ToxSci Advance Access originally published online on May 5, 2006
Toxicological Sciences 2006 92(2):394-408; doi:10.1093/toxsci/kfl001
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cytochrome P450 1A4 and 1A5 in Common Cormorant (Phalacrocorax carbo): Evolutionary Relationships and Functional Implications Associated with Dioxin and Related Compounds
* Center for Marine Environmental Studies, Ehime University, Matsuyama 790-8577, Japan; Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts 02543; and Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA
1 To whom correspondence should be addressed at Center for Marine Environmental Studies, Ehime University, Building "Sogo-Kenkyuto"-1, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan. Fax: +81-89-927-8172. E-mail: iwatah{at}agr.ehime-u.ac.jp.
Received March 7, 2006; accepted April 21, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The present study characterized cytochrome P4501A (CYP1A) isoforms from common cormorant (Phalacrocorax carbo) with regard to their evolutionary relationships and their roles in disposition of dioxin and related compounds (DRCs). Two clones isolated from a cormorant liver cDNA library were named CYP1A4 and CYP1A5 on the basis of greatest overall amino acid identity shared with chicken (Gallus gallus) CYP1A4 (78%) and CYP1A5 (78%), respectively. Spatial heterogeneity in phylogenetic signal along the sequences strongly indicated that cormorant CYP1A4 and CYP1A5 have undergone partial interparalog gene conversion, similar to chicken and mammalian CYP1As. Phylogenetic analysis of a putatively unconverted region produced a tree topology consistent with the orthology of avian CYP1A5s with mammalian CYP1A2s and avian CYP1A4s with mammalian CYP1A1s. Hepatic CYP1A4 and CYP1A5 mRNA levels in wild cormorants from Lake Biwa, Japan, were quantified to examine the effects of DRCs on isoform-specific expression and to evaluate the toxicokinetics of DRCs in which CYP1A expression is involved. Both CYP1A4 and CYP1A5 mRNA levels were positively correlated with total tetrachlorodibenzo-p-dioxin toxic equivalents and concentrations of each congener in most cases in the liver, suggesting the induction of both enzymes through a shared transcriptional mechanism. The lack of correlation of 2,3,7,8-tetrachlorodibenzofuran and 3,3',4,4'-tetrachlorobiphenyl (PCB77) to CYP1A gene expression is likely due to the rapid metabolism of these two congeners. Liver-to-muscle concentration ratios for most DRC congeners except PCB77 and mono-ortho coplanar polychlorinated biphenyls significantly increased with an elevation of CYP1A4 and CYP1A5 mRNA levels. The present data suggest that hepatic sequestration of some DRCs occurs in cormorant via binding to either CYP1A5 or both CYP1A4 and CYP1A5.
Key Words: CYP1A4; CYP1A5; gene conversion; dioxin and related compounds; metabolism; hepatic sequestration.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cytochromes P450 (CYP) comprise a large and growing superfamily of heme-thiolate enzymes that are critical for synthesis and degradation of physiologically important endogenous substrates and for biotransformation of a vast range of xenobiotics. Expression of members of the CYP1A subfamily is induced by dioxin and related compounds (DRCs), including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs). CYP1A expression has been used as a marker in evaluating exposure to mixtures of these chemicals. Since CYP1As can play important roles in either mediating or mitigating the biological effects of DRCs, the expression of CYP1As is a potential determinant of susceptibility to toxicity or disease (Gonzalez and Kimura, 2003
; Teraoka et al., 2003; Uno et al., 2004).
CYP1As play crucial roles in the toxicokinetics of DRCs in laboratory animals. Dose (or internal concentration)-dependent hepatic deposition was found for many 2,3,7,8-chlorine–substituted PCDD/DFs, PCB126, and PCB169 (Abraham et al., 1988; DeVito et al., 1998; Van den Berg et al., 1994), but not for mono-ortho Co-PCBs (DeVito et al., 1998). The hepatic sequestration of DRCs is attributable to involvement of CYP1A2. CYP1A2 has specific binding affinities for those congeners (Kuroki et al., 1986; Voorman and Aust, 1989), and transgenic Cyp1a2(–/–) knockout mice fail to show preferential sequestration of tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-P5CDF in the liver (Diliberto et al., 1999). Both in vitro and in vivo studies have shown that some DRC compounds, such as 2,3,7,8-T4CDF and 3,3',4,4'-tetrachlorobiphenyl (PCB77), are metabolized by rodent and avian hepatic microsomes, apparently by CYP1A enzymes (McKinley et al., 1993; Murk et al., 1994; Olson et al., 1994; Schlezinger et al., 2000). However, the roles of avian CYP1A genes in toxicokinetic behavior of DRCs, and particularly whether they might sequester DRCs, remain unclear.
In chicken (Gallus gallus), an avian model species, there are two TCDD-inducible, CYP1A-like enzymes (Nakai et al., 1992), which were named CYP1A4 and CYP1A5 following their cloning and sequencing (Gilday et al., 1996). Phylogenetic analysis of complete amino acid sequences has shown that chicken CYP1A4 and CYP1A5 cluster together, forming a clade separate from the mammalian CYP1A1s/1A2s (Gilday et al., 1996). This result led to an interpretation that chicken CYP1A4 and CYP1A5 arose via gene duplication after the divergence of avian and mammalian lineages. However, a more recent study (Goldstone and Stegeman, 2006) has indicated that chicken, mammalian, and to a lesser extent, amphibian and fish CYP1A genes lie within an extended genomic region of conserved fine-scale synteny and that chicken and mammalian CYP1As have undergone extensive interparalog gene conversion. These data strongly suggest that avian and mammalian CYP1A paralogs are the result of a single gene duplication, and thus, chicken CYP1A4 and CYP1A5 are orthologous to mammalian CYP1A1 and CYP1A2, respectively.
