TOXICOLOGICAL SCIENCES 88(2), 384–399 (2005)
doi:10.1093/toxsci/kfi326
Advance Access publication September 21, 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
Michio X. Watanabe,* Hisato Iwata,*,1 Mio Okamoto,* Eun-Young Kim,* Kumiko Yoneda,†
Takuma Hashimoto,† and Shinsuke Tanabe*
*Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan; and
†Japan Wildlife Research Center, Shitaya 3-10-10, Taito-ku Tokyo 110-8676, Japan
Received July 13, 2005; accepted September 12, 2005
This study presents concentrations of polychlorinated dibenzop-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like coplanar PCBs (Co-PCBs) in the liver and breast muscle of jungle
crows (JCs; Corvus macrorhynchos) collected from Tokyo, Japan.
2,3,7,8-Tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) derived by WHO bird-TEF were in the range of 23 to 280 pg/g (lipid)
in the liver, which are lower or comparable to the lowest-observedeffect-level of CYP induction in chicken, and 5.6–78 pg/g (lipid) in
the pectoral muscle. Cytochrome P450 (CYP) 1A-, 2B-, 2C-, and
3A-like proteins were detected using anti-rat CYP polyclonal
antibodies in hepatic microsomal fractions. Significant ( p < 0.05)
positive correlations between hepatic TEQs and CYP1A or
CYP3A-like protein expression levels were noticed, implying
induction of these CYP isozymes by TEQs. On the other hand,
there was no significant positive correlation between muscle TEQ
and any one of analyzed CYP isozyme expression levels. CYP1Aand CYP3A-like protein expression levels represented better
correlations with pentoxy- and benzyloxyresorufin-O-dealkylase
activities rather than methoxy- and ethoxyresorufin-O-dealkylase
activities, indicating unique catalytic functions of these CYPs in
JCs. Furthermore, we succeeded in isolating CYP1A5 cDNA from
the liver of JC, having an open reading frame of 531 amino acid
residues with a predicted molecular mass of 60.3 kDa. JC
CYP1A5 mRNA expression measured by real-time RT-PCR had
a significant positive correlation with hepatic TEQs, suggesting
induction of CYP1A5 at the transcriptional level. Ratios of several
Co-PCB congeners to CB-169 in the liver of JCs revealed significant negative correlations with CYP1A protein or CYP1A5
mRNA expression levels, implying metabolism of these congeners
by the induced CYP1A. The liver/breast muscle concentration (L/M)
ratios of PCDDs/DFs and CB-169 increased with an increase in
The nucleotide sequence of JC CYP1A5 has been deposited in the DDBJ/
EMBL/GenBank database under accession number AB220967.
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. Tel./Fax: þ81-89-927-8172.
E-mail: [email protected].
hepatic CYP1A protein or CYP1A5 mRNA expression levels,
suggesting congener-specific hepatic sequestrations by the induced
CYP1A. The present study provides insights into the propensity of
CYP1A induction to the exposure of dioxin-like chemicals, and
unique metabolic and sequestration capacities of CYP1A in JC.
Key Words: jungle crow; dioxin-like compounds; CYP1A5
mRNA; alkoxyresorufin-O-dealkylation activities; metabolism;
hepatic sequestration.
Contamination by dioxin-related compounds (DRCs) such
as polychlorinated-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like coplanar polychlorinated
biphenyls (Co-PCBs) is of a growing concern due to their
ubiquity, biomagnification potential, and highly toxic properties in humans and wildlife. Jungle crow (JC; Corvus macrorhynchos) is considered to be an useful bioindicator for
monitoring contaminants in urban areas, because this species
is residential, occupies the same habitat as humans, and feeds
on a variety of food including domestic waste and garbage.
JCs accumulate environmental contaminants including DRCs
(Watanabe et al., 2005).
Cytochrome P450 (CYP) is a monooxygenase enzyme that
plays a prominent role in the biotransformation of endogenous
and xenobiotic compounds in biological systems. CYP has
been recognized as a marker enzyme to evaluate integrated
exposure to complex mixtures of contaminants, and molecular,
biochemical, and adverse effects in which CYP signaling
cascade is involved. One such response, induction of CYP1A
subfamily, has been extensively used as a sensitive indicator of
exposure and effects of DRCs.
In avian species, chicken CYP1As have been classified as
CYP1A4 and 1A5, based on differences in amino acid
sequences from mammalian CYP1A1 and 1A2, and phylogenetic analysis, showing that the chicken and mammalian
CYP1As form a separate branch in the CYP1A family tree
Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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DIOXIN-LIKE COMPOUNDS IN JUNGLE CROWS
(Gilday et al., 1996). Avian CYP1As have been so far cloned
and sequenced such as CYP1A4/5 in chicken (Gallus gallus)
(accession number X99453/X99454 [Gilday et al., 1996]),
CYP1A4/5 in common cormorant (Pharacrocorax carbo)
(Kubota et al., in preparation), CYP1A4/5 in herring gull
(Larus argentatus) (accession number AY233271/AY330876),
and CYP1A5 in turkey (Meleagris gallopavo) (accession
number AY964644).
There are striking interspecies differences in CYP1A induction to DRCs exposure even among avian species. Kennedy
et al. (1996) reported substantial differences in the sensitivity
of hepatocyte cultures from different avian species for CYP1A
induction. EC50 of EROD induction by 2378-T4CDD was in
the following order: herring gull (280 pg/ml) duck (200–610
pg/ml) turkey (200 pg/ml) > pheasant (45 pg/ml) > chicken
(4.5–15 pg/ml). In an ovo study, Sanderson and Bellward
(1995) reported that ED50 values (ng/g liver) of EROD activity
were about 1–2 for chicken, 20–30 for cormorant, and 30–50
for heron. In view of these large interspecies differences, it is
necessary to investigate the relationship of TEQ-CYP1A
expression in many other species.
Toxicokinetic behavior of DRCs in organisms is, at least
partly, dependent on expression and function of CYP1A, in
addition to chemical structure and number of chlorine substitution in each congener. Low chlorinated congeners such as
2378-T4CDD, 2378-T4CDF, 12378-P5CDD, and 33#44#-PCB
(CB-77) are easily metabolized by CYP1A1/2 in rat liver
microsomes (Hu and Bunce, 1999; Murk et al., 1994; Tai et al.,
1993). In avian species, CB-77 is rapidly metabolized by
hepatic microsomes of eider ducks exposed to CB-77 or
commercial PCB mixture Clophen A50, although wild common tern exposed to PCBs exhibited only limited metabolism
of CB-77 (Murk et al., 1994). In wild common cormorants,
concentrations of 2378-T4CDF and CB-77 in the liver exhibited no significant increase with growth, probably due to
rapid metabolism (Kubota et al., 2004).
PCDDs/DFs accumulate in hepatic tissue to a greater extent
than adipose tissue in rats and mice (Chen et al., 2001; De Vito
et al., 1998; Körner et al., 2002; Van den Berg et al., 1994). A
recent study using transgenic Cyp1a2 knockout mice demonstrated that CYP1A2 is responsible for the sequestration of
2378-T4CDD and 23478-P5CDF in hepatic tissue (Diliberto
et al., 1999). Another study showed that no difference in 2378T4CDD sequestration in liver was found between transgenic
Cyp1a1(-/-) knockout and Cyp1a1/1a2(þ/þ) wild-type mice,
indicating little contribution of CYP1A1 on the sequestration
(Uno et al., 2004). Our studies showed that liver to other tissues
distribution ratio of certain dioxin-like congeners increased
with the total TEQs or CYP1A in the livers of wild seals (Iwata
et al., 2004) and cormorants (Kubota et al., 2004, 2005). These
studies comprehended that the hepatic preference of congeners
is dependent upon the CYP1A induced by TEQs. Comparison
of the limited data available indicates the possibility of marked
species differences in the tissue-distribution of dioxin-like
385
congeners among many other organisms. For example, concentration ratios (liver/muscle or adipose tissue) of most
PCDD/DF and non-ortho PCB congeners were higher in seals
than those of cormorants.
The present study investigates contamination levels of DRCs
in liver and breast muscle of feral JCs from Tokyo, Japan,
and whether hepatic CYPs (mRNA, protein and enzymatic
activities) are induced by such contamination. To elucidate
congener-specific toxicokinetics related to CYP induction,
interactions of DRCs with hepatic CYP will be discussed in
terms of interspecies comparison.
MATERIALS AND METHODS
Sample collection. Thirteen male JCs were collected from Tokyo, Japan in
December 2002 by trapping method and with permission from the Tokyo
Metropolitan Government. JCs were euthanized under deep anesthesia with
ether. Breast muscle and liver samples were immediately removed after the
measurement of biometry. Both tissues were stored in a freezer at 20°C for
chemical analysis. Sub-samples of livers were frozen in liquid nitrogen, and
stored at 80°C until microsomal preparation for enzyme assay and total RNA
extraction.
