Toxicology and Applied Pharmacology 234 (2009) 306 – 313
Contents lists available at ScienceDirect
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y ta a p
a , c a , b , c
d c , d
a
,
b , c , g , h d , e
a
,
c , f , ⁎
b
,
c
f a
Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada b
Department of Zoology, Michigan State University, East Lansing, MI. USA c
National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI. USA d e
Department Animal Science, Michigan State University, East Lansing, MI, USA
ENTRIX, Inc., Okemos, MI, USA
ENTRIX, Inc., Saskatoon, SK, Canada g
Department Biomedical Veterinary Sciences, University of Saskatchewan, Saskatoon, SK, Canada h
Department Biology and Chemistry, City University of Hong Kong, Hong Kong, SAR, China a r t i c l e i n f o
Article history:
Received 2 May 2008
Revised 20 October 2008
Accepted 20 October 2008
Available online 11 November 2008
Keywords:
2,3,4,7,8-PeCDF, 2,3,7,8-TCDF
Response
Correlation
Protein
AhR
Sensitivity a b s t r a c t
As part of an ongoing effort to understand aryl hydrocarbon receptor (AhR) mediated toxicity in mink, cDNAs encoding for CYP1A1 and the CYP1A2 mixed function monooxygenases were cloned and characterized. In addition, the effects of selected dibenzofurans on the expression of these genes and the presence of their respective proteins (P4501A) were investigated, and then correlated with the catalytic activities of these proteins as measured by ethoxyresoru fi n O -deethylase (EROD) and methoxyresoru fi n O -deethylase (MROD) activities. The predicted protein sequences for CYP1A1 and CYP1A2 comprise 517 and 512 amino acid residues, respectively. The phylogenetic analysis of the mink CYP1As with protein sequences of other mammals revealed high sequence homology with sea otter, seals and the dog, with amino acid identities ranging from 89 to 95% for CYP1A1 and 81 to 93% for CYP1A2. Since exposure to both 2,3,7,8-Tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) resulted in dose-dependent increases of CYP1A1 mRNA, CYP1A2 mRNA and CYP1A protein levels an underlying AhR-mediated mechanism is suggested. The up-regulation of CYP1A mRNA in liver was more consistent to the sum adipose TEQ concentration than to the liver TEQ concentration in minks treated with TCDF or PeCDF. The result suggested that the hepatic-sequestered fraction of PeCDF was biologically inactive to the induction of CYP1A1 and
CYP1A2.
© 2008 Elsevier Inc. All rights reserved.
Introduction
Mink ( Mustela vison ) have been suggested as a sentinel or model organism for the risk assessment of exposure and potential effects of environmentally persistent organic chemicals such as polychlorinated-dibenzop -dioxins (PCDDs), -dibenzofurans (PCDFs) and
-biphenyls (PCB) ( Giesy et al., 1994 ). Speci
fi cally, mink have been suggested to be useful in assessing exposure to these compounds both on temporal and spatial scales because of their wide distribution, their abundance in temperate aquatic ecosystems, and their high trophiclevel (
Basu et al., 2007; Heaton et al., 1995; Millsap et al., 2004 ). Mink
have also been shown to accumulate appreciable concentrations of chemicals that cause toxicity mediated through the AhR, such as
PCDDs, PCDFs and PCBs ( Brunstrom et al., 2001; Engelhart et al., 2001;
⁎ Corresponding author. ENTRIX, Inc., Toxicology Centre, University of Saskatchewan,
44 Campus Drive, Saskatoon, SK S7N 5B3, Canada. Fax: +1 306 966 4796.
E-mail address: mhecker@entrix.com
(M. Hecker).
0041-008X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi: 10.1016/j.taap.2008.10.017
Millsap et al., 2004 ). The mink has been used widely in ecological risk
assessments and it is often assumed that use of the toxicity reference values (TRVs) derived for mink will be protective of other species that could be exposed to AhR-active compounds. However, little is known about the molecular and biochemical pathways of AhR-mediated effects in this species, and how the various measures of response are inter-related. Furthermore, it is unknown how the underlying molecular mechanisms of AhR-mediated responses compare to other species.
The cytochrome P450 enzymes are a large group of monooxygenase enzymes located in the endoplasmic reticulum or mitochondrial inner membrane (
Hahn, 1998
). In vertebrates, these enzymes are predominantly found in the liver and small intestine. An important group of cytochrome P450s are the phase I enzymes that insert an oxygen atom into molecules as the fi rst step of excretion,
conjugation and/or further transformation of xenobiotics ( Hahn,
1998
). Of particular interest are the CYP1A genes that can be upregulated by exposure to AhR agonists such as 2,3,7,8-tetrachloro-

X. Zhang et al. / Toxicology and Applied Pharmacology 234 (2009) 306 – 313 dibenzo-p-dioxin (2,3,7,8-TCDD) and result in the production of cytochrome P4501A enzymes. The expression of the genes encoding for these enzymes, as measured by mRNA transcription, protein content or enzyme activities have all been suggested as potential functional indicators of exposure to planar halogenated aromatic
hydrocarbons (PHAHs) ( Kawajir and Fujii-Kuriyama, 2007; Whitlock,
1999
). While these responses have been characterized as adaptive in nature, they have also been suggested to be related to subsequent toxicity, and to serve as important indicators for the exposure to
PHAHs in ecological risk assessments (
Hestermann et al., 2000;
Rifkind, 2006
).
While CYP enzyme activities have previously been measured in
mink ( Shipp et al., 1998; Martin et al., 2007 ), a more complete
characterization of the expression of CYP1A mRNA in relationship to the levels of CYP1A proteins and their associated enzymatic activities had not been done previously. This study was conducted to characterize the CYP1A1 and CYP1A2 genes of mink, to develop methods for measuring CYP1A mRNA levels, to measure CYP1A protein levels and enzyme activities, and to determine the relationships between these three measures of response. The actual adaptive response is the enzyme activity, but it has been previously shown that due to the effects of suicide substrates binding to these proteins, the overall induction of P450 enzyme activities is not necessarily proportional to
concentrations of AhR-active compounds in the tissues ( Hestermann et al., 2000
). Measurement of hepatic CYP1A mRNA and protein levels in parallel with hepatic enzyme activity can help to characterize the chemically induced mechanisms by differentiating between pretranscriptional and post-transcriptional inhibition. Here, we have characterized the CYP1A1 and CYP1A2 genes and their associated proteins, and compare their responses to enzyme activities (EROD and
MROD) in mink exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) singly or in a mixture
via the diet ( Moore et al. in press ). Comparisons were made among
these three methods to monitor functional responses and relationships between exposure to the model PCDFs and the three measures of up-regulation of the CYP1A system.
The speci fi c aims of this study were (i) to clone the cDNA sequence of CYP1As mRNA of mink; (ii) to develop semi-quantitative real-time
PCR methods to determine the mRNA expression in mink tissues; (iii) to develop immuno-reactivity or Western blotting methods to measure the protein expression of CYP1As in mink tissues; (iv) to determine the relationships among mRNA expression, protein synthesis and enzyme activities in vivo and 2,3,7,8-tetrachlorodibenzop dioxin equivalent (TEQ) concentrations in the mink tissues; and (v) to explore the kinetic relationships between chemical exposure and
CYP1As expression at the levels of gene expression, protein expression and enzyme activity.
Materials and Methods
Chemicals and reagents.
2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were obtained from
Accustandard Inc. (New Haven, CT, USA) and dissolved in the least amount of hexane possible to produce a stock solution of known concentration. Working solutions and dilutions were prepared in pesticide residue analysis grade OmniSolv n-hexane from EMD
Chemicals (Lawrence, KS, USA). The key reagents used for the molecular studies include the Agilent Total RNA Isolation Mini Kit
(Agilent Technologies, Palo Alto, CA, USA), Superscript III fi rst-strand synthesis SuperMix (Invitrogen, Carlsbad, CA, USA), BD SMART RACE cDNA ampli fi cation kit (BD-Biosciences Clontech, Palo Alto CA, USA),
SYBR Green master mix (Applied Biosystems, Foster City, CA, USA) and anti-dog CYP1A ELISA kit (Daiichi Pure Chemicals Co., Tokyo, Japan).
Animals and animal care.
The study was conducted at the Michigan
State University (MSU) Experimental Fur Farm. Housing of animals
Experimental design.
Fifty-six fi rst-year female mink were randomly assigned to the control group and the eight treatment groups.
Treatment groups consisted of mink exposed through the diet to three concentrations of each PeCDF and TCDF, and a single mixture of the two congeners. Targeted low, mid, and high spiked feed concentrations of PeCDF and TCDF were 110, 390, and 1600 ng kg
− 1 wet weight (ww) and 500, 2000, and 9700 ng kg
− 1 ww, respectively.
The dietary concentrations were selected to bracket environmental concentrations measured in mink from the Tittabawassee River, MI
USA ( Zwiernik et al., 2008b
). Each morning, 25 g of spiked feed was placed on the cage of each animal. After this feed was consumed, an additional 125 g of “ clean ” feed was given to each animal. This procedure ensured complete ingestion of the spiked feed, eliminating the need to measure daily feed consumption in order to estimate doses. Three animals from each of the TCDF, PeCDF and mixture treatment groups were sampled on days 90 and 180 while control mink were sampled on days 0, 90 and 180. At each sampling time, livers were removed, weighed, and subsamples were appropriately preserved for quanti fi cation of TCDF and PeCDF and for biochemical and molecular analysis. Additional data that was collected during the study including body and organ weights, gross pathological and
histopathological endpoints are reported elsewhere ( Moore et al. in press
). Analytical methods used in the quanti fi cation of TCDF and
PeCDF in the feed, liver and adipose tissue are given elsewhere
( Zwiernik et al., 2008a ). All animal care and use protocols were
approved by the Michigan State University All-University Committee on Animal Use and Care.
307 complied with guidelines speci fi ed in the Standard Guidelines for the
Operation of Mink Farms in the United States (
Fur Commission, USA
1995
). A standard dietary mix was used throughout the study with speci fi c ingredients, mix ratios, and nutritional data presented
elsewhere ( Beckett et al., 2008
). The base diet was used as the control diet with the treatment diets differing in only the supplemental TCDF or PeCDF added. Diets were prepared prior to the start of the study and stored under standard conditions (
Zwiernik et al., 2008a
).
Total RNA isolation and reverse transcription PCR.
extracted from the livers of individual mink by use of the Agilent
Total RNA Isolation Mini Kit (Agilent Technologies, Palo Alto, CA,
USA) according to the manufacture's protocol. Puri fi ed RNA was stored at -80 °C until analysis. First-strand cDNA synthesis was performed using Superscript III fi rst-strand synthesis SuperMix and
Oligo-dT primers (Invitrogen, Carlsbad, CA, USA). Brie fl y, a 0.5 to
2 μ g aliquot of total RNA was combined with 1 μ L of 50 μ M of Oligo
(dT)
20
, 1 μ L of annealing buffer, and RNase-free water to a fi nal volume of 8 μ L. Mixes were denatured at 65 °C for 5 min and then quickly cooled on ice for 2 min. Reverse transcription was performed after adding 10 μ L 2X fi rst-stand reaction mix, and 2 μ L
SuperScript III/RNaseOUT enzyme mix. Reactions were incubated at
50 °C for 50 min and, on completion, were inactivated at 85 °C for
5 min. To digest RNA, 1.25
μ L RNase H (Invitrogen, Inc., Carlsbad, CA,
USA) was added before incubation at 37 °C for 30 min. The cDNA synthesis reactions were stored at -20 °C until further analysis.
PCR cloning and sequencing of partial cDNA.
Total RNA was
The corresponding homologous genes of CYP1A2 and beta-actin in other mammal species (i.e. rat, dog, human, etc.) were fi rst identi fi ed and degenerate primer pairs of each gene were designed from conserved regions
( Table 1
). PCR was performed on the liver cDNA in a volume of 50 μ l consisting of 1 × PCR buffer, 0.2 mM of each dNTP, 2.5 mM of MgCl
2
,
0.2
μ M of each primer, 5 μ L of cDNA template, and 0.75 U Taq polymerase (Invitrogen, Carlsbad, CA, USA). Thirtyfi ve cycles of ampli fi cation were carried out with the reaction pro fi le of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 s, and extension