The distinct resemblance of catalytic specificities between CYP1A4 and CYP1A1, and between CYP1A5 and CYP1A2, likely reflect their common evolutionary origin. Chicken CYP1A4 was previously characterized as catalyzing ethoxyresorufin O-deethylation (EROD) (Rifkind et al., 1994), a reaction which is primarily catalyzed by CYP1A1 in mammals (Burke et al., 1994). The chicken CYP1A5 is mainly responsible for uroporphyrinogen oxidation (Sinclair et al., 1997), a reaction which is primarily catalyzed by mammalian CYP1A2 (Sinclair et al., 1998).
While the evolution and function of CYP1A genes has been investigated extensively in chicken, there are no such comprehensive data available for wild avian species that are useful for monitoring environmental contamination. Additional information on CYP1As in other avian species may improve understanding of the evolutionary relationships among CYP1A isozymes and their functional diversity. Furthermore, characterizing the roles of CYP1As in DRC toxicokinetics in wild avian species is important in terms of building a mechanistic understanding of DRC toxicity in wildlife.
Our previous studies (Kubota et al., 2004, 2005) revealed that common cormorants (Phalacrocorax carbo) from Lake Biwa in Japan accumulated high levels of DRCs (360–50,000 pg/g lipid wt as TCDD toxic equivalents; TEQs) in the liver and manifest the induction of CYP1A-like protein. The induced CYP1A-like protein appears to be responsible for hepatic metabolism and sequestration of particular DRCs. While it seemed likely that there are two CYP1A isoforms in cormorant as well as chicken, only a single CYP1A cross-reactive band was detected in immunoblot analyses. This may be due to the difficulty in resolving two CYP1A isoforms with similar molecular weights using standard SDS-PAGE. Alternatively, differences in the epitopes recognized by the antibody may have resulted in detection of only one isozyme. Thus, such an immunochemical approach may limit isoform-specific detection of multiple CYP1A isozymes.
In the present study, we determined the cDNA sequences of two cormorant CYP1A isoforms. Here we present phylogenetic and statistical sequence analyses with implications regarding the molecular and functional evolution of these avian CYP1As. Moreover, on the basis of correlation analyses between tissue concentrations of DRCs and CYP1A mRNA expression levels, we describe the effects of DRCs on isoform-specific CYP1A expression and suggest a functional role of avian CYP1A isozymes in metabolism and sequestration of DRCs.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sample collection.
Twenty-eight common cormorants were captured under the license from Shiga Prefecture during 2001–2003 from the southern part of Lake Biwa, Japan. The cormorants were immediately dissected on board after biometric measurement. Subsamples of liver were flash frozen in liquid nitrogen and stored at – 80°C until further analyses. The remaining liver and muscle samples were stored at – 20°C for chemical analyses. No adipose tissue was collected in these birds, and the percentages of lipid in liver and breast muscle are essentially the same.
Cloning of full-length cDNAs.
Total RNA from the liver of an adult male cormorant was isolated with ISOGEN (Nippon Gene, Toyama, Japan) and RNeasy (Qiagen, Tokyo, Japan). Poly(A)+ RNA was prepared by using Oligotex-dT30 Super (Roche Diagnostics, Tokyo, Japan). A full-length enriched pME18SFL3 cDNA library was constructed from poly(A)+ RNA using oligo-capping (Maruyama and Sugano, 1994). Randomly selected library clones (6930 in total) were subjected to 5'-end single-pass sequencing using a MegaBACE 1000 capillary sequencer (Amersham Biosciences, Piscataway, NJ). BLAST homology searches against NCBI nucleotide sequence databases were used to identify cormorant cDNAs with strong similarity to chicken CYP1A4 and CYP1A5. The 3'-end sequences of these candidate cormorant CYP1As were determined using ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The resulting full-length cDNA sequences were submitted to the P450 nomenclature committee (D. R. Nelson, University of Tennessee Health Science Center, TN).
Phylogenetic analysis.
Spatial variation in phylogenetic signal was detected with SlidingBayes (Paraskevis et al., 2005), using a window size of 100 bp and step size of 10 bp. For each window, 1.1 million generations of Metropolis-coupled Markov chain Monte Carlo (MC3) simulation was run using a general time-reversible model of nucleotide substitution with substitution rates modeled by a gamma distribution with four rate categories. Trees were sampled every 100 generations. Burn-in was defined at 100,000 generations; remaining trees were used to determine posterior probabilities of selected nodes. Phylogenetic trees were inferred using Mr.Bayes v.3.1; parameter settings were identical to those employed in SlidingBayes analyses. Convergence of all MC3 analyses was confirmed using AWTY (http://king2.csit.fsu.edu/CEBProjects/awty/awty_start.php).
The RDP2 software package (Martin et al., 2005) was used to run simultaneous analyses with five recombination detection algorithms—RDP, GeneConv, MaxChi, Bootscan, and Chimaera. RDP2 was run with default settings modified to use internal and external reference sequences. Default settings were used for GeneConv analysis. MaxChi and Chimaera default settings were modified to use variable window sizes with 10% variable sites and to perform 1000 permutations. Bootscan was run using a window size of 100 bp and a step size of 10 bp; 1000 bootstrap replicates were performed with cutoff percentage set to 95% and the bootstrap value used as p value. The p values from individual algorithms were subjected to Bonferroni correction to account for multiple testing, and all events with corrected p value < 0.01 were considered.
Quantification of mRNAs.