Chemical analysis. Chemical analysis of DRCs was carried out following
the standard method of the Environmental Agency of Japan with some
modifications. About 2–4 g of liver and 20–35 g of muscle samples of JCs
were spiked with 13C-substituted PCDDs/DFs and Co-PCBs as internal
standards (Wellington Laboratories Inc., Guelph, Ontario, Canada), and
extracted with 1.5 molar ethanol-KOH for 1.5 h. The extract was treated with
sulfuric acid for clean-up and then added on to a multilayer column which in
turn was connected to a graphite carbon column. The multilayer column was
packed with anhydrous Na2SO4 (0.5 g), 20% AgNO3/silica gel (4 g), 44%
H2SO4/silica gel (6 g), silica gel (0.5 g), 2% KOH/silica gel (1 g), and
anhydrous Na2SO4 (0.5 g) from top to bottom. The silica gel (Spherical) and
graphite carbon columns (Supelclean ENVI-Carb) were supplied by Kanto
Chemical Co., Inc. (Tokyo, Japan) and Supelco (Bellefonte, PA), respectively.
The columns were connected together and eluted with hexane (90 ml), and the
multilayer silica gel column was removed. The graphite carbon column was
eluted with a mixture of 25% dichloromethane in hexane (90 ml) with a normal
flow. Both the eluates were pooled and passed through an activated basic
alumina column. Mono-ortho Co-PCBs were eluted from the alumina column
using 5% dichloromethane in hexane (40 ml) after discarding the first fraction
eluted with 20 ml hexane. The graphite column eluted with toluene (90 ml) in
reverse flow contained PCDDs/DFs and non-ortho Co-PCBs. For the recovery
check, 13C-labeled CB-138 prepared in decane was added into the final
solutions of mono-ortho Co-PCBs fraction, and 1234-T4CDF, 123469-H6CDF,
1234689-H7CDF and CB-138 into the PCDDs/DFs and non-ortho Co-PCBs
fraction. All the fractions were then micro-concentrated.
Identification and quantification of DRCs were performed using a highresolution gas chromatograph (HP 5890 or 6890, Hewlett-Packard, Wilmington,
DE) coupled with high-resolution mass spectrometric detector (JMS SX-102A,
JEOL JMS-700 or JEOL JMS-700D, JEOL, Tokyo, Japan) at a resolution of
>10,000 (10% valley). SP-2331 (0.20 lm film thickness, 0.25 mm i.d., 60 m
length, Supelco) capillary column was used for quantification of T4- to
H6CDDs/DFs (except 123789-H6CDF), and a SPB-50 (0.25 lm film thickness,
0.25 mm i.d., 30 m length, Supelco) column for H7- and O8CDD/DF and
123789-H6CDF. DB-5 MS (0.25 lm film thickness, 0.25 mm i.d., 60 m length,
J&W Scientific Inc., Folsom, CA) fused silica capillary column was used for
quantification of Co-PCBs. Mass spectrometric detector was operated at an EI
energy of 70 eV and ion current of 800 lA. PCDDs/DFs and Co-PCBs were
monitored by two most intensive ions of the molecular ion cluster, except for
386
WATANABE ET AL.
P5CDD by ions of [M]þ and [Mþ2]þ. All the congeners were quantified using
isotope dilution method to the corresponding 13C-labeled congeners, if isotope
ratio was within 15% of the theoretical value and peak area was more than
10 times to that of noise or procedural blank level. The quantification limits for
PCDDs/DFs and non-ortho Co-PCBs were 4.2–47 and 6.9–37 pg/g (lipid) in
the liver, and 0.54–4.9 and 1.1–3.5 pg/g (lipid) in the pectoral muscle. 2378T4CDD toxic equivalent (TEQ) was calculated using WHO bird-TEF (Van den
Berg et al., 1998).
Cloning of CYP1A5. Total RNA from livers of JCs was isolated using
RNAgent Total RNA Isolation System (Promega, Madison, WI). Poly(A)þ
RNA was purified by PolyATract mRNA Isolation Systems (Promega) or oligo
(dT) spin columns (mRNA Purification Kit; Amersham Biosciences, Piscataway, NJ). CYP1A from JC was cloned using a RT-PCR and RACE (Rapid
Amplification of cDNA Ends) methods. PCR primers were designed from
conserved regions of the chicken and herring gull CYP1A5. Primer sequences
were: Crow-f, 5#-TGGCAACCCKGCTGACTTCATC-3#; Crow-r, 5#-GAGGAGTGCCKGAACRYCTCCA-3; Crow-3#, 5#-GCCCTGTCCTGGAGCCTCAT GTATCTCG-3#; Crow-5#, 5#-CGAGATACATGAGGCTCCAGGACAGGGC-3#. PCR amplification was performed using Crow-f/Crow-r under the
following conditions: 105 s at 95°C, 30 cycles of 15 s at 95°C, 45 s at 50°C,
and 1 min at 72°C. For 3#- and 5#-RACE, double-stranded cDNAs were
synthesized using a Marathon cDNA Amplification kit (BD Biosciences, San
Jose, CA) with DNA polymerases of Advantage 2 PCR Enzyme System (BD
Biosciences) and TaKaRa LA Taq (Takara Bio Inc., Shiga, Japan), respectively.
Gene specific primers (Crow-3# and Crow-5# for 3#- and 5#-RACE, respectively) were coupled with adaptor primers for PCR. Amplification of cDNA
ends was performed under the following conditions: 30 s at 94°C, 5 cycles of
5 s at 94°C, 3 min at 72°C, 5 cycles of 5 s at 94°C, 3 min at 70°C, and 25 cycles
of 5 s at 94°C, 3 min at 68°C. The amplified cDNAs were sequenced using an
ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).
CYP1A amino acid sequence obtained in this study was aligned using
MacVector 7.1.
Quantitative real-time RT-PCR. Expression level of CYP1A mRNA was
acquired by quantitative real-time RT-PCR, which was performed with TaqMan
One-step RT-PCR Master Mix Reagent Kit (Applied Biosystems) using ABI
PRISM 7700 Sequence Detector (Applied Biosystems). For checking the
quality of total RNA, the bands of 28S and 18S in ribosomal RNAs from all
samples were confirmed by gel electrophoresis. Specific primers and probe for
CYP1A were designed by Primer Express software version 1 (PE Applied
Biosystems Inc., Foster City, CA). The 5#- and 3#-end nucleotides of the probe
were labeled with a reporter (FAM) and a quencher dye (TAMRA), respectively. The sequences of the PCR primer pairs and a labeled probe were as
follows: CYP1A forward primer, GACATCACCGACTCCCTCATTC; CYP1A
reverse primer, CAAAGAGGTCATTCACGAGGTTG; CYP1A probe, CAGTGCCTGGACAAAAAAGTGGAAACGA.
Primers and probe labeled with VIC for the endogenous control ribosomal
RNA (rRNA) were purchased from PE Applied Biosystems. Quantitative
values were obtained from the threshold PCR cycle number (Ct) at which the
increase in signal associated with an exponential growth of PCR products were
detected. The relative mRNA levels in each sample were normalized to its
ribosomal RNA content.
Reagents for enzymatic activities and immunoblotting. Reduced nicotinamide adenine dinucleotide phosphate (NADPH), glycerol, dithiothreitol,
and potassium chloride (KCl) were purchased from Nacalai Tesque Inc.
(Kyoto, Japan). Ethylenediaminetetraacetic acid (EDTA), hydrochloric acid
(HCl), and control goat sera were purchased from Wako Pure Chemical
Industries Ltd. (Osaka, Japan). Resorufin, methoxyresorufin, ethoxyresorufin,
pentoxyresorufin, and benzyloxyresorufin were purchased from Sigma
Chemical Co. (St. Louis, MO). Goat anti-rat CYP1A1, CYP2B1, CYP2C6
and rabbit anti-rat CYP3A2 antisera, and horseradish peroxidase (HRP)labeled anti-rabbit IgG were purchased from Daiichi Pure Chemicals Ltd.
(Tokyo, Japan). HRP-labeled anti-goat IgG was purchased from Funakoshi
Ltd. (Tokyo, Japan).
Preparation of microsomes. Hepatic microsomal fractions were prepared
according to the method of Guengerich (1982). Liver tissue (2.4–4.4 g) was
homogenized in 5 vol of cold homogenization buffer (50 mM Tris-HCl, 0.15 M
KCl, pH 7.4–7.5) with a teflon-glass homogenizer (10 passes), and centrifuged
for 10 min at 750 3 g. The nuclear pellets were removed, and the supernatant
was then centrifuged at 12,000 3 g for 10 min. The supernate was further
centrifuged at 105,000 3 g for 70 min. The supernatant (cytosol) fraction was
removed, and microsomal pellets were resuspended in 1 vol of resuspension
buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, 20% (v/v)
glycerol, pH 7.4–7.5). Protein concentration in microsomal fraction was
determined by the bicinchoninic acid method. Bovine serum albumin was
used as a standard (Smith et al., 1985).
Immunoblotting. Immunoblotting of liver microsomal fraction was performed as previously described (Iwata et al., 2002) with some modifications.