308 X. Zhang et al. / Toxicology and Applied Pharmacology 234 (2009) 306 – 313
Table 1
Oligonucleotide primers used in this study
Sequence (5 ′ to 3 ′ ) Primer
Degenerate primers
Beta-actin sense
Beta-actin anti-sense
CYP1A sense
CYP1A sense 2
⁎
CYP1A anti-sense
CCC AAA GCC AAC CG(C/T) GAG
CTC GTT GCC GAT GGT GAT
CAC AAC TGC CAT CTC CTG
GCCACAGA(A/G)CT(G/T)CTCCTGGC
C(C/G/T)(G/T) CTG GCA CGC TGA ACT
RACE and walk primers
Beta-actin 5 ′ RACE
Beta-actin 3 ′ RACE
CYP1A1 5 ′ RACE
CYP1A1 3 ′ RACE
CYP1A1 3 ′ walk
CYP1A1 5 ′ walk
CYP1A2 3 ′ RACE sense
CYP1A2 5 ′ RACE Sense
CYP1A2 5 end walk
CYP1A2 5 end walk 2
CGT CAG GTC CCG GCC AGC CAG GTC CAG
GGG TAC GCC CTG CCC CAC GCC ATC C
CCA GGA AGA GAA AGA CCT CCA GGC GGG C
CGC CTT CCT TCG TCC CCT TCA CCA TCC
CCT AAG CAC CTG GAA AGC
GGT GAA GGG GAC GAA GGA AGC GTG TCG G
GCA ACC CCG GCT CTC CGA CAG ACT CCA G
CCT TTC TAT GCA CCG GCG CTT GCC CAT G
GTA TCG GTG GCT CAG
CCC GCC TCT GCC ATC CGC TGC TGC
Real time PCR primers
Beta-actin sense
Beta-actin anti-sense
CYP1A1 sense
CYP1A1 anti-sense
CYP1A2 sense
CYP1A2 anti-sense
GAT GTG GAT CAG CAA GCA GGA G
GCC AGC AGT CCG TTT AGA AGC
CCT AAG CAC CTG GAA AGC
CTA AGT GTC AGA GGG ATT GG
ACA GCA GTG AGC ACA GAT GG
CCA GAG TAC CAG GCA GAA GAC
⁎
Used to amplify CYP1A1 cDNA using mink lung cDNA template; sequence derived from (
Tanaka et al., 2006 ).
at 72 °C for 1 min. The ampli fi ed PCR fragments were puri fi ed on agarose gel, isolated, cloned into the plasmid were sequenced using the corresponding primers by an ABI 3730 Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA).
Mink CYP1A2 and beta-actin were successfully ampli fi ed from
the liver cDNA using the designed degenerate primers ( Table 1
).
However, the same strategy didn't work for CYP1A1 .
Since it has been demonstrated that CYP1A1 is the dominant CYP1A isoform expressed in lung tissue of several mammal species (cat and rat), a
PCR reaction was performed using mink lung cDNA and an CYP1A1
“ sense ” degenerate primer previously reported to be effective in
the cat ( Tanaka et al., 2006 ) (
Table 1
). A major single band was successfully isolated on agarose gel from ampli fi ed PCR products and then was con fi rmed to be a partial cDNA of mink CYP1A1 by sequencing.
cycle. The thermal cycle pro fi le was denaturizing for 15 s at 95 °C; annealing for 30 s at 60 °C; and extension for 30 s at 72 °C. A total of
45 PCR cycles were used. PCR ef fi ciency, uniformity, and linear dynamic range of each Q-RT-PCR assay were assessed by the construction of standard curves using RNA dilutions. The mRNA expression level of beta-actin was used as a reference in the fold change calculation of CYP1A1 and CYP1A2.
Western blot and ELISA analysis.
Solon, OH, USA).
Mink liver microsome samples were electrophoresed on 12% SDS-PAGE gel, and then electro-blotted onto a 0.45
μ m pore size nitrocellulose membrane at a constant current of 300 mA at 4°C for 1.5 h. The nitrocellulose membrane was blocked in TBS (150 mM NaCl, 20 mM Tris-base, pH 7.4) with 5% (w/v) skimmed milk, and then was incubated with the anti-dog CYP1A serum within a commercially available ELISA kit (Daiichi Pure
Chemicals Co., Tokyo, Japan). The anti-dog anti-CYP1A polyclonal antiserum can detect both CYP1A1 and CYP1A2 but not other CYPs as suggested by the manufacturer. After three washes with TBST (TBS with 0.1% Tween-20), goat-anti-rabbit IgG conjugated with horseradish peroxidase (HRP) was added and incubated for 1 h at room temperature. The membranes were visualized with 3,3 ′ ,5,5 ′ -
Tetramethylbenzidine (TMB) membrane substrate (Amresco Inc.
To quantify mink hepatic CYP1As protein levels in all treatment groups, ELISA analysis was conducted using anti-dog CYP1As antiserum with dog hepatic microsomes as standards. Brie fl y, microtiter plates were coated ( n = 3 for each sample) with microsomal protein
(0.003
– 3.0
μ g/100 μ l) in phosphate-buffered saline (PBS) overnight at 4 °C. The plates then were blocked with PBS containing 1% bovine serum albumin for 2 h at room temperature. Each plate was then incubated with anti-dog CYP1A in PBS containing 0.1% bovine serum albumin for 1 h at 37 °C. After washing with PBS containing 0.2%
Tween 20, the plates were incubated with HRP-labeled anti-rabbit
IgG for 1 h at 37 °C. Subsequently, the plates were incubated for
10 min with 150 μ g/ml 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB) in
0.2 M Na
2
HPO
4 buffer (pH 4.5) containing 0.1 M citric acid. The colorimetric reaction was terminated by the addition of 2 N H
2
SO
4
, and the plates were analyzed by UV-visible spectrophotometry at
450 nm. The amount of each enzyme was calculated by comparison to microsomes of known enzyme levels as per the manufacturer's instructions.
3 ′ and 5 ′ rapid ampli fi cation of cDNA ends (RACE).
Gene-speci fi c primers were designed based on the partial cDNA sequences for each of the studied genes. 5 ′ -RACE and 3 ′ -RACE PCR reactions were performed using a BD SMART RACE cDNA ampli fi cation kit (BD-
Biosciences Clontech, Palo Alto, CA, USA) according to the manufacturer's protocol. Puri fi ed PCR products were cloned into the pCR2.1-TOPO- vectors using a DNA ligation kit (Invitrogen,
Carlsbad, CA, USA), followed by transformation of E. coli cells. DH5 α plasmids were isolated using a Wizard Plus SV minipreps DNA puri fi cation system (Promega, Madison, WI, USA). The PCR products were then sequenced.
Statistical analysis.
All data were expressed as mean ± 1 standard deviation (SD). Exposure data in this study was expressed as 2,3,7,8-
TCDD equivalents (TEQs) that were derived based on the most recent
2,3,7,8-TCDD equivalency factors (TEFs) suggested by the World
Health Organization (WHO) for mammals (
Van den Berg et al.,
2006 ). The quanti
fi cation of target gene expression was based comparative cycle threshold ( C t) method with adjustment of PCR ef fi ciency according to the procedures described in a previous study
(
Zhang et al., 2005 ). The relative expression level of CYP1A1 and
Real-time PCR.
Real-time Q-RT-PCR was performed by using an ABI
7900 high throughput real time PCR System in 384-well PCR plates
(Applied Biosystems, Foster City, CA, USA). PCR reaction mixtures for one hundred reactions contained 500 μ L of SYBR Green master mix
(Applied Biosystems, Foster City, CA, USA), 20 μ L of 10 μ M sense/ anti-sense gene-speci fi c primers, and 260 μ L of nuclease-free distilled water. A fi nal reaction volume of 10 μ L was made up with
2 μ L of diluted cDNA and 8 μ L of PCR reaction mixtures. The PCR reaction mix was denatured at 95 °C for 10 min before the fi rst PCR
Table 2
Comparison of the amino acid identities (%) of the mink CYP1A1 and CYP1A2 to the corresponding isozymes in other species
Organisms
Sea otter
Gray seal
Harp seal
Ribbon seal
Dog
Cat
Human
Rat
Mink CYP1A1
CYP1A1
95
91
91
90
89
86
80
76
CYP1A2
74
72
72
72
70
70
71
66
Mink CYP1A2
CYP1A1
74
73
73
72
70
70
69
64
CYP1A2
93
84
84
85
81
80
78
72