Total RNA was prepared from livers of individual specimens and treated with DNase to eliminate genomic DNA contamination. Quantitative real-time RT-PCR was performed using isoform-specific primers and TaqMan probes (CYP1A4: sense 5'-CACCATGCAGGTCTTTAAGGACA-3', antisense 5'-CCTGATGTGCTCCTTATCAAAGC-3', probe 5'-CAACAGGCGTTTCAGCTTCTTTGTGCAA-3'; CYP1A5: sense 5'-CAGCAGCTGGAATTCAGTGTCT-3', antisense 5'-CTGGAAGTGCTCACATCTTTGGT-3', probe 5'-AATGGCAAGAAGGTAGACATGACCCCACTC-3'), TaqMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems), and ABI PRISM 7700 System (Applied Biosystems) according to the manufacture's instructions. Template was prepared from total RNA of the liver from a specimen. Isoform-specific calibration curves were generated using template dilution series. We also measured 18S rRNA levels using TaqMan Ribosomal RNA Control Reagents (Applied Biosystems), and each sample was normalized by its 18S rRNA content. Expression levels of mRNAs in individual animals were expressed as a relative value calculated from the appropriate calibration curve. The real-time RT-PCR assays were conducted in triplicate for each sample.
DRC concentrations, EROD activity, and CYP1A-like protein level.
Data on the concentrations and TEQs of DRCs in 16 livers and 12 pectoral muscles of common cormorants employed in this study have been reported elsewhere (Kubota et al., 2004). TEQs were calculated using avian toxic equivalency factors reported by Van den Berg et al. (1998). EROD activity and expression level of a protein cross-reacting with an anti-rat CYP1A1 polyclonal antibody in the hepatic microsomes of cormorants collected in 2001 were measured previously (Kubota et al., 2005). In order to verify the accuracy of our observations regarding the birds collected in 2001, we conducted measurements of DRC concentrations, EROD activity, and CYP1A-like protein levels in specimens (n = 12) collected from the same location as in 2001, according to the same methods used previously (Kubota et al., 2004, 2005).
Statistical analyses.
All the statistical analyses were performed using StatView v. 5.0 (SAS Institute, Cary, NC). Spearman's rank correlation test was performed to evaluate the relationships among CYP1A mRNA levels, CYP1A-like protein levels, and EROD activity. Spearman's rank correlation test was also used for the relationships between TEQs and CYP1A mRNA levels, CYP1A protein levels, or EROD activities. For samples with values below quantification limit, half the respective limit was substituted. Relationships between liver-to-muscle concentration ratios (L/M ratios) and CYP1A mRNA levels were examined by simple regression analysis when individual congeners were detected both in the liver and pectoral muscle. When more than 50% of the observations were below the quantification limit, statistical analyses were not conducted for the congener, and those results were shown as "no data available (NA)." A p value of < 0.05 was regarded as statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cloning of Full-Length Cormorant CYP1A4 and CYP1A5 cDNAs
We identified two distinct full-length CYP1A cDNA clones by screening a cDNA library derived from liver of the common cormorant. One of the sequences consisted of a 1581-bp open reading frame that encodes 526 amino acid residues with a predicted molecular mass of 59.5 kDa (Fig. 1a). A second clone consisted of 1593 bp coding for 530 amino acids with a predicted molecular mass of 60.2 kDa (Fig. 1b). The two CYP1As share 85% identity at the level of cDNA coding sequences and 82% identity between deduced amino acid sequences. Alignment of the cormorant CYP1A amino acid sequences with other vertebrate CYP1As revealed that the cormorant sequences share the greatest degree of amino acid identity with chicken CYP1A4 (78%) and CYP1A5 (78%) (Table 1). In addition, several CYP1A functional motifs (i.e., a putative heme-binding motif, conserved threonine residue, proline-rich region, etc.) were well conserved in the deduced amino acid sequences of cormorant CYP1A enzymes (Fig. 2). Thus, the cormorant sequences were named CYP1A4 (Fig. 1a) and CYP1A5 (Fig. 1b) by D. R. Nelson (University of Tennessee, TN).
|
|
|
-helix I which contains threonine residue, and (C) heme-binding motif are boxed. Accession numbers for sequences used are as follows: chicken CYP1A4 (Phylogenetic Analysis of Cormorant CYP1A Genes
Bayesian phylogenetic analysis of full-length coding nucleotide sequences recovered monophyletic avian and mammalian sister clades (Fig. 3a). However, the avian genes were segregated according to species, whereas mammalian orthologs formed monophyletic clusters. Given previously published evidence of gene conversion between CYP1A paralogs in chicken, mouse, and human (Goldstone and Stegeman, 2006
), it seemed plausible that the phylogenetic positions of the cormorant CYP1As could also be artifacts of gene conversion. SlidingBayes (Paraskevis et al., 2005) was used to determine if sections of cormorant CYP1A coding sequences generate mutually exclusive clustering patterns indicative of recombination. We examined a posteriori support for two types of clusters—paralog pairs clustered together by species or clusters consisting of putative orthologs (e.g., avian CYP1A4s or avian CYP1A4s + mammalian CYP1A1s).
|
Variation in posterior probability support for cormorant and chicken paralog clusters largely tracked with one another (Fig. 4, top). With the exception of the region spanning 300–370 bp, the first 720 bp of the alignment showed strong support for both chicken and cormorant CYP1A4 + CYP1A5 clusters. The decrease in paralog clustering support at 300–370 bp coincided with increased support for avian CYP1A4 and CYP1A5 clusters. Similarly, the region from 1140 bp to the end of the alignment showed low paralog clustering support and significant support for avian CYP1A4 and CYP1A5 clusters. RDP2 analyses generally supported these SlidingBayes results, detecting multiple gene conversion tracts in the first 720 bp of avian CYP1As as well as one conversion tract per species in the region 970–1140 bp (Table 2).
|
|
Similar to previously published analyses, significant phylogenetic support for avian CYP1As clustering with their putative mammalian orthologs (i.e., avian CYP1A4s + mammalian CYP1A1s and avian CYP1A5s + mammalian CYP1A2s) was found in the region spanning 720–970 bp (Fig. 4, bottom). Bayesian phylogenetic analysis of just this region recovered a tree topology absolutely supporting a monophyletic tetrapod CYP1A clade and strongly supporting orthology of avian CYP1A5s and mammalian CYP1A2s (Fig. 3b). Monophyly of avian CYP1A4s and mammalian CYP1A1s was not well supported. Rather, this region indicated positioning of avian CYP1A4s intermediate between mammalian CYP1A1s and the CYP1A2/1A5 clade. This may be explained, in part, by strong positive selection forcing differentiation of CYP1A2/5s from CYP1A1/4s and of mammalian CYP1A1s from avian CYP1A4s (Goldstone and Stegeman, 2006
). However, it should be noted that the relatively small amount of unconverted sequence (only 220 bp after masking) limits the power of phylogenetic analyses to resolve relationships between 10 taxa. Overall, this topology is in distinct contrast to that recovered from the full-length alignment (Fig. 3a) and suggests that the results of full-length analyses are distorted by extensive gene conversion.