Protein (40 lg per lane) in microsomal fraction was resolved by electrophoresis
on a sodium dodecyl sulfate polyacrylamide gel (5–20% concentration
gradient; ATTO Co., Tokyo, Japan), and electrophoretically transferred to
a polyvinylidene fluoride (PVDF) membrane. The membranes were reacted
with the polyclonal antibodies against rat CYP1A1, CYP2B1, CYP2C6, or
CYP3A2, and then conjugated with a secondary antibody, anti-goat or -rabbit
IgG-HRP. Detection of the proteins cross-reacted with antibody was performed
using highly sensitive ECL Western blotting detection system (Amersham
Biosciences). The signal intensities of the bands were measured using
a ChemiDoc system (Bio-Rad Laboratories, Hercules, CA). Levels of CYP
proteins in individual animals were expressed as a relative value to staining
intensity from the antibody cross-reactive protein in one specimen.
Alkoxyresorufin-O-dealkylase
activity
assay. Methoxyresofurin-Odemethylase (MROD), ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD)
activities were measured with 2.0 lM substrate and 1.33 mM NADPH
concentrations using a spectrofluorometer (Spectra Fluor Plus, Tecan Group
Ltd., Maennedorf, Switzerland) by a modification of the method described by
Iwata et al. (2002). Approximately 1.0 mg/ml of microsomal protein was used
for the assay. Reactions were initiated by adding NADPH solution, and the
reaction mixture was incubated for 5 min at 37°C. Resorufin formed by the
reaction was detected by excitation wavelength 535 nm and the emission
wavelength 595 nm.
Antibody inhibition of PROD activity. The hepatic microsomal sample in
which relatively high PROD activity was recorded was used for inhibition test.
The microsomes were preincubated with polyclonal antibodies against rat
CYP1A1, 2B1, 2C6, 3A2, or control sera for 30 min at room temperature prior
to the initiation of reaction by adding NADPH. 0, 10, 30, and 50 ll of antisera
were added to 50, 40, 20, and 0 ll of control serum, respectively and PROD
assay was performed in the same method as described.
Statistical analysis. Measurement of alkoxyresorufin-O-dealkylation activities, immunoblotting and antibody inhibition test were done in duplicate.
Quantification of mRNA was conducted in triplicate. Mean values were used for
following statistical analyses. Correlation analyses were carried out by
Spearman’s rank correlation test. Mann-Whitney U-test was applied for
detecting statistical differences among groups. These statistical analyses were
performed using StatView ver. 5.0 (SAS Institute Inc., Cary, NC). For samples
with values below quantification limit, half of the respective limit was
substituted to calculate mean concentrations, SDs, total PCDDs/DFs/Co-PCBs
values and TEQs. Relationships between ratios of congener concentrations in
the liver to those in the breast muscle and CYP1A5 mRNA or CYP1A-like
protein levels were examined when individual congeners were detected both
in the liver and pectoral muscle in more than five specimens. The value of
p < 0.05 was regarded as statistically significant.
387
DIOXIN-LIKE COMPOUNDS IN JUNGLE CROWS
RESULTS
Concentrations and Liver-Muscle Concentration Ratios
The concentrations of PCDDs/DFs in the liver and breast
muscle of JCs were from 100 to 1900, and from 21 to 180 pg/g
(lipid), respectively (Table 1). Concentrations of total Co-PCBs
in the liver and muscle of JCs were 16–130 and 18–220 ng/g
(lipid), respectively. Total TEQ concentrations in the liver and
muscle of JCs were 23–280 pg/g (lipid) (1.1–12 pg/g [wet]) and
5.6–78 pg/g (lipid) (0.15–3.5 pg/g [wet]), respectively. The
TEQs in the livers were mostly lower than those of other bird
species from North American, Asian, and European countries
TABLE 1
Concentration (pg/g lipid) and TEQ (pg/g lipid) of PCDDs/DFs and Co-PCBs in the Liver
and Breast Muscle of JCs from Tokyo, Japan
Tissue
Liver
Lipid (%)
PCDDs
2378-T4CDD
12378-P5CDD
123478-H6CDD
123678-H6CDD
123789-H6CDD
1234678-H7CD
O8CDD
PCDFs
2378-T4CDF
12378-P5CD
23478-P5CDF
123478-H6CD
123678-H6CDF
123789-H6CDF
234678-H6CD
1234678-H7CDF
1234789-H7CDF
O8CDF
Non-ortho Co-PCBs
33#44#-T4CB (77)
344#5-T4CB (81)
33#44#5-P5CB (126)
33#44#55#-H6CB (169)
Mono-ortho Co-PCBs
233#44#-P5CB (105)
2344#5-P5CB (114)
23#44#5-P5CB (118)
2#344#5-P5CB (123)
233#44#5-H6CB (156)
233#44#5#-H6CB (157)
23#44#55#-H6CB (167)
233#44#55#-H7CB (189)
Total PCDDs
Total PCDFs
Total non-ortho Co-PCBs
Total mono-ortho Co-PCBs
PCDDs-TEQs
PCDFs-TEQs
Non-ortho Co-PCBs-TEQs
Mono-ortho Co-PCBs-TEQs
Total TEQs
Breast muscle
4.9 ± 0.73 (3.9–6.5)
2.8 ± 0.97 (1.5–4.5)
5.8
32
63
55
8.0
80
230
2.5
9.5
7.9
9.0
1.3
5.4
8.7
±
±
±
±
±
±
±
3.4 (<4.2–12)
28 (<6.9–74)
58 (<8.3–180)
46 (<12–140)
4.1 (<8.0–18)
60 (<9.3–180)
200 (18–630)
±
±
±
±
±
±
±
1.6 (<0.94–5.3)
9.3 (<1.4–36)
6.4 (1.6–21)
7.0 (<2.4–26)
0.93 (<1.2–4.0)
4.3 (<2.1–16)
6.1 (<2.6–21)
<24a
<22a
45 (<7.9–140)
48 (<13–140)
70 (<14–220)
<39a
37 (<8.5–110)
31 (<9.2–91)
4.4 (<8.1–18)
<47a
2.7
2.0
1.4
2.6
±
±
±
±
2.3
1.1
1.1
2.7
19 ± 16 (<7.2–61)
<37a
8.8 ± 6.4 (<6.9–23)
230 ± 190 (24–610)
14
2.1
20
41
±
±
±
±
9.8 (3.2–39)
1.3 (<1.1–4.4)
12 (4.2–35)
28 (14–94)
2800
2600
34,000
430
20,000
500
1900
6200
44
27
77
74,000
13
8.4
2.9
3.6
27
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
3100 (460–12,000)
1500 (570–6400)
31,000 (7200–130,000)
380 (130–1500)
12,000 (3200–46,000)
3200 (870–11,000)
1300 (420–5300)
6100 (450–22,000)
34 (9.4–130)
16 (11–56)
46 (23–170)
53,000 (18,000–220,000)
11 (1.6–42)
5.3 (2.2–20)
1.7 (0.72–6.0)
2.2 (0.74–8.9)
20 (5.6–78)
56 ±
62 ±
85 ±
47 ±
43 ±
8.2 ±
2400
2600
30,000
700
18,000
4700
1500
6000
470
330
260
65,000
43
82
2.7
3.1
130
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
2000 (360–6900)
1200 (510–4400)
19,000 (7100–78,000)
410 (190–1500)
8300 (2800–29,000)
2500 (820–8800)
790 (430–3000)
6000 (370–21,000)
380 (42–1200)
230 (55–710)
200 (38–660)
33,000 (16,000–130,000)
35 (7.5–91)
60 (11–190)
1.7 (0.93–7.1)
1.5 (0.65–5.1)
96 (23–280)
Note. Values are means ± SD. Numbers in parentheses indicate the range.
a
All the samples had concentrations below quantification limit.
6.3 ± 4.6
5.3 ± 3.7
4.0 ± 2.8
<2.7a
<2.5a
(<1.5–17)
(<2.5–13)
(<2.0–9.1)
<4.4a
(<1.2–8.4)
(<1.3–4.3)
(<1.2–4.7)
(<1.5–9.9)
388
WATANABE ET AL.
(Giesy et al., 1994; Koistinen et al., 1997; Kubota et al., 2004;
Senthilkumar et al., 2002a,b, 2005).
Congener profiles of PCDD/DF concentrations revealed that
12378-P5CDD, 123478-H6CDD, 123678-H6CDD, and O8CDD
were equally predominant in the muscle, and O8CDD was
notably accumulated in the liver (Fig. 1). In the case of nonortho Co-PCBs, CB-169 was more predominant in liver (59–
95%) than muscle (35–69%). Among mono-ortho congeners,
CB-118 (30–61% in muscle, 31–60% in liver) and CB-156
(18–37% in muscle, 18–38% in liver) were predominant
congeners.