CYP1A2 mRNA in animals from different treatment groups were calculated by comparing to the initial mean value of the 0 d control group. Statistical analysis was conducted using the R project language
( http://www.r-project.org/ ). The study was designed for the application of both fi xed effects models (test for differences among exposure groups) and regression analysis. Because of the nature of the parameters, several statistical models were used for the data analyses.
Prior to conducting statistical comparisons a Bartlett Test were performed to check homogeneity of variances of data. If necessary, data were log-transformed to approximate normality. Effects of time, dose, different congeners and their interactions on the quanti fi ed values were examined using Gaussian Linear Models. Differences were evaluated by ANOVA followed by pair-wise t-test with Bonferroni's adjustment. Correlations between quanti fi ed values of individual animals were examined by the Spearman's rank test. Differences with p b 0.05 were considered to be signi fi cant.
Results
X. Zhang et al. / Toxicology and Applied Pharmacology 234 (2009) 306 – 313
Fig. 1.
Western blot analysis of mink hepatic microsomes using anti-dog CYP1A serum.
From left to right, lanes 1, 2, 3 were three individual animals from control group at
180 days; lanes 4, 5, 6: were three individual animals from the 180 d treatment group exposed to the mixture of TCDF and PCDF; lane 7 was dog microsome standards provided in the anti-dog anti-CYP1As ELISA kit.
309 predicted molecular mass of 41.8 kDa. A putative polyadenylation signal AATAAA is located about 836 bp and 83 bp following the Cterminal sequences of CYP1A1 and CYP1A2, respectively.
The phylogenetic analysis showed that mink CYP1A1 and CYP1A2 belonged to carnivores Caniformia CYP1A1 and CYP1A2 clades, respectively ( SM-Fig. 2 ). The deduced amino acid sequences of mink CYP1As were aligned with amino acid sequences of other mammals CYP1A family using CLUSTAL W ( SM-Fig. 3 ). The deduced mink CYP1As amino acid sequences were most closely related to sea otter ( Enhydra lutris ) CYP1As sequences, with 95% and 93% overall
amino acid identities for CYP1A1 and CYP1A2, respectively ( Table 2
).
The mink CYP1As sequences also displayed great similarities with those of seals, such as harp seal ( Pagophilus groenlandicus ), gray seal
( Halichoerus grypus ) and ribbon seal ( Histriophoca fasciata ), with 90 –
91% and 84 – 85% overall amino acid identities for CYP1A1 and
CYP1A2, respectively. Among the commonly-studied non-aquatic mammals, the dog CYP1As amino acid sequences were most closely
Cloning and sequence analysis of mink CYP1A1, CYP1A2, and beta-actin cDNAs
The cDNAs encoding CYP1A1, CYP1A2, and beta-actin were isolated from mink liver or lung by RACE and sequenced. The nucleotide sequence for the mink beta-actin, CYP1A1 and CYP1A2 has been submitted to GenBank/DDBJ/EMBL with the Accession No. EU046492,
EU046493 and EU046494, respectively. The cloned full-length mink
CYP1A1 cDNA consists of 2607 bps with a 1554-bp open reading frame
(ORF) encoding a 517 amino acids residue (See SM-Fig. 1 ). Mink
CYP1A2 cDNA has an ORF of 1539 bp and the deduced protein contains
512 amino acids. The predicted molecular mass of mink CYP1A1 and
1A2 are 58.5 and 57.9 kDa, respectively. The amino acid sequences of mink CYP1A1 and CYP1A2 have 75% identity. The mink beta-actin cDNA consists of 1128 bp ORF encoding 375 amino acid residues with a
Table 3
Daily dietary and tissue concentrations of 2,3,7,8-tetrachlorodibenzofuran (TCDF) and/ or 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) in the liver of mink ( Mustela vison )
a
Treatment Daily dose
Control
TCDF
PeCDF
Mixture
(ng TEQ/kg bw/d) b LOD d LOD
c
0.98
3.8
20
0.62
2.2
9.5
6.9
Adipose
(ng TEQ/kg, ww) b
b LOD d
LOD c
8.9 ± 2.8
22 ± 4.4
62 ± 8.9
74 ± 8.2
200 ± 21
534 ± 104
213 ± 22
Liver
(ng TEQ/kg, ww)
b
b LOD d LOD
1.2 ± 0.27
c
2.3 ± 0.22
7.1 ± 1.1
52 ± 18
270 ± 25
1600 ± 530
360 ± 79
Liver/Adipose ratio
NA
0.15 ± 0.06
0.11 ± 0.024
0.12 ± 0.015
0.70 ± 0.23
1.4 ± 0.24
3.0 ± 0.51
1.6 ± 0.30
NA, not available.
a
Each treatment group had six mink while the control group had eight mink. Control animals were sampled at 0, 90 and 180 d; three treated animals per dose group were sampled at 90 and 180 d. All concentrations were converted to 2,3,7,8-TCDD equivalents
(TEQs) using toxic equivalency factors (TEFs) of 0.3 for PeCDF and 0.1 for TCDF ( Van den et al., 2006
).
b
Tissue concentrations are presented as mean ± SD.
c
LOD = 0.1 ng TEQ/kg, ww.
Fig. 2.
(A) Gel electrophoresis of PCR products using real time PCR primers. From left to right: beta-actin, CYP1A1 and CYP1A2, and DNA ladder . (B) and (C), linear regressions of total RNA amount and the cycle threshold (Ct) in the real time PCR measurement of
CYP1A1 and CYP1A2 respectively.

310 X. Zhang et al. / Toxicology and Applied Pharmacology 234 (2009) 306 – 313
Table 4
Fold-change relative to control of CYP1A1 mRNA, CYP1A2 mRNA and CYP1As protein in
mink from different treatment groups a
Day Treatment Final Diet
0
90
180
Control
Control
TCDF
PeCDF
Mix
Control
TCDF
PeCDF
Mix
(ngTEQ/kg bw/d) b
LOD b
b
LOD b
0.98
3.8
20
3.8
20
0.62
2.2
9.5
6.9
0.62
2.2
9.5
6.9
b
LOD b
0.98
CYP1A1 mRNA
0.99 ± 0.45
1.1 ± 0.21
1.5 ± 0.44
1.7 ± 0.62
3.0 ± 0.99
4.2 ± 0.14
⁎⁎
7.6 ± 3.7
16 ± 5.0
⁎
8.5 ± 0.44
⁎⁎
1.0 ± 0.10
1.5 ± 0.63
2.0 ± 0.49
2.5 ± 0.25
⁎⁎
3.2 ± 0.21
⁎⁎⁎
6.7 ± 3.9
14 ± 1.5
⁎⁎
9.8 ± 1.1
⁎⁎
CYP1A2 mRNA
0.42 ± 0.05
1.3 ± 0.04
1.6 ± 0.30
1.8 ± 0.74
3.7 ± 0.67
⁎
2.2 ± 0.21
⁎
4.6 ± 1.9
8.4 ± 1.0
⁎⁎
4.7 ± 2.6
1.1 ± 0.37
1.6 ± 0.54
1.7 ± 0.13
2.9 ± 0.97
2.5 ± 0.31
⁎⁎
4.5 ± 3.2
8.2 ± 0.60
⁎⁎⁎
5.8 ± 1.3
⁎
CYP1As protein
1.0 ± 0.07
0.63 ± 0.03
0.94 ± 0.28
1.0 ± 0.37
2.0 ± 0.47
⁎
1.2 ± 0.22
⁎
2.6 ± 0.93
4.1 ± 1.4
2.4 ± 0.72
0.91 ± 0.11
1.1 ± 0.23
1.4 ± 0.05
⁎⁎
2.5 ± 0.47
⁎
1.4 ± 0.29
1.9 ± 1.0
4.1 ± 0.64
⁎
2.9 ± 0.81
⁎
a
Statistical comparisons were made by comparing each treatment to its corresponding control group at a given time.
b
LOD = 0.1 ng TEQ/kg, ww.
⁎
⁎⁎ p b 0.01.
⁎⁎⁎ p b 0.05.
p b 0.001.
related to the mink CYP1A1s, with 89% and 81% overall amino acid identities for CYP1A1 and CYP1A2, respectively.
manner over the 180 d exposure. However, no statistically signi fi cant differences were noted between 90 and 180 day TCDF and PeCDF tissue concentrations in mink from any of the individual treatment groups (
Moore et al. in press
).
Chemical- induced effects on mink hepatic CYP1As mRNA level
Real-time PCR method with gene speci fi c primers provided reliable gene expression measurement (
Fig. 2 ). Gel electrophoresis
con fi rmed that the PCR products using the real time PCR primers have different sizes, 141 and 82 for CYP1A1 and CYP1A2 respectively. The linear relationship between ct value and different concentration of cDNA were demonstrated as well. Exposure to either TCDF or PeCDF resulted in a dose-dependent increase of CYP1A1 and CYP1A2 mRNA expression (
Table 4 ). The dose-dependent increase of CYP1A1 induced
by PeCDF was greater than those observed with TCDF. Exposure of the mixture of TCDF and PeCDF signi fi cantly upregulated CYP1A1 mRNA levels in mink liver at both 90 d and 180d. The response pro fi le of the
CYP1A2 gene expression was similar to CYP1A1 for mink induced by the TCDF and PeCDF treatments.
Chemical- induced effects on mink hepatic CYP1As protein level
The response pro fi les of CYP1A protein levels were similar to those observed for CYP1A1 and CYP1A2 mRNA levels at 90 d (
Table 4
).
Consistent with the changes at mRNA and protein level, EROD and MROD activities in mink exposed to TCDF or PeCDF were increased in a dose-
dependent manner over the duration of the study ( Moore et al. in press
).
Immunoblot analysis of mink hepatic CYP1As protein
Consistent with the homologies of mink CYP1A proteins to its dog counterparts, western blotting using an anti-dog anti-CYP1As polyclonal antiserum showed cross-reactivity with mink hepatic micro-
somal samples ( Fig. 1
). The hepatic CYP1A proteins that were detected with this antibody had similar molecular weights and structures as those of the dog. In hepatic microsomes from mink exposed to TCDF and PeCDF for 180 d, the intensity of the CYP1A-associated protein bands were similar with that observed in microsomes from unexposed dog liver but were greater than that observed control mink microsomes (
Fig. 1 ). Since the PHAH have been shown to up-regulate
the expression of CYP1A genes and their associated proteins in other wild mammal species (
Hirakawa et al., 2007; Wilson et al., 2007 ), this
result further con fi rmed the reactivity of anti-dog CYP1As antiserum with mink CYP1As proteins.
Relationships between CYP1As mRNA, protein, and enzyme activities
Spearman Rank correlation analysis indicated that the expression levels of CYP1A1, CYP1A2, P4501A proteins, EROD, and MROD activity in the liver of mink were all signi fi cantly related with each other in a positive manner (
Table 5 ). Furthermore, the mRNA levels
of the two CYP1A genes had the greatest correlation coef fi cient
( R 2 = 0.859) among the combinations.
Relationships between different hepatic CYP1A measurements and dose metrics
The ratio between hepatic TEQ and adipose TEQ was used to represent the hepatic accumulation of different congeners in mink from different treatments. The hepatic accumulation of furans
Dietary concentrations and doses
Concentrations of PeCDF and TCDF in the diet and mink tissues were con fi rmed by instrumental analyses (
Zwiernik et al., 2008b
).
Final dietary concentrations and average daily doses for the various
treatments were calculated ( Table 3 ). Concentrations of TCDF and
PeCDF in the liver and adipose tissues increased in a dose-dependent
Table 5
Spearman rank correlation coef fi cients (numbers) and probabilities ( ⁎ ) between expression levels of CYP1A1 mRNA, CYP1A2 mRNA, CYP1As protein, EROD, and
MROD activity in the liver of mink a,b
CYP1A2 mRNA
CYP1As protein
EROD
MROD
CYP1A1 mRNA
0.915
0.732
0.751
0.820
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
CYP1A2 mRNA
0.742
⁎⁎⁎
0.799
⁎⁎⁎
0.859
⁎⁎⁎
CYP1As protein
0.757
⁎⁎⁎
0.806
⁎⁎⁎
EROD
0.841
⁎⁎⁎ a b
Sample size, N = 49, ⁎⁎⁎ p b 0.001.
EROD and MROD data were reported in a companion manuscript ( Moore et al. in press
).
Fig. 3.
Plot of hepatic accumulation of TEQ versus adipose TEQ concentration (ng/kg, ww). Hepatic accumulation of TEQ was expressed as the ratio of liver TEQ and adipose
TEQ concentration. TEQ was calculated based on single congener (treatment congener).
Adipose and liver TEQ given as wet weights (
Zwiernik et al., 2008b ).