DRC Concentrations and Distribution and EROD Activity
Concentrations and TEQs of DRC congeners and EROD activities in 2002 and 2003 cormorant specimens from Lake Biwa (Table 3) were highly variable, but similar to those in the 2001 specimens (Kubota et al., 2004, 2005). In liver of common cormorants, PCDD/DFs and Co-PCBs were detected at concentrations of 640–19,000 pg/g lipid wt and 100,000–19,000,000 pg/g lipid wt, respectively. The pectoral muscle contained 260–2800 pg/g of PCDD/DFs and 210,000–28,000,000 pg/g of Co-PCBs. Sums of TEQs of PCDDs, PCDFs, and Co-PCBs ranged from 360 to 23,000 pg/g lipid wt in the liver and from 310 to 7100 pg/g lipid wt in the pectoral muscle. L/M ratios for most PCDD/DFs were > 1.0 in all specimens. O8CDD had the greatest L/M ratios, reaching a maximum value of 30. In contrast, L/M ratios for Co-PCBs were generally < 1.0, with the exceptions of PCB126 and PCB169. EROD activities were 36–620 pmol/min/mg protein.
|
Relationships between Tissue Concentrations of DRCs and CYP1A Expression Levels
Quantitative real-time RT-PCR data for cormorant CYP1A4 and CYP1A5 were used to assess the isoform-specific relationships of mRNA expression with DRC concentrations in wild populations. Spearman's rank correlation test showed that total TEQs were positively correlated with mRNA levels of both CYP1A4 (r = 0.66, p < 0.001) and CYP1A5 (r = 0.74, p < 0.001) (Fig. 5). Relationships between TEQs of individual congeners and CYP1A mRNA levels were also analyzed by Spearman's rank correlation test. Congener-specific analyses revealed significant positive correlations between TEQs for most individual congeners and mRNA levels of both isoforms (0.47 < r < 0.77) (Table 4). In contrast, neither 2,3,7,8-T4CDF nor PCB77 showed any significant correlation with mRNA levels of either CYP1A isoform (Table 4). Similar correlation profiles were obtained from the analyses of hepatic TEQs and CYP1A-like protein levels or EROD activity (Table 4).
|
|
Spearman's rank correlation test was used to evaluate the relationship between CYP1A mRNA levels, protein levels, and enzymatic activity in cormorant liver. The mRNA levels of CYP1A4 and CYP1A5 were positively correlated (r = 0.94, p < 0.0001) with each other. Spearman's rank correlation test also demonstrated that both CYP1A-like protein level and EROD activity were positively correlated with mRNA levels of both CYP1A4 (CYP1A-like protein: r = 0.67, p < 0.001 [Fig. 6a], EROD: r = 0.56, p < 0.01) and CYP1A5 (CYP1A-like protein: r = 0.75, p < 0.0001 [Fig. 6b], EROD: r = 0.67, p < 0.001). Thus, CYP1A mRNA levels can be used as a proxy for CYP1A protein and enzymatic activity levels as well as an indicator of transcriptional activation.
|
We postulated that the lack of significant correlation between levels of 2,3,7,8-T4CDF or PCB77 and CYP1A expression is due to more rapid metabolism of these compounds by CYP1A enzymes. CYP1A metabolism of these congeners has also been suggested by residue analyses in wild crow (Watanabe et al., 2005
) and Baikal seal (Iwata et al., 2003) populations. To provide more support for this hypothesis, the ratio of 2,3,7,8-T4CDF or PCB77 to PCB169, which appears to be recalcitrant to metabolism in this species (Guruge and Tanabe, 1997), was compared with CYP1A mRNA levels. Spearman's rank correlation analyses revealed that mRNA levels of both CYP1A4 and CYP1A5 were negatively correlated with the concentration ratios of both compounds (Fig. 7), suggesting metabolism of both 2,3,7,8-T4CDF and PCB77 by cormorant CYP1As.
|
CYP1A-related toxicokinetic behavior of DRCs was investigated further by regression analysis between CYP1A mRNA levels and L/M ratios. The results showed that L/M ratios for many PCDD/DF and non-ortho Co-PCB congeners were positively correlated with both CYP1A4 and CYP1A5 mRNA levels (Table 5). This includes all 2,3,7,8-substituted PCDDs; 2,3,7,8-T4CDF; 2,3,4,7,8-P5CDF; 1,2,3,4,7,8-H6CDF; 1,2,3,6,7,8-H6CDF; 1,2,3,4,6,7,8-H7CDF; PCB81; PCB126; and PCB169. This relationship suggests that CYP1As may be involved in sequestration of these congeners in the liver. In contrast, among the mono-ortho Co-PCBs, the only significant linear relationship was between the L/M ratio for PCB114 and CYP1A5 mRNA level.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Characterization of Cormorant CYP1A4 and CYP1A5
We have cloned and characterized full-length CYP1A4 and CYP1A5 cDNAs from common cormorant. As the expected molecular masses of the two enzymes differed by less than 1 kDa, it seems plausible that detection of only a single cross-reactive band in previous Western blot analysis (Kubota et al., 2005
) was due to lack of resolution in SDS-PAGE. High levels of pairwise amino acid identity and phylogenetic analyses indicate that these genes are orthologous to chicken CYP1A4 and CYP1A5. Furthermore, SlidingBayes and RDP2 results demonstrate that cormorant CYP1A genes, like their chicken and mammalian counterparts, have undergone interparalog gene conversion. Hence, greater similarity between avian CYP1A paralogs (e.g., cormorant CYP1A4 and CYP1A5) may not be the result of convergent evolution, but that of concerted evolution via interparalog gene conversion. Gene conversion homogenizes sequence variation between paralogs (Goldstone and Stegeman, 2006; Teshima and Innan, 2004) and, in essence, creates chimeric gene structures (Fig. 8). The two sequence regions where there was strong support for avian, but not pan-tetrapod, ortholog clustering (i.e., 300–370 bp and from 1140 bp to the end of the alignment) may be attributable to avian-specific gene conversion activities predating the divergence of the chicken and cormorant lineages. The remaining gene conversion events appear to be species specific and indicate a gene conversion rate of 
8 x 10–8 in avian lineages.