Comparison of lipid-normalized congener concentrations
between liver and breast muscle showed that concentrations of
123478-H6CDD, 123678-H6CDD, 1234678-H7CDD, O8CDD,
23478-P5CDF, 123478-H6CDF, 123678-H6CDF, CB-169, and
CB-123 were significantly higher in the liver than those in the
breast muscle ( p < 0.05). In contrast, no statistical difference in
concentration between liver and muscle was found for CB-77
and mono-ortho Co-PCBs except CB-123 (Fig. 1). Table 2
shows mean and range of lipid-normalized concentration ratios
of DRCs between liver and breast muscle (L/M ratios) in JCs.
The ratios tended to increase with the number of chlorine
substitution of PCDD/DF and non-ortho Co-PCB congeners,
but no such a trend has been observed for mono-ortho CoPCBs. In addition, Figure 2 exhibits comparison between the
ratios in JCs and those in cormorants (Kubota et al., 2004). All
the L/M ratios of PCDD/DF and non-ortho Co-PCB congeners
analyzed were higher in JCs than those of cormorants.
As for congener profiles of TEQs, sum of 12378-P5CDD
and 23478-P5CDF accounted for 33–76% and 26–70% to
total TEQs in the liver and muscle of JCs, respectively.
CB126, which is a dominant congener in many avian species
(Senthilkumar et al., 2002a), contributed only 4.3–14% and
0.21–2.3% to total TEQs in the muscle and liver, respectively,
whereas contributions of PCDDs/DFs were 63–89% and 84–
98% in the muscle and liver, respectively.
Isolation of CYP1A5 in JC
Full-length open reading frame of CYP1A cDNA of JC was
1596 bp (531 aa). 173 bp of 5#-UTR and 130 bp of 3#-UTR
with a polyA tail were also cloned. The predicted molecular
mass was 60.3 kDa (Fig. 3). In the alignment of the amino acid
sequence, JC CYP1A cloned was most closely related to the
cormorant CYP1A5 (81%; Kubota et al., in preparation) and
shared 74, 73, and 66% of overall amino acid identities with
chicken CYP1A5, cormorant CYP1A4 and chicken CYP1A4,
respectively. This new full-length CYP from JC has been
designated as JC CYP1A5 by Dr. D. Nelson (University of
Tennessee), and has been submitted to GenBank with Accession No. AB220967.
CYP Isozymes Responsible for AROD Activities
Alkoxyresorufin-O-dealkylation (AROD) activities in the
liver microsomes of JCs were characterized by high MROD
(350 ± 160 [mean ± SD] pmol/min/mg prot.) and EROD (130 ±
40 pmon/min/mg prot.) activities followed by PROD (3.6 ± 1.0
pmol/min/mg prot.) or BROD (6.5 ± 3.4 pmol/min/mg prot.)
activities.
CYP1A-, 2B-, 2C-, and 3A-like proteins were immunochemically detected using anti-rat CYP polyclonal antibodies
in hepatic microsomes of JCs. Figure 4 represents the results
of Western blot analyses. Regarding CYP1A-like proteins, two
bands with higher (HMW) and lower molecular weights (LMW)
FIG. 1. Concentrations of PCDDs/DFs and Co-PCBs in liver and breast muscle of jungle crows from Tokyo. Triangles and circles show average concentrations
in liver and muscle of jungle crows, respectively. Bars indicate the range of concentrations. Dotted line indicates that there were specimens in which congeners
were not detected. ND means that congeners were not detected in all specimens. * and ** show that the concentrations in liver were significantly higher than those
of muscle at a level of p < 0.05 and p < 0.01, respectively.
DIOXIN-LIKE COMPOUNDS IN JUNGLE CROWS
TABLE 2
Liver to Pectoral Muscle Concentration Ratios of DRCs in JCs
Liver/muscle concentration ratio
Congener
PCDDs
2378-T4CDD
12378-P5CDD
123478-H6CDD
123678-H6CDD
123789-H6CDD
1234678-H7CDD
O8CDD
PCDFs
2378-T4CDF
12378-P5CDF
23478-P5CDF
123478-H6CDF
123678-H6CDF
123789-H6CDF
234678-H6CDF
1234678-H7CDF
1234789-H7CDF
O8CDF
Non-ortho Co-PCBs
33#44#-T4CB (77)
344#5-T4CB (81)
33#44#5-P5CB (126)
33#44#55#-H6CB (169)
Mono-ortho Co-PCBs
233#44#-P5CB (105)
2344#5-P5CB (114)
23#44#5-P5CB (118)
2#344#5-P5CB (123)
233#44#5-H6CB (156)
233#44#5#-H6CB (157)
23#44#55#-H6CB (167)
233#44#55#-H7CB (189)
na
Mean
Range
4
10
10
10
0
10
11
2.7
3.8
8.7
6.8
NA
14
26
1.7–4.3
0.51–7.7
0.94–19
1.0–18
NA
1.9–20
3.3–47
0
0
11
10
10
0
6
5
1
0
NA
NA
9.8
10
20
NA
16
20
11
NA
NA
NA
1.3–19
1.2–16
1.7–29
NA
1.4–24
3.2–29
11
NA
9
0
2
13
1.7
NA
0.61
5.2
0.64–3.6
NA
0.54–0.68
0.47–17
13
13
13
13
13
13
13
13
0.78
0.92
0.82
1.6
0.80
0.76
0.74
0.84
0.19–1.2
0.29–1.6
0.24–1.2
0.36–2.5
0.27–1.2
0.29–1.1
0.25–1.1
0.27–1.4
Note. NA, no data available.
The number of specimens in which the particular congener was detected
both in the liver and pectoral muscle.
a
were recognized. Table 3 shows rho values of Spearman’s rank
correlations among expression levels of CYP1A5 mRNA,
CYP1A-, 2B-, 2C-, and 3A-like proteins and AROD activities
in liver microsomes of JC. CYP1A5 mRNA expression levels
had a significant positive correlation with protein level of
HMW CYP1A and not with LMW CYP1A. As for other CYP
proteins, anti-rat polyclonal antisera of other CYP isozymes
emerged as a single band. Table 3 presents that PROD and
BROD activities had higher positive correlations with HMW
CYP1A and 3A-like proteins than other AROD activities and
CYP isozyme protein levels, implying the involvement of these
CYP isozymes in PROD or BROD activities.
An antibody inhibition test of PROD activity was conducted
using anti-rat CYP1A1, 2B1, 2C6, and 3A2 antisera to further
specify the CYP isozyme responsible for the catalytic activity
389
(Fig. 5). Magnitude of inhibition of PROD activity was in the
following order: anti-rat CYP1A1 > 3A2 > 2B1 > 2C6 at lower
dose of antibody, and anti-rat CYP3A2 > 1A1 > 2B1 > 2C6 at
the highest dose of antibody. Anti-rat CYP3A2 and CYP1A1
antisera inhibited PROD activity by 43 and 44% at 30 ll
serum, and by 84 and 34% at 50 ll serum, respectively.
Although anti-rat CYP3A2 antisera showed dose-dependent
inhibition of PROD activity, the inhibition rate by anti-rat
CYP1A1 antisera was constant at any dose of the serum.
CYP Induction by TEQs
Relationships between TEQ of each congener and CYP1A5
mRNA, CYP protein contents or AROD activities were shown
in Table 4. Hepatic total TEQs in JCs exhibited significant
positive correlations with CYP1A5 mRNA, HMW CYP1A- or
CYP3A-like protein expression levels, and PROD and BROD
activities, whereas MROD and EROD activities, and CYP2B/
2C-like protein expression levels revealed no significant
positive correlations with hepatic TEQ concentrations. Some
of these relationships were also shown in Figure 6.
Individual TEQs (wet) derived from all the PCDDs/DFs,
CB-169, CB156, CB-157, and CB189 had significant ( p <
0.05) positive correlation with CYP1A5 mRNA or HMW
CYP1A-like protein contents (Table 4). We conducted the
correlation analysis using TEQ expressed on lipid weight basis
(data not shown) also, which showed a similar result in the case
of TEQ on wet weight basis. Table 5 shows relationships
between expression levels of CYP1A5 mRNA, CYP1A-, 2B-,
2C-, or 3A-like proteins, M-, E-, P-, or BROD activity in livers
and TEQ (wet) of each congener in muscle of JCs. In contrast
to TEQs in the livers, TEQ of most congeners in the muscle
showed no significant positive correlation with hepatic
CYP1A5 mRNA and HMW CYP1A protein contents.
Hepatic Metabolism and Sequestration of Dioxin-Like
Congeners by Induced CYP1A
To investigate the metabolic potential of each congener by
hepatic CYP of JCs, correlation analyses were conducted
between expression levels of CYP1A5 mRNA, CYP1A-, 2B-,
2C-, or 3A-like protein, M-, E-, P-, or BROD activities and
concentration ratios of each congener to CB-169 which was
shown to be resistant to CYP metabolism (Guruge and Tanabe,
1997). The ratios of several Co-PCB congeners revealed
significant ( p < 0.05) negative correlations with CYP1A5
mRNA and HMW CYP1A protein levels (Table 6).