X. Zhang et al. / Toxicology and Applied Pharmacology 234 (2009) 306 – 313 signi fi cantly raised following the increase of adipose TEQ in minks treatment ( p b
0.001) ( Fig. 3
). The treatments of TCDF were located at the “ fl at ” region of the curve and PeCDF treatments were at the increasing region of the curve. ANOVA analysis indicated that the hepatic accumulation of TEQ was signi fi cantly different between TCDF and PeCDF treatment ( p b 0.001).
Although changes of CYP1As at mRNA or protein levels in livers were correlated with sum TEQ in adipose or liver of minks from 180 d
exposure ( Fig. 4 ), such changes were more consistent with adipose
TEQ than with hepatic TEQ in the same animals. The regressions between CYP1A1 mRNA and sum TEQ concentrations in liver were marginally different for TCDF treatment and PeCDF treatment.
However, the chemical effect was not signi fi cant when CYP1A1 mRNA was correlated to adipose TEQ concentrations. Similarly, the regressions between CYP1A2 mRNA and sum TEQ concentrations in livers were signi fi cantly different between TCDF and PeCDF treatments, but were not signi fi cantly different when CYP1A2 mRNA was correlated to adipose TEQ concentrations. The regressions between
CYP1As protein production in livers and TEQ concentrations in liver or adipose were statistically signi fi cant between PeCDF and TCDF. The difference between the two regressions was less expressed when the
CYP1As protein was correlated to adipose TEQ level.
Discussion cDNAs encoding for the mixed function monooxygenase genes
CYP1A1 and CYP1A2 were cloned from mink tissue. The sequence information of CYP1As facilitated the development of CYP1As mRNA and protein expression as biomarker of exposure. Using these biomarkers, the dose- and time-related effects of environmentally relevant concentrations of PeCDF and TCDF were examined in mink.
Molecular characterization of mink CYP1A1 and CYP1A2
311
Mink have preserved several important traits of CYP1As identi fi ed in other mammal species, including the heme-binding motif
Fig. 4.
Regression analysis of hepatic expression levels of CYP1A1 mRNA or CYP1A2 mRNA, or production of CYP1As protein versus TEQ concentrations (ng/kg, ww) in liver or adipose
of mink treated with TCDF or PeCDF for 180 d. TEQs were expressed as the sum of all 17 congeners ( Zwiernik et al., 2008b
). Slopes of the regression models are given and the p-values indicate signi fi cance level of differences between the slopes for TCDF and PCDF.

312 X. Zhang et al. / Toxicology and Applied Pharmacology 234 (2009) 306 – 313
(FxxGxxxCxG) (
Porter and Coon, 1991 ), the proline-rich region
(PPGPxxxP) (
Kusano et al., 2001 ) and the six substrate recognition
sites (SRS) for the CYP1 family (
Gotoh, 1992 ). The mink CYP1As protein
sequences displays great sequence homology with sea otter, seals, and the dog. This result is consistent with the phylogeny of carnivores and
evolutionary history of pinnipeds ( Hirakawa et al., 2007; Koep fl i and
Wayne, 1998 ). Immunochemical analysis demonstrated that a poly-
clonal antibody against dog CYP1As cross-reacted with proteins from mink liver. Although an antibody inhibition assay was not conducted in this study, the similar size of the mink cross-reactive proteins to that observed for dog CYP1As and the induction of these mink proteins by Ah receptor agonists, TCDF and PeCDF, support the contention that the protein band identi fi ed by the polyclonal antibodies represented CYP1As.
Effect of PeCDF and TCDF on CYP1A related endpoints in Mink low as 1.5 ng TEQ/kg liver tissue. This is in accordance with previous studies that have shown that mink are a highly susceptible to dioxin and related compounds (
Hochstein et al., 1998, 2001 ).
Overall, the results in this study suggest that the basic mechanism of CYP1A induction through AhR mediated pathway is evolutionarily conserved in mink. The positive correlations between hepatic TEQ concentrations and hepatic expression of CYP1A mRNAs, protein,
EROD and MROD activities suggest that hepatic CYP1A were induced by both TCDF and PeCDF. The linear relationship between mRNA expression of CYP1A and adipose TEQ concentration suggest that the hepatic-sequestered fraction of PeCDF was biologically inactive to
AhR-mediated pathways. However, long-term exposure investigations, such as multi-generation studies, are needed to examine whether inhibition of CYP1A induction and hepatic sequestration of
PeCDF results in overall protection of the whole organism or whether it could lead to chronic effects in other organs.
Acknowledgments
This research was supported by an unrestricted grant from The
Dow Chemical Company to John Giesy and Matthew Zwiernik at
Michigan State University. All aspects of this study that involve the use of animals were conducted in the most humane manner. The protocols for this study were approved by the Michigan State University
Institutional Animal Care and Use Committee (AUF# 12/05-165-000).
Sustained induction of CYP1A mRNA, protein, and enzymatic activities by TCDF and PeCDF in mink from this study has been previously observed in fi sh, birds, and other mammals (
Abnet et al.,
1999; Fiedler et al., 1998; Hahn and Stegeman, 1994; Lorenzen et al.,
1997
). Furthermore, it has been shown that TCDF and PeCDF behaved as full Ah receptor agonists and displayed high-intrinsic ef fi cacy in the
induction of CYP1A ( Heid et al., 2001; Hestermann et al., 2000 ). These
observations suggest that induction of CYP1A enzymes in the mink liver can be assumed to be induced by a share transcriptional mechanism, through activation of Ah receptor by both TCDF and PeCDF.
Relationships between Hepatic TEQ levels and CYP1A responses
The sum TEQ concentration in either liver or adipose tissue was correlated to the CYP1A responses in livers of mink treated with TCDF
or PeCDF ( Fig. 4 ). This result indicated that the body concentration
could possibly be used to predict the activation of CYP1A enzymes and ultimately the toxicity associated with this exposure. However, distribution of PeCDF, but not TCDF, demonstrated increased hepatic accumulation (hepatic/adipose TEQ ratio) with increasing dose. The different tissue distribution was likely due to the different metabolism rate of the two congeners. Elimination half-times of TCDF were b 15 h and were inversely proportional to dose, while those for 4-PeCDF were approximately 7 to 9 d with no clear dose-dependency in the
tested dose range ( Zwiernik et al., 2008b ). The role of metabolism as a
modi fi er of congener tissue accumulation is also supported by the fi nding of
Kitmaura et al. (2001)
who reported that in mammals, TCDF is metabolized and eliminated more rapidly than PeCDF. An alternative hypothesis for the explanation of differential accumulation of PeCDF and TCDF in liver is sequestration of PeCDF. Hepatic sequestration of PeCDF has been previously reported in mammalian liver where it can bind to CYP1A2 (
Budinsky et al., 2006 ).
The CYP1A responses in liver of minks treated with TCDF or PeCDF were more related to the sum adipose TEQ concentration than liver
TEQ concentration (
Fig. 4 ). Especially, up-regulation of CYP1A1 and
CYP1A2 mRNA was solely dependent on the increase of adipose TEQ concentration, which suggests the sequestrated fraction of PeCDF in liver was biologically inactive to the AhR mediated pathway. If the sequestered fraction was biologically active, one would expect much greater CYP1A related responses in PeCDF exposed mink which did not occur in this study. However, the toxicological signi fi cance of
PeCDF being sequestered in the liver is unknown at this time. The dose of TEQ required for induction of mink CYP1As is less than that in other well-examined experimental animals such as mouse and rat. For example, in B6C3F1 mice liver CYP1As were inducible in the range between 400 and 1000 μ g TEQ /Kg tissue as indicated by EROD activity
(
DeVito et al., 1997
). Wistar Rat CYP1As were induced in maternal liver at concentrations of 100 ng TEQ/kg tissue (
Bell et al., 2007
). In contrast, the induction of mink CYP1As occurred at concentrations as
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.taap.2008.10.017
.
References
Abnet, C.C., Tanguay, R.L., Heideman, W., Peterson, R.E., 1999. Transactivation activity of human, zebra fi sh, and rainbow trout aryl hydrocarbon receptors expressed in COS-
7 cells: greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners. Toxicol. Appl. Pharmacol.
159, 41 – 51.
Basu, N., Scheuhammer, A.M., Bursian, S.J., Elliott, J., Rouvinen-Watt, K., Chan, H.M.,
2007. Mink as a sentinel species in environmental health. Environ. Res. 103,
130 – 144.
Beckett, K.J., Yamini, B., Bursian, S.J., 2008. The effects of 3,3,4,4,5-pentachlorobiphenyl
(PCB 126) on mink ( Mustela vison ) reproduction and kit survivability and growth.
Arch. Environ. Contam. Toxicol. 54, 123 – 129.
Bell, D.R., Clode, S., Fan, M.Q., Fernandes, A., Foster, P.M.D., Jiang, T., Loizou, G., MacNicoll,
A., Miller, B.G., Rose, M., Tran, L., White, W., 2007. Relationships between tissue levels of 2,3,7,8-Tetrachlorodibenzop -dioxin (TCDD), mRNAs, and toxicity in the developing male wistar(Han) rat. Toxicol. Sci. 99, 591 – 604.
Brunstrom, B., Lund, B.O., Bergman, A., Asplund, L., Athanassiadis, I., Athanasiadou, M.,
Jensen, S., Orberg, J., 2001. Reproductive toxicity in mink ( Mustela vison ) chronically exposed to environmentally relevant polychlorinated biphenyl concentrations.
Environ. Toxicol. Chem. 20, 2318 – 2327.
Budinsky, R., Paustenbach, D., Fontaine, D., Landenberger, B., Starr, T.B., 2006.
Recommended relative potency factors for 2,3,4,7,8-pentachlorodibenzofuran: the impact of different dose metrics. Toxicol. Sci. 91, 275 – 285.
DeVito, M.J., Diliberto, J.J., Ross, D.G., Menache, M.G., Birnbaum, L.S., 1997. Doseresponse relationships for polyhalogenated dioxins and dibenzofurans following subchronic treatment in mice. I. CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin. Toxicol. Appl. Pharmacol. 147, 267 – 280.
Engelhart, A., Behnisch, P., Hagenmaier, H., Apfelbach, R., 2001. PCBs and their putative effects on polecat ( Mustela putorius ) populations in Central Europe. Ecotoxicol.
Environ. Safety 48, 178 – 182.
Fiedler, H., Cooper, K., Bergek, S., Hjelt, M., Rappe, C., Bonner, M., Howell, F., Willett, K.,
Safe, S., 1998. PCDD, PCDF, and PCB in farm-raised cat fi sh from southeast United
States — concentrations, sources, and CYP1A induction. Chemosphere 37,
1645 – 1656.
Fur Commission USA, 1995. Standard guidelines for the operation of mink farms in the
United States. Fur Commission USA, St. Paul, MN.
Giesy, J.P., Verbrugge, D.A., Othout, R.A., Bowerman, W.W., Mora, M.A., Jones, P.D.,
Newsted, J.L., Vandervoort, C., Heaton, S.N., Aulerich, R.J., Bursian, S.J., Ludwig, J.P.,
Dawson, G.A., Kubiak, T.J., Best, D.A., Tillitt, D.E., 1994. Contaminants in fi shes from
Great Lakes-in fl uenced sections and above dams of three Michigan rivers. II:
Implications for health of mink. Arch. Environ. Contam. Toxicol. 27, 213 – 223.
Gotoh, O., 1992. Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences.
J. Biol. Chem. 267, 83 – 90.