|
Phylogenetic analysis of a putatively unconverted region (720–970 bp) recovered the orthology of avian CYP1A5s and mammalian CYP1A2s. The positioning of avian CYP1A4s is intermediate between mammalian CYP1A1s and the CYP1A2/1A5 clade. The failure to recover a monophyletic CYP1A1/1A4 clade may be an artifact of strong positive selection forcing rapid evolution of mammalian CYP1A1s (Goldstone and Stegeman, 2006
). Overall, the current data are consistent with and support previously published analyses, suggesting the orthology of avian CYP1A4 and mammalian CYP1A1 and of avian CYP1A5 and mammalian CYP1A2 (Goldstone and Stegeman, 2006).
The inclusion of cormorant CYP1A sequences in recombination analyses improved the resolution of chicken gene conversion tracts. The algorithms implemented by RDP2 require closely related orthologous gene sequences for comparisons. In the absence of the cormorant sequences, both SlidingBayes and RDP2 slightly overestimated the extent of gene conversion between chicken CYP1A4 and CYP1A5. Gene conversion boundaries shared by chicken and cormorant may indicate recombination hotspots in the avian lineage. Nevertheless, the frequency of gene conversion in cormorant and chicken is higher than that in mammals, which supports the idea that the intergenic distance between CYP1A paralogs may affect the frequency of gene conversion. Total intergenic distance could be as great as 3 kb in chicken CYP1A4 and CYP1A5, whereas CYP1A1 and CYP1A2 are separated by 13–17 kb in rodents and 21–24 kb in primates, resulting in the lesser frequency of gene conversion in mammalian species.
CYP1A Expression and Tissue DRC Levels
Our recent studies (Kubota et al., 2005; Watanabe et al., 2005) have demonstrated that both EROD activity and expression of a hepatic protein that cross-reacts with an anti-rat CYP1A1 polyclonal antibody increase in relation to DRC accumulation in cormorant and crow. These results suggest the induction of CYP1A-like protein by the current environmental DRC levels. The sequence data on cormorant CYP1A4 and CYP1A5 obtained in the present study enabled further examination of isoform-specific relationships to DRC levels. Significant relationships between total TEQs and CYP1A4 or CYP1A5 mRNA expression levels indicate the induction of both enzymes by DRCs in cormorant liver. Considering that expression levels of CYP1A4 mRNA were strongly correlated with those of CYP1A5, these CYP1A enzymes may be coordinately induced by DRCs, ostensibly through activation of the aryl hydrocarbon receptor (AHR). In chick embryos and liver, CYP1A4 and CYP1A5 were transcriptionally coinduced by the treatment with TCDD (Gilday et al., 1996; Mahajan and Rifkind, 1999). The palindromic arrangement of chicken CYP1A paralogs may result in sharing of the transcriptional mechanism, which leads to the coordinate induction of both CYP1A enzymes by DRCs. Our recent study (Yasui et al., 2004) isolated two distinct AHR isoforms in common cormorant, but the regulatory mechanisms of the two CYP1A genes by these AHRs remain to be determined. In any case, the positive relationship between CYP1A4 and CYP1A5 mRNA levels and DRC levels implies effects of chronic exposure to DRCs in wild cormorants. To our knowledge, this is the first study indicating the induction of CYP1A4 and CYP1A5 mRNAs by chronic exposure to DRCs in a wild avian population. Previous studies have reported a significant concentration-dependent induction of proteins homologous to mammalian or fish CYP1A and CYP1A catalytic activity in various wild avian species, including great blue herons (Bellward et al., 1990), black-crowned night herons (Rattner et al., 1994), double-crested cormorants (Sanderson et al., 1994), common terns (Bosveld et al., 1995), bald eagles (Elliott et al., 1996), and ospreys (Elliott et al., 2001). However, CYP isozymes were not determined in those studies.
Despite the significant positive correlations between CYP1A mRNA expression levels and TEQs for most of the individual AHR agonists among the PCDDs, PCDFs, or PCBs, no significant relationship was observed for 2,3,7,8-T4CDF and PCB77. We suggest that this may be due to preferential metabolism of these congeners, as the concentration ratios of 2,3,7,8-T4CDF and PCB77 to a stable congener, PCB169, was inversely related to CYP1A4 and CYP1A5 mRNA levels in the liver (Fig. 7). The relative importance of the two CYP1A isozymes in metabolizing these lower chlorinated congeners could not be inferred from the correlation analyses. Without assigning CYP1A isoform specificity, several studies have shown that the metabolism of these two congeners is increased when CYP1A is induced. In vitro studies have shown that hepatic CYP1A and microsomal metabolism of PCB77 is induced in several bird species, including the double-crested cormorant (Phalacrocorax auritus), a congeneric species to the common cormorant studied here (Murk et al., 1994; Schlezinger et al., 2000). Studies have also shown that TCDD pretreatment in rats, which increases hepatic CYP1A1 and 1A2, increases metabolism of 2,3,7,8-T4CDF (McKinley et al., 1993; Olson et al., 1994; Tai et al., 1993).