Table 7 shows relationships between the L/M ratios of each
congener and expression levels of CYP1A5 mRNA or HMW
CYP1A-like proteins. The L/M ratios of individual PCDD/DF
congeners and CB-169 increased with an increase in hepatic
CYP1A5 mRNA or HMW CYP1A protein expression levels
(Table 7). The L/M ratios for higher chlorinated congeners
were greater than those for lower chlorinated congeners. In
contrast, no elevation was found in the L/M ratios for CB-77
390
WATANABE ET AL.
FIG. 2. Concentration ratios of PCDD/DF and Co-PCB congeners in livers to those in muscle of jungle crows. Average (plots) and range (bars) are shown.
Data on cormorants is taken from Kubota et al. (2004). * and ** show that the ratios of jungle crows were significantly higher than those of cormorants at a level of
p < 0.05 and p < 0.01, respectively.
and mono-ortho Co-PCBs, which were nearly equal to 1.0 in
all specimens.
DISCUSSION
In this study, concentrations of DRCs in the liver and muscle
of JCs were analyzed. The TEQs in the livers were mostly
lower than those of other bird species so far reported. Lower
TEQs and higher contributions of PCDDs/DFs to total TEQs in
JCs are consistent with observations in terrestrial birds from the
Great Lakes (Jones et al., 1993). These results in terrestrial
birds were contrastive to those in fish-eating birds. Efficient
trophic transfer of Co-PCBs in aquatic food web would reduce
the proportion of PCDDs/DFs in fish-eating birds (Giesy et al.,
1994; Jones et al., 1993; Wan et al., 2005). As a unique feature
of congener profile in JC, 2378-T4CDD, 123789-H6CDD,
2378-T4CDF, 12378-P5CDF, 123789-H6CDF, O8CDF, CB81, and CB-126, which were accumulated in a variety of avian
species in Japan (Senthilkumar et al., 2002a, 2005), were not
detected in most livers. This implies a high metabolic potential
of these congeners in JC liver.
L/M ratios in each specimen increased with the number of
chlorine substitution of PCDD/DF and non-ortho Co-PCB
congeners (Table 2, Fig. 2). Several laboratory studies have
demonstrated that PCDDs/DFs and non-ortho Co-PCBs are
sequestered in the rodent liver following their subchronic
exposures (Chen et al., 2001; De Vito et al., 1998; Körner et al.,
2002; Van den Berg et al., 1994). A recent study revealed
that CYP1A2 is a target binding protein responsible for hepatic sequestration of 2378-T4CDD and related compounds
(Diliberto et al., 1999). Regarding wildlife, our studies also
exhibited that distribution ratios of certain dioxin-like congeners between liver and other tissues increased with the total
TEQs in the livers of seals (Iwata et al., 2004) and cormorants
(Kubota et al., 2004), suggesting that the hepatic preference of
congeners is dependent on CYP1A induced by TEQs. Considering these reports, CYP1A induced by TEQs may be involved
in the hepatic sequestration of PCDDs/DFs and CB-169 in JCs.
In addition, comparison of the L/M ratios in JCs with our
previous data on cormorants (Kubota et al., 2004) revealed that
all the L/M ratios of PCDD/DF and non-ortho Co-PCB
congeners analyzed were higher in JCs than those of cormorants (Fig. 2), implying higher sequestration capacity of JC liver
than that of cormorant.
JC CYP1A5 cDNA was cloned and sequenced in this study.
Highly conserved amino acid motif (FxxGxxxCxG) with the
heme-binding cysteine was maintained in JC (Fig. 3). Amino
acid residues (FGAGFDT) in which threonine is located in the
center of a-helix I (Edwards et al., 1989) at the oxygen-binding
pocket, and is part of proton delivery network (Imai et al.,
1989; Yeom et al., 1995) were also conserved. The proline-rich
region (PPGP), which plays a key role in protein folding, was
observed in the JC CYP1A5. A hydrophobic N-terminal
sequence that anchors the protein to endoplasmic reticulum
membrane was longer than that of mammalian or fish CYP1A
enzymes, and similar in length to that of chicken (Gilday et al.,
1996).
The hepatic CYP1A-, 2B-, 2C-, and 3A-like proteins of JCs
were immunochemically detected using anti-rat CYP polyclonal antibodies in hepatic microsomal fractions. Regarding
CYP1A-like proteins, two bands with higher (HMW) and
lower molecular weight (LMW) were recognized (Fig. 4). This
implies the presence of CYP1A5- and CYP1A4-like proteins as
DIOXIN-LIKE COMPOUNDS IN JUNGLE CROWS
391
FIG. 3. The nucleotide and deduced amino acid sequence for JC CYP1A5. The start codon (ATG) and stop codon (TGA) for translation are underlined. The
heme-binding ligand (FxxGxxxCxG), the center of a-helix I (FGAGFDT) and the proline-rich region (PPGP) are boxed.
previously suggested in chicken (Gilday et al., 1996). CYP1A5
mRNA expression levels acquired by quantitative real-time RTPCR had a significant positive correlation with protein level of
HMW CYP1A (Table 3) but no correlation with LMW protein,
implying that HMW CYP1A protein is CYP1A5.
JC livers showed high MROD and EROD activities followed
by PROD and BROD activities. Such a catalytic profile was
similar to that in herring gull (Verbrugge et al., 2001). Not only
EROD but PROD activity was also found to increase by
treatment with b-naphthoflavone (BNF) or 3-methylcholanthrene (3-MC) in mallard ducks (Leffin and Riviere, 1992),
black-crowned night heron (Rattner et al., 1993), and herring
gull (Verbrugge et al., 2001). In addition, induction of BROD
activity by BNF was observed in black-crowned night heron
(Rattner et al., 1993) and chicken (Verbrugge et al., 2001).
Correlation analysis suggested that TEQ induce hepatic PROD
activities in avian species (Bosveld et al., 1995, 2000; Guruge
and Tanabe, 1997; Kubota et al., 2005). Together with these
392
WATANABE ET AL.
FIG. 5. Result of antibody inhibition of PROD activity in hepatic
microsome of a jungle crow. The reactions were carried out in the presence of
varying volumes of control serum, anti-rat CYP1A1, 2B1, 2C6, and 3A2 serum.
FIG. 4. Results of Western blot analyses of jungle crow hepatic microsomes using anti-rat CYP1A1, 2B1, 2C6, and 3A2 serum. *HMW and LMW
mean higher and lower molecular weight, respectively.
Considering the effective levels of biochemical responses in
other avian species, the TEQs in JC livers were found to be
lower than the ED50 (1–2 ng/g liver) of hepatic EROD
induction in chicken (Sanderson and Bellward, 1995), and
comparable with lowest-observed-effect-level (LOEL) of aryl
hydrocarbon hydroxylase (AHH) induction (10 pg/g egg) in
chicken embryo (Poland and Glover, 1973) and EC50 (4.5–
15 pg/ml) of EROD induction in chicken hepatocyte cultures
(Kennedy et al., 1996). Although total TEQ in the liver of JCs
(5.9 ± 4.0 pg/g [wet]) were comparable or lower than the
estimated LOEL of CYP1A induction in chicken embryo,
hepatic CYP1A induction by TEQ was suggested in JC (Table
4 and Fig. 6). These results imply that JC may be as sensitive to
TEQ exposure as chicken. On the other hand, the previous life
history of these birds is unknown. Differences in habitat and
food consumption may have an impact on background CYP1A
results, our data revealing higher positive correlations between
PROD or BROD activities and expression levels of CYP1A5
mRNA or HMW CYP1A (Table 3) clearly suggests that avian
CYP1A is responsible for PROD and BROD activities, and the
substrate specificity is different between birds and mammals;
PROD and BROD activities are specifically catalyzed by
phenobarbital-induced CYP2B in rat, not by 3-MC-induced
CYP1A (Burke et al., 1994). The antibody inhibition test of
PROD activity indicate that CYP3A and CYP1A may be
responsible for PROD activity in JCs, but CYP2B and CYP2C
isozymes may be less potential. Interestingly, MROD activities
had a significant positive correlation with the expressions of
CYP2C-like protein. However, to our knowledge, there is no
supporting information on the involvement of CYP2C in
MROD activity.
TABLE 3
Spearman’s Rank Correlations (rho Values) among Expression Levels of CYP1A5 mRNA, CYP1A-, 2B-, 2C-, and 3A-like
Proteins and Alkoxyresorufin-O-dealkylation Activities in Liver Microsomes of JC
Protein content
HMW CYP1Aa
LMW CYP1Aa
CYP2B
CYP2C
CYP3A
Alkoxyresorufin-O-dealkylation
MROD
EROD
PROD
BROD
CYP1A5 mRNA
HMW CYP1A
LMW CYP1A
CYP2B
CYP2C
0.70*
0.080
0.37
0.33
0.67*
0.56
0.50
0.11
0.52
0.59*
0.57
0.24
0.36
0.16
0.76**
0.37
0.14
0.51
0.74*
0.099
0.38
0.72*
0.53
0.30
0.38
0.41
0.069
0.082
0.016
0.22
0.18
0.65*
0.31
0.041
0.42
a
HMW and LMW indicate higher and lower molecular weight, respectively.