Hahn, M.E., 1998. The aryl hydrocarbon receptor: a comparative perspective. Comp.
Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 121, 23 – 53.
Hahn, M.E., Stegeman, J.J., 1994. Regulation of cytochrome P4501A1 in teleosts: sustained induction of CYP1A1 mRNA, protein, and catalytic activity by 2,3,7,8tetrachlorodibenzofuran in the marine fi sh Stenotomus chrysops . Toxicol. Appl.
Pharmacol. 127, 187 – 198.
Heaton, S.N., Bursian, S.J., Giesy, J.P., Tillitt, D.E., Render, J.A., Jones, P.D., Verbrugge, D.A.,
Kubiak, T.J., Aulerich, R.J., 1995. Dietary exposure of mink to carp from Saginaw Bay,
Michigan. 1. Effects on reproduction and survival, and the potential risks to wild mink populations. Arch. Environ. Contam. Toxicol. 28, 334 – 343.
Heid, S.E., Walker, M.K., Swanson, H.I., 2001. Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation.
Toxicol. Sci. 61, 187 – 196.
Hestermann, E.V., Stegeman, J.J., Hahn, M.E., 2000. Relative contributions of af fi nity and intrinsic ef fi cacy to aryl hydrocarbon receptor ligand potency. Toxicol. Appl.
Pharmacol. 168, 160 – 172.
Hirakawa, S., Iwata, H., Takeshita, Y., Kim, E.Y., Sakamoto, T., Okajima, Y., Amano, M.,
Miyazaki, N., Petrov, E.A., Tanabe, S., 2007. Molecular characterization of cytochrome P450 1A1, 1A2, and 1B1, and effects of polychlorinated dibenzo-pdioxin, dibenzofuran, and biphenyl congeners on their hepatic expression in Baikal seal ( Pusa sibirica ). Toxicol. Sci. 97, 318 – 335.
Hochstein, J.R., Bursian, S.J., Aulerich, R.J., 1998. Effects of dietary exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin in adult female mink (Mustela vison). Arch. Environ.
Contam. Toxicol. 35, 348 – 353.
Hochstein Jr., M.S., Render, J.A., Bursian, S.J., Aulerich, R.J., 2001. Chronic toxicity of dietary 2,3,7,8-tetrachlorodibenzo-p-dioxin to mink. Vet. Hum. Toxicol. 43, 134 – 139.
Kawajir, K., Fujii-Kuriyama, Y., 2007. Cytochrome P450 gene regulation and physiological functions mediated by the aryl hydrocarbon receptor. Arch. Biochem. Biophys.
464, 207 – 212.
Kitamura, K., Nagahashi, M., Sunaga, M., Watanabe, S., Nagao, M., 2001. Balance of intake and excretion of 20 congeners of polychlorinated dibenzo-p-dioxin, polychlorinated dibenzofuran and coplanar polychlorinated biphenyl in healthy Japanese men. J. Health Sci. 47, 145 – 154.
Koep fl i, K.P., Wayne, R.K., 1998. Phylogenetic relationships of otters (Carnivora:
Mustelidae) based on mitochondrial cytochrome b sequences. J. Zool. 246, 401 – 416.
Kusano, K., Sakaguchi, M., Kagawa, N., Waterman, M.R., Omura, T., 2001. Microsomal p450s use speci fi c proline-rich sequences for ef fi cient folding, but not for maintenance of the folded structure. J. Biochem. (Tokyo) 129, 259 – 269.
Lorenzen, A., Kennedy, S.W., Bastien, L.J., Hahn, M.E., 1997. Halogenated aromatic hydrocarbon-mediated porphyrin accumulation and induction of cytochrome
P4501A in chicken embryo hepatocytes. Biochem. Pharmacol. 53, 373 – 384.
Martin, P.A., Mayne, G., Bursian, S.J., Tomy, G.T., Palace, V.P., Pekarik, C., Smits, J.E., 2007.
Immunotoxicity of the commercial polybrominated diphenyl ether mixture DE-71 in ranch mink (Mustela vision). Environ. Toxicol. Chem. 26, 9880997.
X. Zhang et al. / Toxicology and Applied Pharmacology 234 (2009) 306 – 313 313
Millsap, S.D., Blankenship, A.L., Bradley, P.W., Jones, P.D., Kay, D., Neigh, A., Park, C.,
Strause, K.D., Zwiernik, M.J., Giesy, J.P., 2004. Comparison of risk assessment methodologies for exposure of mink to PCBs on the Kalamazoo River, Michigan.
Environ. Sci. Technol. 38, 6451 – 6459.
Moore, J.N., Zwiernik, M.J., Bursian, S.J., Newsted, J., Zhang, X., Budinsky, R., Higley, E.B.,
Alward, L., Fitzgerald, S.D., Giesy, J.P., Hecker, M., in press Relationships between P450
Enzyme Induction, Jaw Histology and Tissue Morphology in mink ( Mustela vison )
Exposed to Polychlorinated Dibenzofurans (PCDFs). Arch. Environ. Toxicol. Chem.
Porter, T.D., Coon, M.J., 1991. Cytochrome P-450. Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J. Biol. Chem. 266, 13469 – 13472.
Rifkind, A.B., 2006. CYP1A in TCDD toxicity and in physiology-with particular reference to CYP dependent arachidonic acid metabolism and other endogenous substrates.
Drug Metab. Rev. 38, 291 – 335.
Shipp, E.B., Restum, J.C., Giesy, J.P., Bursian, S.J., Aulerich, R.J., Helferich, W.G., 1998.
Multigenerational study of the effects of consumption of PCB-contaminated carp from Saginaw Bay, Lake Huron on mink. 2. Liver PCB concentration and induction of hepatic cytochrome P450 activity as a potential biomarker of PCB exposure.
J. Toxicol. Environ. Health 54A, 377 – 401.
Tanaka, N., Miyasho, T., Shinkyo, R., Sakaki, T., Yokota, H., 2006. cDNA cloning and characterization of feline CYP1A1 and CYP1A2. Life Sci. 79, 2463 – 2473.
Van den Berg, M., Birnbaum, L.S., Denison, M., De Vito, M., Farland, W., Feeley, M.,
Fiedler, H., Hakansson, H., Hanberg, A., Haws, L., Rose, M., Safe, S., Schrenk, D.,
Tohyama, C., Tritscher, A., Tuomisto, J., Tysklind, M., Walker, N., Peterson, R.E., 2006.
The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93,
223 – 241.
Whitlock Jr., J.P., 1999. Induction of cytochrome P4501A1. Annu. Rev. Pharmacol. Toxicol.
39, 103 – 125.
Wilson, J.Y., Wells, R., Aguilar, A., Borrell, A., Tornero, V., Reijnders, P., Moore, M.,
Stegeman, J.J., 2007. Correlates of cytochrome P450 1A1 expression in bottlenose dolphin ( Tursiops truncatus ) integument biopsies. Toxicol. Sci. 97, 111 – 119.
Zhang, X., Yu, R.M., Jones, P.D., Lam, G.K., Newsted, J.L., Gracia, T., Hecker, M.,
Hilscherova, K., Sanderson, T., Wu, R.S., Giesy, J.P., 2005. Quantitative RT-PCR methods for evaluating toxicant-induced effects on steroidogenesis using the
H295R cell line. Environ. Sci. Technol. 39, 2777 – 2785.
Zwiernik, M.J., Kay, D.P., Moore, J.N., Beckett, K.J., Khim, J.S., Newsted, J.L., Roark, S., Giesy,
J.P., 2008a. Exposure and effects assessment of resident mink exposed to polychlorinated dibenzofurans and other dioxin-like compounds in the Tittabawassee River Basin, Midland, MI, USA on wild mink ( Mustela vison ). Environ.
Toxicol. Chem. 27, 2076 – 2087.
Zwiernik, M.J., Bursian, S.J., Aylward, L.L., Kay, D.P., Moore, J.N., Rowlands, C., Woodburn,
K., Shotwell, M.S., Khim, J.S., Giesy, J.P., Budinsky, R.A., 2008b. Toxicokinetics of
2,3,7,8-TCDF and 2,3,4,7,8-PECDF in mink (Mustela vison) at ecologically relevant exposures. Toxicol. Sci. 105, 33 – 43.

1,2
2,4,6
2,5
1,2,6,7,8
2,3
1,2
2,6,
3
2
1
3
4
5
6
7
8

CYP1A1
1 ttatattactccccacaagacaagcagcggtgtcatcgcagcgtaggcggggatcctgaa 60
61 ggtgacagctcttaggagcctcccctgatcttctttggggtgtcaggcgtaagaggccac 120
121 tcgtcccagctcacttcagtacctcctcacagcagcagcagttatctggagaactctaca 180
181 ctgatgatgtcctctgtgtccagactctccatccccatctcggccacagagtttcttctg 240
M M S S V S R L S I P I S A T E F L L
241 gcctctgccgtcttctgcctagtgctctgggtggtcagggcctggcagcctcgggttcct 300
A S A V F C L V L W V V R A W Q P R V P
301 aaaggcctgaagagtccaccggggccgtggggctggcctctgctggggaacgtgctgacc 360
K G L K S P P G P W G W P L L G N V L T
361 ttggggaagaccccacacctggcactgacgaggctgagccagcggtacggggacgtgttg 420
L G K T P H L A L T R L S Q R Y G D V L
421 caaatccacattggctccacacccgtgctggtgctcagtggcctggacaccatccggcag 480
Q I H I G S T P V L V L S G L D T I R Q
481 gccctggtgcggcagggggatgatttcaagggccggcctgacctctacagcttcactctg 540
A L V R Q G D D F K G R P D L Y S F T L
541 gttactgatggccaaagcatgaccttcaacccggactctggaccagcgtgggctgcccgc 600
V T D G Q S M T F N P D S G P A W A A R
601 aggcgcctggcccagaatgccctgaagagcttctccaccgcatcagacccggcttcctcg 660
R R L A Q N A L K S F S T A S D P A S S
661 tgcacctgctacctggaagagcacgtgagcaaggaggcagaggccctcctcggcaggctg 720
C T C Y L E E H V S K E A E A L L G R L
721 cagcagcggatggcagaggctgggtgctttgaaccctaccgatatgtagtggtatcagtg 780
Q Q R M A E A G C F E P Y R Y V V V S V
781 gctaatgtcatctgtgccatgtgctttggcaagcgctatgaccatgatgaccaagagcta 840
A N V I C A M C F G K R Y D H D D Q E L
841 cttagcttagtgaacctgagtaatgagtttggggaggcggttgcctctgggagccccatg 900
L S L V N L S N E F G E A V A S G S P M
901 gacttcttccccatccttcgttacttgcccaaccccaccctggattccttcaaggacctg 960
D F F P I L R Y L P N P T L D S F K D L
961 aataagaagttctataacttcatgcaaaagctggtcaaggaacactacagaacatttgag 1020
N K K F Y N F M Q K L V K E H Y R T F E
1021 aagggccacattcgggatatcacagacagcctgatcgagcactgccaggagaagaggcta 1080
K G H I R D I T D S L I E H C Q E K R L
1081 gatgaaaatgccaacatccagctgtctgacgagaagatcgttaacgttgtcttggacctg 1140
D E N A N I Q L S D E K I V N V V L D L
1141 tttggagcaggatttgacacggtcacaactgccatctcctggagcctcctgtacctggtg 1200
F G A G F D T V T T A I S W S L L Y L V
1201 acgaaccccaatgtgcagaaaaagatccaggaggagctggacacagtaattggcagggcc 1260
T N P N V Q K K I Q E E L D T V I G R A
1261 cggcagccccggctctccgacaggccccagctgccctatatggaggccttcatcctggag 1320
R Q P R L S D R P Q L P Y M E A F I L E
1321 atattccgacacgcttccttcgtccccttcaccatccctcatagcaccacaagagacaca 1380
I F R H A S F V P F T I P H S T T R D T
1381 agcctcagtggcttttacatccccaagggacgttgtgtcttcgtgaaccagtggaagatc 1440
S L S G F Y I P K G R C V F V N Q W K I
1441 aaccatgaccagaagctctggggtgacccgtctaagttccgaccagaacgatttctcaat 1500
N H D Q K L W G D P S K F R P E R F L N