Previously published data demonstrated that most of the PCDD/DFs were better retained in cormorant liver than in the pectoral muscle, as inferred from the L/M ratios of individual compounds (Kubota et al., 2004). The L/M ratios for the sum of the PCDD/DF and Co-PCB concentration in cormorants (Table 3) are similar to the structure-activity relationship for mammalian CYP1A2 binding. For most DRC congeners, the liver deposition increased in a TEQ-dependent manner. Our previous study unraveled the possible involvement of CYP1A-like protein in the concentration-dependent hepatic sequestration not only in cormorants (Kubota et al., 2005) but also in crows (Watanabe et al., 2005) and kites (Kubota et al., 2006). To further evaluate the isoform specificity of sequestration, the present study examined relationships between CYP1A mRNA levels and the L/M ratios. L/M ratios for most PCDD/DFs increase with CYP1A4 and CYP1A5 mRNA levels (Table 5). The slopes progressively increase with degree of chlorination for PCDDs, suggesting congener-specific hepatic sequestration by CYP1A induction. However, it is difficult to determine which CYP isozyme is more preferentially involved in the sequestration of these congeners. L/M ratios for Co-PCBs were generally less than or nearly equal to 1.0 (Table 3). Among Co-PCB congeners, only two non-ortho congeners, PCB126 and PCB169, showed higher ratios (up to 2.4 for PCB126 and 3.9 for PCB169) in some specimens, suggesting that these compounds are sequestered in a CYP1A-dependent manner, albeit to a lesser extent than PCDD/DFs.
Considering the results of correlation analyses between CYP1A levels and tissue DRC concentrations, 2,3,7,8-T4CDF may be subject to not only the metabolism but also the sequestration in the liver of cormorants (Table 5, Fig. 7). When the toxicokinetics of DRCs is controlled mainly by a balance of metabolism and sequestration by CYP1A, 2,3,7,8-T4CDF metabolism by CYP1A would be efficient but relatively weak, resulting in the hepatic deposition of this congener.
The orthology of CYP1A5s and CYP1A2s, as suggested by our phylogenetic analyses, lends weight to the idea that particular DRC congeners in cormorant livers might be sequestered by CYP1A5, as mammalian CYP1A2 is exclusively associated with hepatic sequestration of DRCs (Diliberto et al., 1999; Uno et al., 2004). If the capacity for hepatic sequestration is conferred by sequences within the unconverted region (e.g., substrate recognition sites 3 or 4), this function could be conserved between avian CYP1A5 and mammalian CYP1A2. However, the involvement of CYP1A4 in DRCs sequestration in the liver cannot be ruled out, as L/M ratios of DRCs were also positively correlated with CYP1A4 mRNA. Involvement of both cormorant CYP1A isoforms in sequestration would suggest that this function is determined by sequence features that have been subject to gene conversion only in avian lineages (e.g., 550–720 bp or 975–1100 bp).
For most PCDD/DF congeners, the R2 values for the relationships of CYP1A mRNA levels with L/M ratios are pretty weak, even if it is statistically significant (Table 5). The fact that CYP1A levels only account for 20–40% of the sequestration effect suggests additional sequestration mechanisms. However, it is to be noted that the relationships between CYP1A protein levels and L/M ratios seem to have greater R2 values (0.47 < R2 < 0.63) (data not shown) than those between CYP1A mRNA levels and L/M ratios.
In summary, common cormorant possesses two paralogous CYP1A genes that are orthologous to chicken CYP1A4 and CYP1A5 and likely resulted from a single gene duplication event in the tetrapod lineage. Concerted evolution via extensive interparalog gene duplication has resulted in a high level of sequence identity between the cormorant CYP1A paralogs, and consequently, true ortholog/paralog relationships may be obscured. Indeed, the orthology of CYP1A5s and CYP1A2s is recovered by phylogenetic analysis of the region where no statistical support for gene conversion was obtained. Cormorant hepatic CYP1A4 and CYP1A5 appear to be coinduced by DRCs possibly through a shared transcriptional mechanism. Moreover, CYP1A induction is correlated with greater ratios of liver-to-muscle PCDD/DF levels, and to a lesser extent, PCB126 and PCB169 levels. We suggest that hepatic sequestration of these congeners in cormorant occurs via binding to one or both CYP1As. Further studies with cormorant CYP1A4 and CYP1A5 proteins would be necessary to characterize the functional similarities or differences between two CYP1As, in terms of metabolism and sequestration of individual DRC congeners.
|
ACKNOWLEDGMENTS |
|---|
The authors thank Ms K. Yoneda, senior scientist of Japan Wildlife Research Center, for collection of common cormorants used in this study. 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 (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), by "21st Century COE Program" from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by NIH grant 2-P42-ES07381 to J.J.S. The award of the Doctoral Fellowship for Researchers from the Japan Society for the Promotion of Science to A.K. (no. 00407) is acknowledged. The nucleotide sequences of common cormorant CYP1A4 and CYP1A5 have been deposited in the DDBJ/EMBL/GenBank database under accession number AB239444 and AB239445, respectively.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Abraham, K., Krowke, R., and Neubert, D. (1988). Pharmacokinetics and biological activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Dose-dependent tissue distribution and induction of hepatic ethoxyresorufin O-deethylase in rats following a single injection. Arch. Toxicol. 62, 359–368.[CrossRef][ISI][Medline]
Bellward, G. D., Norstrom, R. J., Whitehead, P. E., Elliott, J. E., Bandiera, S. M., Dworschak, C., Chang, T., Forbes, S., Cadario, B., Hart, L. E., et al. (1990). Comparison of polychlorinated dibenzodioxin levels with hepatic mixed-function oxidase induction in great blue herons. J. Toxicol. Environ. Health 30, 33–52.[Medline]
Bosveld, A. T. C., Gradener, J., Murk, A. J., Brouwer, A., van Kampen, M., Evers, E. H. G., and Van den Berg, M. (1995). Effects of PCDDs, PCDFs and PCBs in common tern (Sterna hirundo) breeding in estuarine and coastal colonies in The Netherlands and Belgium. Environ. Toxicol. Chem. 14, 99–115.