*p < 0.05.
**p < 0.01.
CYP3A
MROD
EROD
PROD
0.49
0.12
0.50
0.75*
0.39
0.008
0.54
0.78**
0.31
0.72*
TABLE 4
Spearman’s Rank Correlations between Relative Expression Levels of CYP1A5 mRNA, CYP1A-, 2B-, 2C-, or 3A-like Proteins,
M-, E-, P-, or BROD Activities and TEQ (Wet) of Each Congener in JC Livers
CYP1A5 mRNA
Congener
na
0.51
0.72
0.77
na
0.55
0.61
na
na
0.52
0.69
0.70
na
0.60
0.55
na
na
0.47
na
na
0.65
p
0.075
0.012
0.0076
0.057
0.034
0.072
0.017
0.012
0.038
0.059
0.10
r
na
0.60
0.67
0.71
na
0.57
0.65
na
na
0.65
0.73
0.64
na
0.63
0.58
na
na
p
0.038
0.021
0.014
0.048
0.025
0.025
0.011
0.026
0.030
0.046
LMW CYP1Aa
r
na
0.088
0.033
0.016
na
0.093
0.030
na
na
0.17
0.099
0.044
na
0.038
0.10
na
na
0.016
0.019
na
na
0.072
0.14
0.36
0.40
0.45
0.60
0.53
0.37
0.63
0.63
0.21
0.17
0.13
0.039
0.066
0.20
0.030
0.58
0.67
0.52
0.55
0.66
0.044
0.021
0.074
0.057
0.022
0.024
0.45
na
na
0.69
0.098
0.31
0.32
0.28
0.54
0.60
0.38
0.74
0.73
0.28
0.28
0.33
0.060
0.038
0.19
0.010
0.51
0.55
0.53
0.47
0.58
0.075
0.055
0.069
0.11
0.044
0.12
p
0.76
0.91
0.95
0.75
0.92
0.57
0.73
0.88
0.89
0.72
CYP2B
r
CYP2C
p
r
na
0.28
0.39
0.35
na
0.099
0.19
na
0.34 0.50
0.18 0.56
0.23 0.59
na
0.73 0.65
0.51 0.63
na
na
0.26
0.26
0.24
na
0.10
0.017
na
na
na
na
0.42
0.55
0.64
na
0.61
0.65
na
na
0.36
0.38
0.41
0.72
0.95
CYP3A
p
0.087
0.052
0.040
0.025
0.029
0.14
0.055
0.026
0.035
0.024
0.32 0.25
na
na
0.42 0.51
0.38
0.80
0.29
na
na
0.23
0.19
0.15
0.18
0.24
0.063
0.099
0.15
0.094
0.51
0.61
0.54
0.41
0.83
0.73
0.60
0.75
0.17
0.23
0.14
0.085
0.28
0.23
0.19
0.28
0.55
0.42
0.64
0.77
0.34
0.42
0.52
0.34
0.18
0.080
0.72
0.13
0.45
0.57
0.25
0.51
0.033
0.15
0.016
0.13
0.077
0.91
0.59
0.95
0.66
0.79
0.23
0.25
0.34
0.31
0.17
0.44
0.39
0.24
0.29
0.56
0.50
0.46
0.26
0.30
0.51
0.95
r
MROD
p
r
na
0.64
0.70
0.77
na
0.70
0.72
na
0.026 0.36
0.016 0.57
0.0077 0.42
na
0.016 0.41
0.012 0.45
na
na
0.54
0.71
0.73
na
0.71
0.73
na
na
na
na
0.31
0.41
0.52
na
0.39
0.46
na
na
0.060
0.014
0.011
0.014
0.011
EROD
p
0.22
0.050
0.14
0.15
0.12
0.29
0.15
0.071
0.18
0.12
0.14
na
na
0.33
0.64
0.53 0.006 0.98
0.78
0.20 0.49
0.80
0.25 0.39
0.65
0.18 0.54
0.12
0.59 0.041
0.049 0.60 0.039
0.38
0.40 0.17
0.080 0.57 0.048
0.28
0.008
0.18
0.13
0.44
0.50
0.030
0.55
0.083
0.11
0.36
0.30
0.077
0.37
0.33
0.17
0.33
0.33
0.076
0.31
na
na
0.66
0.65
0.60
0.39
0.46
0.65
0.28
0.022
0.024
0.038
0.18
0.11
0.025
na
0.30
0.15
0.19
na
0.19
0.074
na
na
0.36
0.24
0.15
na
0.24
0.23
na
na
0.30
0.59
0.52
0.51
0.80
0.21
0.41
0.61
0.41
0.43
na
0.66
0.55
0.58
na
0.57
0.55
na
na
0.65
0.62
0.56
na
0.57
0.57
na
na
p
0.022
0.057
0.044
0.048
0.056
0.024
0.031
0.052
0.048
0.050
r
na
0.66
0.73
0.76
na
0.70
0.72
na
na
0.57
0.68
0.73
na
0.58
0.60
na
na
p
0.022
0.011
0.0086
0.016
0.013
0.050
0.019
0.011
0.044
0.039
0.55 0.058
na
na
0.63 0.028
0.33
0.79 0.0064
0.98
0.52 0.069
0.55
0.72 0.013
0.66
0.79 0.0061
0.13
0.23 0.43
0.085 0.006 0.98
0.92
0.51 0.075
0.055 0.028 0.92
0.66
0.63
0.73
0.82
0.52
0.33
0.61
0.37
0.023
0.030
0.011
0.0046
0.073
0.26
0.034
0.20
0.20
0.31
0.37
0.40
0.52
0.46
0.37
0.52
0.49
0.29
0.21
0.17
0.071
0.11
0.20
0.071
0.20
0.25
0.57
0.25
0.25
0.63
0.68
0.59
0.56
0.65
0.029
0.019
0.042
0.055
0.024
0.65
0.62
0.57
0.43
0.62
0.024
0.032
0.050
0.13
0.032
0.29
0.36
0.32
0.32
0.31
0.50
r
0.51 0.075
na
na
0.63 0.030
0.26
0.20
na
na
0.26
p
BROD
0.37
0.31
0.22
0.27
0.26
0.28
393
Note. na indicates no analysis was conducted due to below the detection limit of concentrations in more than 50% of samples.
a
HMW and LMW indicates higher and lower molecular weight, respectively.
r
PROD
DIOXIN-LIKE COMPOUNDS IN JUNGLE CROWS
PCDDs
2378-T4CDD
12378-P5CDD
123478-H6CDD
123678-H6CDD
123789-H6CDD
1234678-H7CDD
O8CDD
PCDFs
2378-T4CDF
12378-P5CDF
23478-P5CDF
123478-H6CDF
123678-H6CDF
123789-H6CDF
234678-H6CDF
1234678-H7CDF
1234789-H7CDF
O8CDF
Non-ortho Co-PCBs
33#44#-T4CB (77)
344#5-T4CB (81)
33#44#5-P5CB (126)
33#44#55#-H6CB (169)
Mono-ortho Co-PCBs
233#44#-P5CB (105)
2344#5-P5CB (114)
23#44#5-P5CB (118)
2#344#5-P5CB (123)
233#44#5-H6CB (156)
233#44#5#-H6CB (157)
23#44#55#-H6CB (167)
233#44#55#-H7CB
(189)
PCDDs
PCDFs
Non-ortho Co-PCBs
Mono-ortho Co-PCBs
Total TEQs
r
HMW CYP1Aa
394
WATANABE ET AL.
FIG. 6. Relationships between TEQ concentrations and relative expression levels of CYP1A5 mRNA, HMW CYP1A or 3A-like protein, EROD, PROD, and
BROD activities in jungle crow livers.
expression level. Additional unknown compounds may alter
hepatic CYP level and thus lead to a significant, but low
correlation ( p < 0.05). The significant positive correlation of
CYP3A with TEQ may be due to the parallel accumulation
pattern of TEQ and CYP3A inducers.
Individual TEQs derived from PCDDs/DFs, CB-169,
CB156, and CB189 had significant positive correlation with
HMW CYP1A protein contents (Table 4). Almost the same
trend was found in CYP1A5 mRNA. Relatively higher R and
lower P values of PCDDs/DFs than those of mono-ortho CoPCBs indicate that PCDDs/DFs are mainly involved in the
induction of CYP1A(5). CB-77 showed no significant positive
correlation with expression levels of CYP1A5 mRNA and
HMW CYP1A, which is probably due to rapid biotransformation of this congener. An in vitro study using hepatic microsomes prepared from two avian species exposed to PCBs
demonstrated that the metabolism of CB-77 depends on
CYP1A induction (Murk et al., 1994). No significant positive
correlation between TEQ of most congeners in the muscle and
hepatic CYP1A5 mRNA or HMW CYP1A protein contents
(Table 5) suggest that concentrations of DRCs in extra-hepatic
tissues are less reflected by hepatic CYP1A expression levels.