1501 ctcgatggcaccatcaataaggcactgagtgagaaggtgattctctttggtatgggcaag 1560
L D G T I N K A L S E K V I L F G M G K
1561 cggaagtgcatcggtgagaccattgcccgcctggaggtctttctcttcctggccatcctg 1620
R K C I G E T I A R L E V F L F L A I L
1621 ctgcagcaggtggaattcagtgtgccccagggcacaagggtggacatgacccccatctat 1680
L Q Q V E F S V P Q G T R V D M T P I Y
1681 gggctgaccatgaagcatgcccgctgtgagcacttccaagtgcgggtgcgcacttagggg 1740
G L T M K H A R C E H F Q V R V R T
1741 actgagagccctgcagcctagactctgcctacctggcctgtctgggcagccaggacaggg 1800
1801 ggctggcccaggtctggggggttttctaagcctcagctagaaacccacagatactggtct 1860
1861 ggctggattttgctgtctgggctgcaggcaaggctgaaggatcgtgcttgcccaccagcc 1920
1921 tggacttgctgctctgcgtactgatcatggacagtgggcacagcaggagccccaaaggag 1980
1981 cagatggagccttctggaattgtgcttgatccaggaggcttggttggtttgggagatcct 2040
2041 aagcacctggaaagcttgagggaatgcactaaaaggccgtaaacccagtggctggctggc 2100
2101 tttctgtgggggtggattggctggagaaacaattagagcaggccacaccggggcctgtcc 2160
2161 aatccctctgacacttaggagctgtcactaatgatgggaagaagcatcttcagatctgcc 2220
2221 gagcgatctgaccacggggttctgggccactgtgatgccttttgtgttctccgtgcatca 2280
2281 gcaacagggctgcctcccagccttggaaggcccctgttcctgtccaggaggggctgggga 2340
2341 cttggacatgtctaagaggagtagagagcagagggagggcccagattcagacaccagaga 2400
2401 ccagttctctatgccctatataaaaatgccttttaaaaatatttgtatgcaagaagcttt 2460
2461 ttatttgtctgcatttatcatacataagagcttaagaacaacttactgagtgcaatagag 2520
2521 aaaaagtctaacccaagtatccagaaatgtgtaagaaacacctacctaagcta
g 2580
2 581 atattatccaggaaaaaaaaaaaaaaa
CYP1A2
1 ttattagtccccctttggggccaacaggggttttcaccgaggagtcggggggagctctgt 60
61 accagcctccacaaccctgctcatctcaagcacctgcctctacagcagtgagcacagatg 120
M
121 gcctggtccccgatagctacagagctgctcctggtctccgccgtcttctgcctggtactc 180
A W S P I A T E L L L V S A V F C L V L
181 tgggtggccagggcctggcagcctcgggttcccaaaggcctgaagagacccccagggcca 240
W V A R A W Q P R V P K G L K R P P G P
241 tggggatggcccttgctggggaacgtgctgaccttggggaagaccccacacctggcgctg 300
W G W P L L G N V L T L G K T P H L A L
301 acgagactgagccaccaatacggggatgtgctgcaaatccacattggctccacacccgtg 360
T R L S H Q Y G D V L Q I H I G S T P V
361 ctggtgctcaccggcatggacaccatccggcaggccctagtgcaacaaggggatgatttc 420
L V L T G M D T I R Q A L V Q Q G D D F
421 aagggccggcctgacctctacaacttcactctggttactgatggccaaagcatgaccttc 480
K G R P D L Y N F T L V T D G Q S M T F
481 aacccagactctggaccactgtgggctgcgcgcaggcgcctggcccagaacgccctgaag 540
N P D S G P L W A A R R R L A Q N A L K
541 agcttctccatcgcgtcagacccggcttccttgtgcacctgctacctggaagagcacgtg 600
S F S I A S D P A S L C T C Y L E E H V
601 agcaaggaggcagaggccctcctcggcaggctgcagcagcggatggcagaggcggggcgc 660
S K E A E A L L G R L Q Q R M A E A G R
661 tttgacccctacaacgaggtgctcttctccgtggccagtgtcattggtgccatgtgcttt 720
F D P Y N E V L F S V A S V I G A M C F
721 gggaagcgcttccctcagagcagtgaggagatgctcagccttataagcagcaccaatgac 780
G K R F P Q S S E E M L S L I S S T N D
781 tttgtggagacggcctcctctgggaaccctgtggacttcttccccattctccggtatctg 840
F V E T A S S G N P V D F F P I L R Y L
841 ccgaacccttccctgcagaaattcaaggccttcaaccagaagttctttcggttcctgcag 900
P N P S L Q K F K A F N Q K F F R F L Q

901 Aatattgtccaggagcactaccaggactttgacgagagcaacattcaggacatcacaggc 960
N I V Q E H Y Q D F D E S N I Q D I T G
961 gccctcctgaagcacaatgagaagggctccagaactagtgctggtcacatcccccatgag 1020
A L L K H N E K G S R T S A G H I P H E
1021 aagatcgtcaacattatcaacgacatttttggggccggatttgacaccatcacaacggcc 1080
K I V N I I N D I F G A G F D T I T T A
1081 atctcctggagcatcatgtatcttgtgacaaaccctgagatacagagaaagatccaggaa 1140
I S W S I M Y L V T N P E I Q R K I Q E
1141 gagttggacagagtgattggcagggcccggcaaccccggctctccgacagactccagctg 1200
E L D R V I G R A R Q P R L S D R L Q L
1201 ccctacctggaggccttcatcctggagctcttccgacacacctccttcgtcccctttacc 1260
P Y L E A F I L E L F R H T S F V P F T
1261 atcccccacagcacaacaagggacacgatactgaaaggcttctacatccctaaagaacgc 1320
I P H S T T R D T I L K G F Y I P K E R
1321 tgtgtctttgtaaaccagtggcaggtcaatcatgacccaaaggtgtggggagatccatcc 1380
C V F V N Q W Q V N H D P K V W G D P S
1381 aagttccgaccagagagattccttaccgctgatggcactgcaatcaataagaccttgagt 1440
K F R P E R F L T A D G T A I N K T L S
1441 gagaaggtgatactcttcggcatgggcaagcgccggtgcataggagaggtcatggccaag 1500
E K V I L F G M G K R R C I G E V M A K
1501 tcggagatcttcctcttcctagctatcttgctgcagcggctggagttcagtgtgccagcc 1560
S E I F L F L A I L L Q R L E F S V P A
1561 ggtgtgaaagtagacctaacccctatctatgggctgaccatgaagcacccccactgtgag 1620
G V K V D L T P I Y G L T M K H P H C E
1620 catgtccaggcacggccacgcttctccaccaagtgaagaaggcaccagcatgccaaggca 1680
H V Q A R P R F S T K
1681 gggggaggggtaaggatcatctatggggcctcaacccttattttttttcctttttctta
1740
1741
agctttatggaggtataatacacaa beta-actin
1 tctcactcgccgccgccggcccgtgctccgcgttctcctcccgctcgtccttcgcgtcca 60
61 ccgccgccacctccgccgaccgcaatggaagaggagatcgccgcgctcgtcatcgacaat 120
M E E E I A A L V I D N
121 ggctccggcatgtgcagggcgggctttgctggggacgacgccccccgggccgtgttcccg 180
G S G M C R A G F A G D D A P R A V F P
181 tccatcgtcgggcgaccccggcaccagggcgtgatggtgggaatgggccagaaggactcc 240
S I V G R P R H Q G V M V G M G Q K D S
241 tacgtgggcgacgaggcccagagcaagcggggcatcctgaccctgaagtaccccatcgag 300
Y V G D E A Q S K R G I L T L K Y P I E
301 cacggcatcgtcaccaactgggacgacatggagaagatctggcaccacaccttctacaat 360
H G I V T N W D D M E K I W H H T F Y N
361 gagctgcgcgtggcgcccgaggagcaccccgtgctgctgaccgaggcccccctgaacccc 420
E L R V A P E E H P V L L T E A P L N P
421 aaagccaaccgcgagaagatgacccagatcatgttcgagaccttcaacaccccagccatg 480
K A N R E K M T Q I M F E T F N T P A M
481 tacgtggccatccaggctgtgctgtccctgtacgcctctggccgcaccactggtattgtc 540
Y V A I Q A V L S L Y A S G R T T G I V
541 atggactctggggacggggtcacccgcactgtgcccatctacgaggggtacgccctgccc 600
M D S G D G V T R T V P I Y E G Y A L P
601 cacgccatcctgcgtctggacctggctggccgggacctgacggactacctcatgaagatc 660
H A I L R L D L A G R D L T D Y L M K I
661 cttaccgagcgcggctacagcttcaccaccaccgccgagcgggagatcgtgcgtgacatc 720
L T E R G Y S F T T T A E R E I V R D I
721 aaggagaagctctgctacgtcgccctggacttcgagcaggagatggccacggccgcgtcc 780

