like protein level was negatively correlated with the 2,3,7,8-T4CDF/PCB169 (p ) 0.0008) and PCB77/ PCB169 (p ) 0.0002) ratios (Figure 5). On the contrary, no correlation with PCB126/PCB169 (p > 0.05) was obtained. These results indicate that less chlorinated congeners, 2,3,7,8T4CDF and PCB77, were preferentially metabolized by CYP1A that was induced by PCDD/DFs and Co-PCBs. Several other studies using avian and rodent species also support our data. Murk et al. (42) demonstrated that PCB77 was rapidly metabolized by the eider duck hepatic microsomes treated with this congener, and consequently, three hydroxylated metabolites (4-OH-3,3′,4,5-, 5-OH-3,3′,4,4′-, and 6-OH-3,3′,4,4′-tetrachlorobiphenyl) were identified. In rats, 2,3,7,8-T4CDD pretreatment, with an increase in CYP1A, produced an increase in the metabolism of 2,3,7,8-T4CDF (43). Preferential metabolism of these lower chlorinated congeners related to CYP1A induction was also found in wild populations of black-footed albatross (Diomedea nigripes) collected from the North Pacific (44). CYP1A Expression Responsible for Hepatic Sequestration. In our previous study (17), significant positive correlations were found between hepatic total TEQs and concentration ratios for most individual congeners in the liver to pectoral muscle. This implies concentration-dependent hepatic sequestration of dioxin-like compounds in the wild cormorant population. On the basis of these results, we hypothesized that cormorant hepatic tissue expressed an inducible binding protein(s), and consequently, hepatic sequestration of congeners occurred. In mammalian species, dose-dependent hepatic sequestration of congeners appears to be due to a liver-specific inducible species, namely, CYP1A2 (18, 45-47), as the enzyme has a specific binding affinity for certain 2,3,7,8-substituted PCDD/DF and Co-PCB congeners (48-50). Furthermore, a study using Cyp1a2(-/-) knockout mice demonstrated much less accumulation of 2,3,7,8-T4CDD and 2,3,4,7,8-P5CDF in the liver (19). A more recent study showed that no difference in 2,3,7,8-T4CDD sequestration in liver was found between transgenic Cyp1a1(-/-) knockout and Cyp1a1/1a2(+/+) wild-type mice, indicating a minor contribution of CYP1A1 on sequestration (10).

TABLE 5. Spearman’s Rank Correlations between the Expression Level of CYP1A-like Protein and Liver to Pectoral Muscle Concentration Ratios of PCDDs, PCDFs, and Co-PCBs in Common Cormorantsa PCDD/DFs

nb

G value

Co-PCBs

nb

G value

2,3,7,8-T4CDD 1,2,3,7,8-P5CDD 1,2,3,4,7,8-H6CDD 1,2,3,6,7,8-H6CDD 1,2,3,7,8,9-H6CDD 1,2,3,4,6,7,8-H7CDD 1,2,3,4,6,7,8,9-O8CDD

20 20 19 20 18 19 20

0.63d 0.51c 0.49c 0.70d 0.52c 0.54c 0.74d

PCB77 PCB81 PCB126 PCB169

20 20 20 20

-0.072 0.68d 0.67d 0.74d

2,3,7,8-T4CDF 1,2,3,7,8-P5CDF 2,3,4,7,8-P5CDF 1,2,3,4,7,8-H6CDF 1,2,3,6,7,8-H6CDF 1,2,3,7,8,9-H6CDF 2,3,4,6,7,8-H6CDF 1,2,3,4,6,7,8-H7CDF 1,2,3,4,7,8,9-H7CDF 1,2,3,4,6,7,8,9-O8CDF

18 7 20 19 19 4 19 17 3 4

0.73d NA 0.49c 0.53c 0.56c NA 0.25 0.38 NA NA

PCB105 PCB114 PCB118 PCB123 PCB156 PCB157 PCB167 PCB189

20 20 20 20 20 20 20 20

0.16c 0.49 0.41 0.47c 0.34 0.41 0.39 0.39

a NA denotes no data available because more than 50% of the observations were below the quantification limit (17). b The number of specimens in which the particular congener was detected both in the liver and pectoral muscle. c p < 0.05. d p < 0.01.

To further provide a plausible explanation of the concentration-dependent deposition of certain dioxin-like congeners in cormorant livers (17), relationships between CYPlike protein levels and liver/muscle concentration ratios for individual congeners were evaluated. For all 2,3,7,8substituted PCDDs, 2,3,7,8-T4CDF, 2,3,4,7,8-P5CDF, 1,2,3,4,7,8H6CDF, 1,2,3,6,7,8-H6CDF, PCB81, PCB126, PCB169, PCB114, and PCB123, the liver/muscle ratios significantly increased with the CYP1A-like protein level (Table 5, Figure 6), implying CYP1A-dependent hepatic sequestration of these congeners. Our results, therefore, indicate the possible role of an avian CYP1A isoform(s), corresponding to mammalian CYP1A2, as a hepatic binding protein involved in PHAH sequestration in the liver of cormorants. Results from correlation analysis between the CYP1A-like protein level and liver/muscle ratios were roughly consistent with correlation profiles between hepatic total TEQs and liver/muscle ratios except for monoortho-Co-PCBs (17). No significant correlation (p > 0.05) between expression levels of CYP2B-, CYP2C-, and CYP3Alike proteins and the liver/muscle ratios for individual congeners was observed. Therefore, certain PHAH congeners might be sequestered by their binding to CYP1A induced in the liver of cormorants. This paper is the first report suggesting CYP1A-dependent hepatic sequestration of individual PCDD/ DF and Co-PCB congeners in wild avian species. In conclusion, the overall toxicity of dioxin-like compounds in wild avian species may be determined by a balance of (1) the potential for CYP1A induction, (2) metabolism by CYP1A, and (3) sequestration by CYP1A. It has been reported that white leghorn chicken, the most common avian model species, has two distinct forms of CYP1A, namely, CYP1A4 and CYP1A5 (26). However, while comparing the amino acid sequences and functions of chicken CYP1A isoforms with those of mammalian CYP1A isoforms, neither chicken CYP1A4 nor chicken CYP1A5 appears to be directly orthologous to CYP1A1 or CYP1A2. Hence, cormorant CYP1A isoforms corresponding to chicken CYP1A4 and CYP1A5 would be involved in congener-specific metabolism and sequestration of dioxin-like compounds. Further research is necessary to determine the presence of CYP1A isoforms and to characterize their isoform-specific function in the liver of cormorants. This approach may unravel the isoform-specific

capacities of CYP1As in metabolizing and sequestering individual PHAH congeners in avian species.

Acknowledgments We thank Prof. An. Subramanian, Ehime University, for critical reading of this manuscript. Financial assistance was provided by the “Survey on the State of Dioxin Accumulation in Wildlife” from the Ministry of the Environment, Japan. This study was also supported by Grants-in-Aid for Scientific Research (B) (No. 13480170) from the Japan Society for the Promotion of Science (JSPS), and for Scientific Research on Priority Areas (A) (No. 13027101), and by the “21st Century COE Program” from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The award of the JSPS Doctoral Fellowship for Researchers in Japan to A.K. (Grant No. 00407) is acknowledged.

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Received for review August 6, 2004. Revised manuscript received February 27, 2005. Accepted March 3, 2005. ES048771G

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