Concentration ratios of several Co-PCB congeners to CB169 revealed significant negative correlations with CYP1A5
mRNA and HMW CYP1A protein levels (Table 6). This
implies high potential of CYP1A(5) to metabolize these
congeners in JC. The ratios of CB-77 especially had a strong
negative correlation ( p < 0.01) with HMW CYP1A protein
contents, suggesting rapid metabolism of CB-77 by the induced
CYP1A in JC. This may be somewhat surprising since CYP1A,
which is induced by low levels of DRCs, might be responsible
for the metabolism of some congeners. Murk et al. (1994)
reported that CB-77 is rapidly metabolized by hepatic microsomes of eider ducks exposed to 50 mg/kg body weight of CB77, although the exposed concentration was very high.
Metabolism of CB-77 has also been proposed in wild
cormorant (Guruge et al., 1997, 2000; Kubota et al., 2004,
2005; Senthilkumar et al., 2005), common tern (Bosveld et al.,
2000), and bald eagle (Senthilkumar et al., 2002b) with high
TEQs.
The L/M ratios of multiple PCDD/DF congeners and CB169 increased with an increase in hepatic CYP1A5 mRNA or
HMW CYP1A protein expression levels (Table 7). The slopes
for higher chlorinated congeners revealed a tendency to be
TABLE 5
Spearman’s Rank Correlations between Relative Expression Levels of CYP1A5 mRNA, CYP1A-, 2B-, 2C-, or 3A-like Proteins, M-, E-, P-,
or BROD Activities in Livers and TEQ (Wet) of Each Congener in Muscle of JCs
CYP1A5 mRNA
Congener
p
0.26
0.099
0.32
0.29
na
0.003
0.029
0.36
0.73
0.27
0.32
na
na
0.18
0.10
0.058
na
0.16
na
na
na
0.99
0.92
0.54
0.73
0.84
0.59
r
0.40
0.14
0.23
0.18
na
0.014
0.17
na
na
0.052
0.033
0.055
na
0.011
na
na
na
LMW CYP1Aa
p
r
p
0.16
0.63
0.43
0.53
0.055
0.28
0.39
0.39
na
0.34
0.19
0.85
0.33
0.18
0.18
0.96
0.55
0.86
0.91
0.85
0.97
na
na
0.16
0.34
0.25
na
0.15
na
na
na
0.23
0.52
0.57
0.24
0.39
0.59
CYP2B
r
0.055
0.30
0.11
0.19
na
0.25
0.24
na
na
0.38
0.41
0.30
na
0.29
na
na
na
p
CYP2C
r
0.58
0.65
0.77
0.70
na
0.40 0.64
0.41 0.54
0.85
0.30
0.72
0.51
0.19
0.15
0.30
0.31
na
na
0.28
0.62
0.66
na
0.62
na
na
na
CYP3A
MROD
p
r
p
r
0.043
0.024
0.0075
0.015
0.47
0.48
0.58
0.54
na
0.41
0.45
0.10
0.097
0.043
0.0062
0.028
0.062
0.33
0.031
0.022
0.031
na
na
0.16
0.36
0.42
na
0.36
na
na
na
0.16
0.12
0.59
0.21
0.14
0.21
0.45
0.28
0.44
0.36
na
0.30
0.20
na
na
0.047
0.23
0.30
na
0.28
na
na
na
EROD
p
r
0.12
0.33
0.13
0.22
0.15
0.16
0.055
0.12
na
0.036
0.055
0.29
0.49
0.87
0.43
0.30
0.34
na
na
0.40
0.055
0.044
na
0.014
na
na
na
PROD
p
0.59
0.57
0.85
0.67
0.90
0.85
0.17
0.85
0.88
0.96
r
0.40
0.28
0.17
0.25
na
0.15
0.30
na
na
0.29
0.088
0.15
na
0.025
na
na
na
p
0.17
0.33
0.57
0.39
0.61
0.30
0.32
0.76
0.61
0.93
BROD
r
p
0.38
0.27
0.33
0.33
na
0.26
0.28
0.19
0.36
0.25
0.26
na
na
0.022
0.061
0.16
na
0.077
na
na
na
0.37
0.33
0.94
0.83
0.58
0.79
0.35
0.35
0.43
0.094
0.23
0.23
0.14
0.75
0.50
0.33
0.47
0.12
0.083
0.25
0.10
0.69
0.23
0.041
0.017
0.14
0.44 0.14
0.89 0.30
0.95 0.072
0.63
0.27
0.62
0.30
0.80
0.35
0.022
0.17
0.27
0.46
0.94
0.55
0.36
0.11
0.31
0.31
0.47
0.23
0.29
0.28
0.11
0.42
0.027
0.38
0.26
0.10
0.92
0.19
0.37
0.72
0.76
0.38
0.56
0.12
0.0086
0.19
0.052
0.68
0.84
0.48
0.66
0.14
0.0038 0.48
0.099
0.47
0.023
0.48
0.62
0.049
0.094
0.11
0.099
0.86
0.15
0.26
0.13
0.011
0.35
0.38
0.063
0.58
0.17
0.21
0.42
0.30
0.036
0.6
0.37
0.65
0.97
0.23
0.19
0.83
0.043
0.56
0.47
0.15
0.30
0.90
0.096
0.29
0.23
0.31
0.48
0.40
0.20
0.50
0.20
0.071
0.47
0.39
0.18
0.74
0.31
0.42
0.29
0.097
0.17
0.48
0.083
0.48
0.80
0.11
0.18
0.53
0.15
0.033
0.097
0.28
0.10
0.039
0.049
0.18
0.28
0.21
0.022
0.044
0.11
0.61
0.91
0.74
0.33
0.72
0.89
0.86
0.54
0.34
0.47
0.94
0.88
0.70
0.42
0.99
0.92
0.76
0.74
0.67
0.70
0.97
0.40
0.14
0.91
0.91
0.46
0.17
0.058
0.17
0.14
0.20
0.33
0.027
0.52
0.70
0.38
0.25
0.10
0.43
0.55
0.84
0.56
0.62
0.50
0.25
0.92
0.071
0.015
0.19
0.38
0.72
0.14
0.030
0.15
0.061
0.11
0.31
0.34
0.099
0.57
0.54
0.18
0.26
0.26
0.34
0.92
0.60
0.83
0.71
0.28
0.24
0.73
0.048
0.062
0.53
0.37
0.37
0.24
0.31
0.0030
0.25
0.23
0.29
0.34
0.18
0.57
0.36
0.027
0.25
0.12
0.082
0.28
0.92
0.39
0.42
0.32
0.24
0.53
0.050
0.22
0.92
0.39
0.68
0.78
0.77
0.70
0.78
0.83
0.43
0.35
0.68
0.093
0.099
0.32
0.58
0.57
0.38
0.0078
0.016
0.0072
0.0040
0.14
0.23
0.019
0.75
0.73
0.26
0.044
0.050
0.19
0.59
0.60
0.64
0.74
0.51
0.47
0.52
0.39
0.28
0.20
0.66
0.54
0.39
0.041
0.038
0.027
0.011
0.080
0.10
0.071
0.18
0.33
0.49
0.022
0.062
0.18
0.77
0.40
0.55
0.46
0.32
0.22
0.83
0.098
0.29
0.94
0.11
0.40
0.54
0.23
0.003
0.030
0.088
0.096
0.12
0.11
0.011
0.24
0.43
0.033
0.033
0.21
395
Note. na indicates no analysis was conducted due to below the detection limit of concentrations in more than 50% of samples.
a
HMW and LMW indicates higher and lower molecular weight, respectively.