K E K L C Y V A L D F E Q E M A T A A S
781 tcctcctccctggagaagagctacgagcttcccgacgggcaggtcatcaccatcggcaac 840
S S S L E K S Y E L P D G Q V I T I G N
841 gagcggttccgctgcccggaggcgctcttccagccttccttcctgggcatggaatcctgc 900
E R F R C P E A L F Q P S F L G M E S C
901 ggcatccacgagaccaccttcaactccatcatgaagtgtgacgtcgacatccggaaggat 960
G I H E T T F N S I M K C D V D I R K D
961 ctctacgccaacacggtgctgtccggcgggaccaccatgtaccccggcatcgctgaccgg 1020
L Y A N T V L S G G T T M Y P G I A D R
1021 atgcagaaggagatcaccgctctggcgcccagcacgatgaagatcaagatcatcgcgccc 1080
M Q K E I T A L A P S T M K I K I I A P
1081 cccgagcgcaagtactccgtgtggatcgggggctccatcctggcctcgctgtccaccttc 1140
P E R K Y S V W I G G S I L A S L S T F
1141 cagcagatgtggatcagcaagcaggagtacgacgagtccggcccctccattgtccaccgc 1200
Q Q M W I S K Q E Y D E S G P S I V H R
1201 aaatgcttctaaacggactgctggcaagctaaccctgagtaccttctgccccggcagatg 1260
K C F
1261 tgcacacctcacgccggcctcagagactggaatcaacctttgaaaagaaatttgtccttg 1320
1321 aagcttgtatctgatatcagcactggattgtagaacttgttgctgatcttgaccttgtat 1380
1381 cccagttaactgttcccttggtatatgtttaataccctgtacatatctttgattt

Mink_CYP1A2 ---MAWSP----IATELLLVSAVFCLVLWVARAWQPRVPKGLKRPPGPWGWPLLGNVLTL 53
Sea_otter_CYP1A2 ---MAMSQ----TATELLLASSVFCLVLWVVRAWQPRVPKGLKSPPGPWGWPLLGNVLTL 53
Dog_CYP1A2 ---MALSQ----MATGLLLASTIFCLILWVVKAWQPRLPKGLKSPPGPWGWPLLGNVLTL 53
Grey_seal_CYP1A2 ---MALSQ----MATELLLASAVFCLVLWVVRAWQPRVPKGLKSPPGPWGWPLLGNVLTL 53
Human_CYP1A2 ---MALSQSVPFSATELLLASAIFCLVFWVLKGLRPRVPKGLKSPPEPWGWPLLGHVLTL 57
Mink_CYP1A1 MMSSVSRLSIPISATEFLLASAVFCLVLWVVRAWQPRVPKGLKSPPGPWGWPLLGNVLTL 60
Sea_otter_CYP1A1 MMFSAFGLSIPISATEFLLASSVFCLVLWVVRAWQPRVPKGLKSPPGPWGWPLLGNVLTL 60
Dog_CYP1A1 -MMSMFRLSIPISASELLLASTVFCLVLWVVKAWQPRLPKGLKSPPGPWGWPVLGNVLTL 59
Grey_seal_CYP1A1 MMFSASRLSIPISATELLLASAVFCLMPWVVRAWQPRVPKGLKSPPGPWGWPLLGNVLTL 60
Rat_CYP1A1 -MPSVYGFPAFTSATELLLAVTTFCLGFWVVRVTRTWVPKGLKSPPGPWGLPFIGHVLTL 59
Human_CYP1A1 -----MLFPISMSATEFLLASVIFCLVFWVIRASRPQVPKGLKNPPGPWGWPLIGHMLTL 55
Rat_CYP1A2 ---MAFSQYIS-LAPELLLATAIFCLVFWVLRGTRTQVPKGLKSPPGPWGLPFIGHMLTL 56
*. :**. *** ** : :. :***** ** *** *.:*::***
SRS-1
Mink_CYP1A2 GKTPHLALTRLSHQYGDVLQIHIGSTPVLVLTGMDTIRQALVQQGDDFKGRPDLYNFTLV 113
Sea_otter_CYP1A2 GKTPHLALTRLSQRYGDVLQIHIGSTPVLVLSGLDTIRQALVQQGDDFKGRPDLYSFTLV 113
Dog_CYP1A2 GKSPHLALSRLSQRYGDVLQIRIGSTPVLVLSGLDTIRQALVRQGDDFKGRPDLYSLSLI 113
Grey_seal_CYP1A2 GKNPHLALSRLSQRYGDVLQIHIGSTPVLVLSGLRTVRQALVRQGEDFKGRPDLYSFTLI 113
Human_CYP1A2 GKNPHLALSRMSQRYGDVLQIRIGSTPVLVLSRLDTIRQALVRQGDDFKGRPDLYTSTLI 117
Mink_CYP1A1 GKTPHLALTRLSQRYGDVLQIHIGSTPVLVLSGLDTIRQALVRQGDDFKGRPDLYSFTLV 120
Sea_otter_CYP1A1 GKTPHLALTRLSQRYGDVLQIHIGSTPVLVLSGLDTIRQALVQQGNDFKGRPDLYSFTLV 120
Dog_CYP1A1 GKSPHLALSRLSQRYGDVLQIRIGSTPVLVLSGLDTIRQALVRQGDDFKGRPDLYSFSLV 119
Grey_seal_CYP1A1 GKNPHLALSRLSQRYGDVLQIHIGSTPVLVLSGPDTVRQALVRQGEDFKGRPDLYSFTLI 120
Rat_CYP1A1 GKNPHLSLTKLSQQYGDVLQIRIGSTPVVVLSGLNTIKQALVKQGDDFKGRPDLYSFTLI 119
Human_CYP1A1 GKNPHLALSRMSQQYGDVLQIRIGSTPVVVLSGLDTIRQALVRQGDDFKGRPDLYTFTLI 115
Rat_CYP1A2 GKNPHLSLTKLSQQYGDVLQIRIGSTPVVVLSGLNTIKQALVKQGDDFKGRPDLYSFTLI 116
**.***:*:::*::*******:******:**: *::****:**:*********. :*:
Mink_CYP1A2 TDGQSMTFNPDSGPLWAARRRLAQNALKSFSIASDPASLCTCYLEEHVSKEAEALLGRLQ 173
Sea_otter_CYP1A2 TDGQSMSFSPDSGPVWAARRRLAQNVLKSFSIASDPASSCTCYLEEHVSKEAEALLGRLQ 173
Dog_CYP1A2 TDSQSMSFSPDSGPVWAAGRRLAQNALNTFSIASDPASSCSCYLEEHVSKEAEALLSRLQ 173
Grey_seal_CYP1A2 TNGQSMSFSPDSGPVWAARRRLAQNALKSFSIASDPGSSSSCYLEEHVSKEAEALLSRLQ 173
Human_CYP1A2 TDGQSLTFSTDSGPVWAARRRLAQNALNTFSIASDPASSSSCYLEEHVSKEAKALISRLQ 177
Mink_CYP1A1 TDGQSMTFNPDSGPAWAARRRLAQNALKSFSTASDPASSCTCYLEEHVSKEAEALLGRLQ 180
Sea_otter_CYP1A1 TDGQSMSFSPDSGPVWAARRRLAQSALKSFSTASDPASSCTCYLEEHVSKEAEALLGRLQ 180
Dog_CYP1A1 TDGQSLTFSPDSGPVWAARRRLAQNALKSFSIASDPASSCSCYLEEHVSKEAEVLLSRLQ 179
Grey_seal_CYP1A1 TNGQSMSFSPDSGPVWAARRRLAQNALKSFSIASDPGSSSSCYLEEHVSKEAEALLSRLQ 180
Rat_CYP1A1 ANGQSMTFNPDSGPLWAARRRLAQNALKSFSIASDPTLASSCYLEEHVSKEAEYLISKFQ 179
Human_CYP1A1 SNGQSMSFSPDSGPVWAARRRLAQNGLKSFSIASDPASSTSCYLEEHVSKEAEVLISTLQ 175
Rat_CYP1A2 TNGKSMTFNPDSGPVWAARRRLAQDALKSFSIASDPTSVSSCYLEEHVSKEANHLISKFQ 176
::.:*::*..**** *** *****. *::** **** :***********: *:. :*
SRS-2
Mink_CYP1A2 QRMAEAGRFDPYNEVLFSVASVIGAMCFGKRFPQSSEEMLSLISSTNDFVETASSGNPVD 233
Sea_otter_CYP1A2 QRMAEAGRFDPYNEVMLSVTGVIGAMCFGRRFPQSSEDMLSLISSTNDFVEMASSGNPVD 233
Dog_CYP1A2 EQMAEVGRFDPYNQVLLSVANVIGAMCFGHHFSQRSEEMLPLLMSSSDFVETVSSGNPLD 233
Grey_seal_CYP1A2 EQMAEVGHFDPYNQVLLSVANVIGAMCFGQHFPQSNEEMLSLIKSSNDFVETASSGNPVD 233
Human_CYP1A2 ELMAGPGHFDPYNQVVVSVANVIGAMCFGQHFPESSDEMLSLVKNTHEFVETASSGNPLD 237
Mink_CYP1A1 QRMAEAGCFEPYRYVVVSVANVICAMCFGKRYDHDDQELLSLVNLSNEFGEAVASGSPMD 240
Sea_otter_CYP1A1 EQMAEAGRFEPYRYVVVSVANVICAMCFGKRYDHDDQELLSLVNLSNEFGEAVASGSPTD 240
Dog_CYP1A1 EQMAEVGRFDPYRYIVVSVANVICAMCFSKRYDHDDQELLSLVNLSNEFGEGVASANPLD 239
Grey_seal_CYP1A1 EQMAEVGHFDPYRYVVVSVANVVCAMCFGKRYDHDDQELLSLINLNNEFGEAVASGNPVD 240
Rat_CYP1A1 KLMAEVGHFDPFKYLVVSVANVICAICFGRRYDHDDQELLSIVNLSNEFGEVTGSGYPAD 239
Human_CYP1A1 ELMAGPGHFNPYRYVVVSVTNVICAICFGRRYDHNHQELLSLVNLNNNFGEVVGSGNPAD 235
Rat_CYP1A2 KLMAEVGHFEPVNQVVESVANVIGAMCFGKNFPRKSEEMLNLVKSSKDFVENVTSGNAVD 236
: ** * *:* . :: **:.*: *:**.:.: . :::* :: . :* * . *. . *