Burke, M. D., Thompson, S., Weaver, R. J., Wolf, C. R., and Mayer, R. T. (1994). Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem. Pharmacol. 48, 923–936.[CrossRef][ISI][Medline]
DeVito, M. J., Ross, D. G., Dupuy, A. E., Jr, Ferrario, J., McDaniel, D., and Birnbaum, L. S. (1998). Dose-response relationships for disposition and hepatic sequestration of polyhalogenated dibenzo-p-dioxins, dibenzofurans, and biphenyls following subchronic treatment in mice. Toxicol. Sci. 46, 223–234.
Diliberto, J. J., Burgin, D. E., and Birnbaum, L. S. (1999). Effects of CYP1A2 on disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 2,2',4,4',5,5'-hexachlorobiphenyl in CYP1A2 knockout and parental (C57BL/6N and 129/Sv) strains of mice. Toxicol. Appl. Pharmacol. 159, 52–64.[CrossRef][ISI][Medline]
Elliott, J. E., Norstrom, R. J., Lorenzen, A., Hart, L. E., Philibert, H., Kennedy, S. W., Stegeman, J. J., Bellward, G. D., and Cheng, K. M. (1996). Biological effects of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls in bald eagle (Haliaeetus leucocephalus) chicks. Environ. Toxicol. Chem. 15, 782–793.[CrossRef]
Elliott, J. E., Wilson, L. K., Henny, C. J., Trudeau, S. F., Leighton, F. A., Kennedy, S. W., and Cheng, K. M. (2001). Assessment of biological effects of chlorinated hydrocarbons in osprey chicks. Environ. Toxicol. Chem. 20, 866–879.[CrossRef][ISI][Medline]
Gilday, D., Gannon, M., Yutzey, K., Bader, D., and Rifkind, A. B. (1996). Molecular cloning and expression of two novel avian cytochrome P450 1A enzymes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem. 271, 33054–33059.
Goldstone, H. M. H., and Stegeman, J. J. (2006). A revised evolutionary history of the CYP1A subfamily: Gene duplication, gene conversion, and positive selection. J. Mol. Evol. (Apr 28; Epub ahead of print).
Gonzalez, F. J., and Kimura, S. (2003). Study of P450 function using gene knockout and transgenic mice. Arch. Biochem. Biophys. 409, 153–158.[CrossRef][ISI][Medline]
Guruge, K. S., and Tanabe, S. (1997). Congener specific accumulation and toxic assessment of polychlorinated biphenyls in common cormorants, Phalacrocorax carbo, from Lake Biwa, Japan. Environ. Pollut. 96, 425–433.[CrossRef][Medline]
Iwata, H., Kim, E. Y., Sakamoto, T., Ebisuda, K., Okajima, Y., Watanabe, M., Tanabe, S., Amano, M., and Miyazaki, N. (2003). Implications of AHR- and CYP1A/1B-mediated effects by PCDDs/DFs and coplanar PCBs in Baikal seal. Organohalogen Compd. 62, 216–219.
Kubota, A., Iwata, H., Tanabe, S., Yoneda, K., and Tobata, S. (2004). Levels and toxicokinetic behaviors of PCDD, PCDF, and coplanar PCB congeners in common cormorants from Lake Biwa, Japan. Environ. Sci. Technol. 38, 3853–3859.[Medline]
Kubota, A., Iwata, H., Tanabe, S., Yoneda, K., and Tobata, S. (2005). Hepatic CYP1A induction by dioxin-like compounds, and congener-specific metabolism and sequestration in wild common cormorants from Lake Biwa, Japan. Environ. Sci. Technol. 39, 3611–3619.[Medline]
Kubota, A., Iwata, H., Tanabe, S., Yoneda, K., and Tobata, S. (2006). Congener-specific toxicokinetics of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls in black-eared kites (Milvus migrans): Cytochrome P4501A-dependent hepatic sequestration. Environ. Toxicol. Chem. 25, 1007–1016.[Medline]
Kuroki, J., Koga, N., and Yoshimura, H. (1986). High affinity of 2,3,4,7,8-pentachlorodibenzofuran to cytochrome P-450 in the hepatic microsomes of rats. Chemosphere 15, 731–738.[CrossRef]
Mahajan, S. S., and Rifkind, A. B. (1999). Transcriptional activation of avian CYP1A4 and CYP1A5 by 2,3,7,8-tetrachlorodibenzo-p-dioxin: Differences in gene expression and regulation compared to mammalian CYP1A1 and CYP1A2. Toxicol. Appl. Pharmacol. 155, 96–106.[Medline]
Martin, D. P., Williamson, C., and Posada, D. (2005). RDP2: recombination detection and analysis from sequence alignments. Bioinformatics 21, 260–262.
Maruyama, K., and Sugano, S. (1994). Oligo-capping: A simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171–174.[CrossRef][ISI][Medline]
McKinley, M. K., Kedderis, L. B., and Birnbaum, L. S. (1993). The effect of pretreatment on the biliary excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 3,3',4,4'-tetrachlorobiphenyl in the rat. Fundam. Appl. Toxicol. 21, 425–432.[CrossRef][ISI][Medline]
Murk, A., Morse, D., Boon, J., and Brouwer, A. (1994). In vitro metabolism of 3,3',4,4'-tetrachlorobiphenyl in relation to ethoxyresorufin-O-deethylase activity in liver microsomes of some wildlife species and rat. Eur. J. Pharmacol. 270, 253–261.[ISI][Medline]
Nakai, K., Ward, A. M., Gannon, M., and Rifkind, A. B. (1992). Beta-naphthoflavone induction of a cytochrome P-450 arachidonic acid epoxygenase in chick embryo liver distinct from the aryl hydrocarbon hydroxylase and from phenobarbital-induced arachidonate epoxygenase. J. Biol. Chem. 267, 19503–19512.
Olson, J. R., McGarrigle, B. P., Gigliotti, P. J., Kumar, S., and McReynolds, J. H. (1994). Hepatic uptake and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran. Fundam. Appl. Toxicol. 22, 631–640.[CrossRef][ISI][Medline]
Paraskevis, D., Deforche, K., Lemey, P., Magiorkinis, G., Hatzakis, A., and Vandamme, A. M. (2005). SlidingBayes: exploring recombination using a sliding window approach based on Bayesian phylogenetic inference. Bioinformatics 21, 1274–1275.
Rattner, B. A., Hatfield, J. S., Melancon, M. J., Custer, T. W., and Tillitt, D. E. (1994). Relation among cytochrome P450, Ah-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron. Environ. Toxicol. Chem. 13, 1805–1812.
Rifkind, A. B., Kanetoshi, A., Orlinick, J., Capdevila, J. H., and Lee, C. (1994). Purification and biochemical characterization of two major cytochrome P-450 isoforms induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in chick embryo liver. J. Biol. Chem. 269, 3387–3396.
Sanderson, J. T., Norstrom, R. J., Elliott, J. E., Hart, L. E., Cheng, K. M., and Bellward, G. D. (1994). Biological effects of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls in double-crested cormorant chicks (Phalacrocorax auritus). J. Toxicol. Environ. Health 41, 247–265.[Medline]
Schlezinger, J. J., Keller, J., Verbrugge, L. A., and Stegeman, J. J. (2000). 3,3',4,4'-Tetrachlorobiphenyl oxidation in fish, bird and reptile species: Relationship to cytochrome P450 1A inactivation and reactive oxygen production. Comp. Biochem. Physiol. C 125, 273–286.
Sinclair, P. R., Gorman, N., Tsyrlov, I. B., Fuhr, U., Walton, H. S., and Sinclair, J. F. (1998). Uroporphyrinogen oxidation catalyzed by human cytochromes P450. Drug Metab. Dispos. 26, 1019–1025.
Sinclair, P. R., Gorman, N., Walton, H. S., Sinclair, J. F., Lee, C. A., and Rifkind, A. B. (1997). Identification of CYP1A5 as the CYP1A enzyme mainly responsible for uroporphyrinogen oxidation induced by AH receptor ligands in chicken liver and kidney. Drug Metab. Dispos. 25, 779–783.
Tai, H. L., McReynolds, J. H., Goldstein, J. A., Eugster, H. P., Sengstag, C., Alworth, W. L., and Olson, J. R. (1993). Cytochrome P4501A1 mediates the metabolism of 2,3,7,8-tetrachlorodibenzofuran in the rat and human. Toxicol. Appl. Pharmacol. 123, 34–42.[CrossRef][ISI][Medline]
Teraoka, H., Dong, W., Tsujimoto, Y., Iwasa, H., Endoh, D., Ueno, N., Stegeman, J. J., Peterson, R. E., and Hiraga, T. (2003). Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish. Biochem. Biophys. Res. Commun. 304, 223–228.[CrossRef][ISI][Medline]
Teshima, K. M., and Innan, H. (2004). The effect of gene conversion on the divergence between duplicated genes. Genetics 166, 1553–1560.
Uno, S., Dalton, T. P., Sinclair, P. R., Gorman, N., Wang, B., Smith, A. G., Miller, M. L., Shertzer, H. G., and Nebert, D. W. (2004). Cyp1a1(–/–) male mice: Protection against high-dose TCDD-induced lethality and wasting syndrome, and resistance to intrahepatocyte lipid accumulation and uroporphyria. Toxicol. Appl. Pharmacol. 196, 410–421.[CrossRef][ISI][Medline]
Van den Berg, M., Birnbaum, L., Bosveld, A. T. C., Brunström, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., et al. (1998). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health Perspect. 106, 775–792.[ISI][Medline]
Van den Berg, M., De Jongh, J., Poiger, H., and Olson, J. R. (1994). The toxicokinetics and metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and their relevance for toxicity. Crit. Rev. Toxicol. 24, 1–74.[ISI][Medline]
Voorman, R., and Aust, S. D. (1989). TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a tight binding inhibitor of cytochrome P-450d. J. Biochem. Toxicol. 4, 105–109.[ISI][Medline]
Watanabe, M. X., Iwata, H., Okamoto, M., Kim, E. Y., Yoneda, K., Hashimoto, T., and Tanabe, S. (2005). Induction of cytochrome P450 1A5 mRNA, protein and enzymatic activities by dioxin-like compounds, and congener-specific metabolism and sequestration in the liver of wild jungle crow (Corvus macrorhynchos) from Tokyo, Japan. Toxicol. Sci. 88, 384–399.
Yasui, T., Kim, E. Y., Iwata, H., and Tanabe, S. (2004). Identification of aryl hydrocarbon receptor 2 in aquatic birds; cDNA cloning of AHR1 and AHR2 and characteristics of their amino acid sequences. Mar. Environ. Res. 58, 113–118.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Yasui, E.-Y. Kim, H. Iwata, D. G. Franks, S. I. Karchner, M. E. Hahn, and S. Tanabe Functional Characterization and Evolutionary History of Two Aryl Hydrocarbon Receptor Isoforms (AhR1 and AhR2) from Avian Species Toxicol. Sci., September 1, 2007; 99(1): 101 - 117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hirakawa, H. Iwata, Y. Takeshita, E.-Y. Kim, T. Sakamoto, Y. Okajima, M. Amano, N. Miyazaki, E. A. Petrov, and S. Tanabe 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) Toxicol. Sci., June 1, 2007; 97(2): 318 - 335. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||