SRS-3
Mink_CYP1A2 FFPILRYLPNPSLQKFKAFNQKFFRFLQNIVQEHYQDFDESNIQDITGALLKHNEKGSRT 293
Sea_otter_CYP1A2 FFPILRYLPNPSLQKFKAFNQKFFRFLQNIVREHYRDFDESNIQDITGALLKHNEKGSRT 293
Dog_CYP1A2 FFPILQYMPNSALQRFKNFNQTFVQSLQKIVQEHYQDFDERSVQDITGALLKHNEKSSRA 293
Grey_seal_CYP1A2 FFPILQYMPNPALQRFKAFNQKLVQFLQKIVQEHYQDFDESSIQDVTGALLKHNEKGSRA 293
Human_CYP1A2 FFPILRYLPNPALQRFKAFNQRFLWFLQKTVQEHYQDFDKNSVRDITGALFKHSKKGPRA 297
Mink_CYP1A1 FFPILRYLPNPTLDSFKDLNKKFYNFMQKLVKEHYRTFEKGHIRDITDSLIEHCQEKRLD 300
Sea_otter_CYP1A1 FFPILRYLPNPALDSFKDLNKKFYSFMQKLVKEHYRTFEKGHIRDITDSLIEHCQEKRLD 300
Dog_CYP1A1 FFPILRYLPNPALDFFKDLNKRFYSFMQKMVKEHYKTFEKGQIRDVTDSLIEHCQDKRLD 299
Grey_seal_CYP1A1 FFPILRYLPNPALDFFKDLNKRFYSFMQKLVKEHYKTFEKGHIRDITDSLIKHCQDKRLD 300
Rat_CYP1A1 FIPILRYLPNSSLDAFKDLNKKFYSFMKKLIKEHYRTFEKGHIRDITDSLIEHCQDRRLD 299
Human_CYP1A1 FIPILRYLPNPSLNAFKDLNEKFYSFMQKMVKEHYKTFEKGHIRDITDSLIEHCQEKQLD 295
Rat_CYP1A2 FFPVLRYLPNPALKRFKNFNDNFVLFLQKTVQEHYQDFNKNSIQDITGALFKHSEN-YKD 295
*:*:*:*:**.:*. ** :*. : ::: ::***: *:: ::*:*.:*::* :.
SRS-4/Center of α -helix I
Mink_CYP1A2 SAG--HIPHEKIVNIINDIFGAGFDTITTAISWSIMYLVTNPEIQRKIQEELDRVIGRAR 351
Sea_otter_CYP1A2 SAG--HIPHEKIVNIINDIFGAGFDTITTAISWSIMYLVTNPEIQRKIQEELDRVIGRAR 351
Dog_CYP1A2 SDG--HIPQEKIVNLINDIFGAGFDTVTTAISWSLMYLVANPEIQRQIQKELDTVIGRAR 351
Grey_seal_CYP1A2 GGG--HIPHEKIVSLINDIFGAGFEPITTAISWSLIYLVANPEIQRKIQEELDTVTGRAR 351
Human_CYP1A2 SGN--LIPQEKIVNLVNDIFGAGFDTVTTAISWSLMYLVTKPEIQRKIQKELDTVIGRER 355
Mink_CYP1A1 ENANIQLSDEKIVNVVLDLFGAGFDTVTTAISWSLLYLVTNPNVQKKIQEELDTVIGRAR 360
Sea_otter_CYP1A1 ENANILLSDEKIVNVVLDLFGAGFDTVTTAISWSLLYLVTNPSVQKKIQEELDTVIGRAR 360
Dog_CYP1A1 ENANIQLSDEKIVNVVLDLFGAGFDTVTTAISWSLLYLVTNPNVQKKIQKELDTVIGRAR 359
Grey_seal_CYP1A1 ENANIQLSDEKIVNVVLDLFGAGFDTVTTAISWSLLYLVTSPSVQKKIQEELDTVIGRAR 360
Rat_CYP1A1 ENANVQLSDDKVITIVFDLFGAGFDTITTAISWSLMYLVTNPRIQRKIQEELDTVIGRDR 359
Human_CYP1A1 ENANVQLSDEKIINIVLDLFGAGFDTVTTAISWSLMYLVMNPRVQRKIQEELDTVIGRSR 355
Rat_CYP1A2 NGG--LIPQEKIVNIVNDIFGAGFETVTTAIFWSILLLVTEPKVQRKIHEELDTVIGRDR 353
:..:*::.:: *:*****:.:**** **:: ** .* :*::*::*** * ** *
SRS-5
Mink_CYP1A2 QPRLSDRLQLPYLEAFILELFRHTSFVPFTIPHSTTRDTILKGFYIPKERCVFVNQWQVN 411
Sea_otter_CYP1A2 QPRLSDRLQLPYLEAFILEIFRHTSFVPFTIPHSTTRDTTLKGFYIPKERCVFVNQWQVN 411
Dog_CYP1A2 QPRLSDRPQLPLMEAFILEIFRHTSFVPFTIPHSTTKDTTLKGFYIPKECCVFINQWQVN 411
Grey_seal_CYP1A2 QPRLSDRPQLPYMEAFILEIFRHTSFVPFTIPHSTTRDTTLKGFYIPKERCVFINQWHVN 411
Human_CYP1A2 RPRLSDRPQLPYLEAFILETFRHSSFLPFTIPHSTTRDTTLNGFYIPKKCCVFVNQWQVN 415
Mink_CYP1A1 QPRLSDRPQLPYMEAFILEIFRHASFVPFTIPHSTTRDTSLSGFYIPKGRCVFVNQWKIN 420
Sea_otter_CYP1A1 QPRLSDRPQLPYMEAFILEIFRHASFVPFTIPHCTTKDTSLSGFYIPKGRCVFVNQWQID 420
Dog_CYP1A1 QPRLSDRPQLPYMEAFILETFRHASFVPFTIPHSTTRDTSLSGFYIPKGRCVFVNQWQIN 419
Grey_seal_CYP1A1 QPRLSDRPQLPYLEAFILETFRHASFVPFTIPHSTTKDTSLSGFYIPKGRCVFVNQWQIN 420
Rat_CYP1A1 QPRLSDRPQLPYLEAFILETFRHSSFVPFTIPHSTIRDTSLNGFYIPKGHCVFVNQWQVN 419
Human_CYP1A1 RPRLSDRSHLPYMEAFILETFRHSSFVPFTIPHSTTRDTSLKGFYIPKGRCVFVNQWQIN 415
Rat_CYP1A2 QPRLSDRPQLPYLEAFILEIYRYTSFVPFTIPHSTTRDTSLNGFHIPKECCIFINQWQVN 413
:****** :** :****** :*::**:******.* :** *.**:*** *:*:***:::
Heme-binding
Mink_CYP1A2 HDPKVWGDPSKFRPERFLTADGTAINKTLSEKVILFGMGKRR C IGEVMAKSEIFLFLAIL 471
Sea_otter_CYP1A2 HDRKVWGDPSEFRPERLLTADGTAINKILSEKVILFSMGKRR
C
IGEVMAKSEIFLFLAIL 471
Dog_CYP1A2 HDQQVWGDPFAFRPERFLTADGTTINKTLSEKVMLFGMGKRR C IGEVLAKWEIFLFLAIL 471
Grey_seal_CYP1A2 HDQKVWGDPFEFRPERFLTADGTSINKILSEKVMIFGMGKRR
C
IGELLAKWEIFLFLAIL 471
Human_CYP1A2 HDPELWEDPSEFRPERFLTADGTAINKPLSEKMMLFGMGKRR C IGEVLAKWEIFLFLAIL 475
Mink_CYP1A1 HDQKLWGDPSKFRPERFLNLDG-TINKALSEKVILFGMGKRK C IGETIARLEVFLFLAIL 479
Sea_otter_CYP1A1 HDQKLWGDPSEFRPERFLTLDG-TINKALSEKVILFGMGKRK
C
IGETIARLEVFLFLAIL 479
Dog_CYP1A1 HDQKLWGNPSEFQPERFLTLDG-TINKALSEKVILFGLGKRK C IGETIARLEVFLFLAIL 478
Grey_seal_CYP1A1 HDQELWGDPSEFRPERFLTLDG-TINKALSEKVILFGMGKRK
C
IGETIARLEVFLFLAIL 479
Rat_CYP1A1 HDQELWGDPNEFRPERFLTSSG-TLDKHLSEKVILFGLGKRK C IGETIGRLEVFLFLAIL 478
Human_CYP1A1 HDQKLWVNPSEFLPERFLTPDG-AIDKVLSEKVIIFGMGKRK
C
IGETIARWEVFLFLAIL 474
Rat_CYP1A2 HDEKQWKDPFVFRPERFLTNDNTAIDKTLSEKVMLFGLGKRR C IGEIPAKWEVFLFLAIL 473
** : * :* * ***:*. .. :::* ****:::*.:***:**** .: *:*******
SRS-6
Mink_CYP1A2 LQRLEFSVPAGVKVDLTPIYGLTMKHPHCEHVQARPRFSTK----- 512
Sea_otter_CYP1A2 LQRLEFSVPACVKVDLTPIYGLTMKHPHCEHVQARPRFSTK----- 512
Dog_CYP1A2 LQRLEFSVPAGVKVDLTPIYGLTMKHTRCEHVQARPRFSIK----- 512
Grey_seal_CYP1A2 LQRLEFSVPDGVKVDLTPIYGLTMKHTRCEHVQARPRFSTK----- 512
Human_CYP1A2 LQQLEFSVPPGVKVDLTPIYGLTMKHARCEHVQAR-RFSIN----- 515
Mink_CYP1A1 LQQVEFSVPQGTRVDMTPIYGLTMKHARCEHFQVRVRT-------- 517
Sea_otter_CYP1A1 LQQVEFSVPQGTRVDMTPIYGLTMKHARCEHVQVRVRA-------- 517
Dog_CYP1A1 LQQVEFSVPEGTKVDMTPIYGLTMKHARCEHFQVRVRTEGAESPAA 524
Grey_seal_CYP1A1 LQQVEFSVPQGTKVDMTPIYGLTMKHARCEHVQVRVRA-------- 517
Rat_CYP1A1 LQQMEFNVSPGEKVDMTPAYGLTLKHARCEHFQVQMRSSGPQHLQA 524
Human_CYP1A1 LQRVEFSVPLGVKVDMTPIYGLTMKHACCEHFQMQLRS-------- 512
Rat_CYP1A2 LHQLEFTVPPGVKVDLTPSYGLTMKPRTCEHVQAWPRFSK------ 513
*:::**.*. :**:** ****:* ***.* *

Phylogenetic tree of CYP1A1 and CYP1A2. The tree was constructed by Jukes-Cantor genetic distance model and neighbor-joining method using Geneious 3.0 (
). Blosum62 was used as the cost matrix. The gap open and gap extension penalty was 12 and 3, respectively. The sequences accession numbers are NP_001020030 (cat CYP1A1), NP_001041478
(cat CYP1A2), P56590 (dog CYP1A1); P56592 (dog CYP1A2); Q00557 (golden hamster CYP1A1); P24453 (golden hamster
CYP1A2); CAF18539 (gray seal CYP1A1); CAF18540 (gray seal CYP1A2); Q06367 (guinea pig CYP1A1); Q64391 (guinea pig
CYP1A2); CAF18541 (harp seal CYP1A1); CAF18542 (harp seal CYP1A2); P04798 (human CYP1A1); P05177 (human CYP1A2);
BAE46562 (minke whale CYP1A1); BAE46563 (minke whale CYP1A2); P33616 (monkey CYP1A1); BAA33789 (monkey
CYP1A2); P00184 (mouse CYP1A1); P00186 (mouse CYP1A2); P00185 (rat CYP1A1); P04799 (rat CYP1A2); BAB20377 (ribbon seal CYP1A1); BAB20378 (ribbon seal CYP1A2); P05176 (rabbit CYP1A1); P00187 (rabbit CYP1A2) ABQ53137 (sea otter
CYP1A2) and ABR10295 (sea otter CYP1A1).