0.085
0.25
0.17
0.21
0.29
0.36
0.060
0.48
0.31
0.022
0.47
0.24
0.18
DIOXIN-LIKE COMPOUNDS IN JUNGLE CROWS
PCDDs
2378-T4CDD
12378-P5CDD
123478-H6CDD
123678-H6CDD
123789-H6CDD
1234678-H7CDD
O8CDD
PCDFs
2378-T4CDF
12378-P5CDF
23478-P5CDF
123478-H6CDF
123678-H6CDF
123789-H6CDF
234678-H6CDF
1234678-H7CDF
1234789-H7CDF
O8CDF
Non-ortho Co-PCBs
33#44#-T4CB (77)
344#5-T4CB (81)
33#44#5-P5CB (126)
33#44#55#-H6CB (169)
Mono-ortho Co-PCBs
233#44#-P5CB (105)
2344#5-P5CB (114)
23#44#5-P5CB (118)
2#344#5-P5CB (123)
233#44#5-H6CB (156)
233#44#5#-H6CB (157)
23#44#55#-H6CB (167)
233#44#55#-H7CB (189)
PCDDs
PCDFs
Non-ortho Co-PCBs
Mono-ortho Co-PCBs
Total TEQs
r
HMW CYP1Aa
396
TABLE 6
Spearman’s Rank Correlations between Relative Expression Levels of CYP1A5 mRNA, CYP1A-, 2B-, 2C-, or 3A-like Proteins, M-, E-, P-,
or BROD Activities and Concentration Ratios of each Congener to CB-169 in JC Livers
CYP1A5 mRNA
Congener
na
0.050
0.44
0.13
na
0.094
0.34
na
na
0.37
0.12
0.29
na
0.083
0.11
na
na
p
0.86
0.13
0.65
0.75
0.24
0.20
0.69
0.31
0.78
0.72
r
na
0.35
0.15
0.17
na
0.20
0.22
na
na
0.39
0.20
0.14
na
0.25
0.40
na
na
p
0.23
0.59
0.56
0.48
0.45
0.18
0.49
0.62
0.38
0.16
LMW CYP1Aa
r
na
0.34
0.13
0.29
na
0.63
0.24
na
na
0.10
0.24
0.50
na
0.60
0.60
na
na
p
CYP2B
r
na
0.022
0.40
0.13
na
0.029
0.58
0.41
0.093
0.25
0.66
0.31
0.72
0.41
0.087
0.038
0.039
CYP2C
p
r
na
0.18
0.38
0.35
na
0.044 0.46
0.75 0.43
0.94
0.16
0.66
CYP3A
p
0.53
0.19
0.23
0.11
0.13
r
na
0.21
0.41
0.16
na
0.43
0.56
na
na
0.41
0.016
0.12
na
0.45
0.54
na
na
na
na
0.16
0.069
0.95 0.20
0.69 0.49
na
0.12 0.36
0.063 0.25
na
na
na
na
0.81 0.088
0.49
0.20
0.092 0.38
na
0.21
0.32
0.40
0.18
na
na
MROD
p
r
EROD
p
r
na
0.40
0.75
0.41
na
0.14 0.088
0.052 0.37
na
0.17
0.12
0.0091 0.15
0.16
0.32
na
0.76
0.12
0.20
0.088
na
na
0.022
0.51
0.72
na
0.19
0.066
na
na
na
na
0.13
0.20
0.30
na
0.038
0.14
na
na
0.46
0.16
0.58
0.76
0.49
0.19
0.27
0.54
0.32
na
na
—
0.26
0.77
na
na
—
0.0077
0.50
na
na
—
0.083
0.21
na
na
—
0.46
0.28
na
na
—
0.34
0.48
na
na
—
0.094
0.67
0.44
0.62
0.61
0.46
0.22
0.64
0.036
0.020
0.13
0.033
0.035
0.11
0.45
0.026
0.90
0.58
0.62
0.66
0.71
0.60
0.61
0.67
0.27
0.044
0.032
0.022
0.014
0.036
0.035
0.020
0.35
0.060
0.20
0.066
0.10
0.14
0.42
0.12
0.24
0.83
0.48
0.82
0.72
0.62
0.14
0.69
0.40
0.51
0.24
0.32
0.29
0.15
0.082
0.31
0.038
0.077
0.41
0.26
0.32
0.59
0.78
0.28
0.89
0.46
0.43
0.54
0.61
0.45
0.14
0.42
0.061
0.11
0.14
0.063
0.034
0.12
0.63
0.14
0.83
0.43
0.52
0.49
0.64
0.57
0.29
0.49
0.21
0.13
0.52
0.074 0.060
0.090 0.35
0.027 0.41
0.050 0.071
0.32 0.12
0.090 0.38
0.47 0.35
Note. na indicates no analysis was conducted due to below the detection limit of concentrations in more than 50% of samples.
a
HMW and LMW indicate higher and lower molecular weight, respectively.
0.94
0.080
0.013
0.51
0.82
0.027 0.92
na
na
—
0.071
0.83
0.22
0.15
0.80
0.69
0.19
0.22
PROD
p
r
BROD
p
r
na
0.69 0.008 0.98
0.61 0.11 0.70
0.26 0.34 0.23
na
0.69 0.088 0.76
0.76 0.23 0.43
na
0.24
0.56
0.12
na
0.31
0.48
na
na
0.083
0.17
0.14
na
0.008
0.19
na
na
na
na
0.13
0.20
0.38
na
0.13
0.072
na
na
0.65
0.49
0.30
0.89
0.64
0.41
na
na
—
0.15 0.56
na
na
—
0.34
0.049
0.19
0.027
0.049
0.31
0.044
0.46
0.24
0.86
0.51
0.92
0.86
0.29
0.88
0.11
0.061
0.36
0.17
0.35
0.39
0.51
0.39
0.52
0.78
0.55
0.63
0.98
0.52
0.052 0.30
na
na
—
0.83
0.21
0.55
0.22
0.18
0.080
0.18
0.073
0.38
0.38
0.39
0.51
0.42
0.29
0.54
0.28
p
0.41
0.054
0.68
0.29
0.093
0.65
0.49
0.19
0.65
0.80
0.30
0.19
0.19
0.18
0.075
0.14
0.31
0.063
0.33
WATANABE ET AL.
PCDDs
2378-T4CDD
12378-P5CDD
123478-H6CDD
123678-H6CDD
123789-H6CDD
1234678-H7CDD
O8CDD
PCDFs
2378-T4CDF
12378-P5CDF
23478-P5CDF
123478-H6CDF
123678-H6CDF
123789-H6CDF
234678-H6CDF
1234678-H7CDF
1234789-H7CDF
O8CDF
Non-ortho Co-PCB
33#44#-T4CB (77)
344#5-T4CB (81)
33#44#5-P5CB (126)
33#44#55#-H6CB (169)
Mono-ortho Co-PCBs
233#44#-P5CB (105)
2344#5-P5CB (114)
23#44#5-P5CB (118)
2#344#5-P5CB (123)
233#44#5-H6CB (156)
233#44#5#-H6CB (157)
23#44#55#-H6CB (167)
233#44#55#-H7CB (189)
r
HMW CYP1Aa
397
DIOXIN-LIKE COMPOUNDS IN JUNGLE CROWS
TABLE 7
Relationships between the Concentration Ratios of Each Congener (Liver/Breast Muscle), and Relative Expression
Levels of CYP1A5 mRNA and HMW CYP1A-like Protein in Hepatic Microsomes in JCs
CYP1A5 mRNA
Congener
PCDDs
2378-T4CDD
12378-P5CDD
123478-H6CDD
123678-H6CDD
123789-H6CDD
1234678-H7CDD
O8CDD
PCDFs
2378-T4CDF
12378-P5CDF
23478-P5CDF
123478-H6CDF
123678-H6CDF
123789-H6CDF
234678-H6CDF
1234678-H7CDF
1234789-H7CDF
O8CDF
Non-ortho Co-PCBs
33#44#-T4CB (77)
344#5-T4CB (81)
33#44#5-P5CB (126)
33#44#55#-H6CB (169)
Mono-ortho Co-PCBs
233#44#-P5CB (105)
2344#5-P5CB (114)
23#44#5-P5CB (118)
2#344#5-P5CB (123)
233#44#5-H6CB (156)
233#44#5#-H6CB (157)
23#44#55#-H6CB (167)
233#44#55#-H7CB (189)
n
NA
10
10
10
NA
10
11
NA
NA
11
10
10
NA
6
5
NA
NA
CYP1A protein
aa
b
r2
p
aa
b
r2
p
1.4
3.1
3.1
0.45
1.3
0.59
0.62
0.54
0.59
0.025
0.39
0.26
0.37
1.0
0.95
1.1
1.3
0.21
0.62
0.68
0.79
0.056
0.025
0.028
3.3
9.9
6.8
3.4
0.83
0.83
0.045
0.0045
1.4
2.3
5.3
10
0.65
0.63
0.010
0.016
3.6
3.1
6.3
1.5
3.9
6.3
0.73
0.50
0.61
0.0074
0.098
0.015
0.98
1.2
1.6
3.0
3.6
9.8
0.76
0.55
0.32
0.0089
0.034
0.13
9.8
12
2.4
1.4
0.76
0.89
0.28
0.046
2.0
2.7
4.3
2.3
0.57
0.90
0.064
0.072
0.75
0.24
0.52
0.043
2.0
0.043
0.67
0.99
0.60
0.011
0.89
0.32
0.76
0.0061
0.49
0.59
0.46
0.98
0.47
0.47
0.37
0.39
0.26
0.21
0.37
0.28
0.37
0.37
0.53
0.51
0.12
0.069
0.067
0.072
0.069
0.069
0.026
0.041
0.024
0.033
0.039
0.048
0.034
0.031
0.039
0.039
0.63
0.72
0.58
1.3
0.58
0.57
0.49
0.60
0.11
0.13
0.27
0.10
0.24
0.25
0.36
0.23
0.13
0.11
0.094
0.28
0.11
0.11
0.040
0.095
9
NA
NA
13
0.39
3.0
13
13
13
13
13
13
13
13
0.14
0.16
0.17
0.30
0.16
0.14
0.18
0.22
Note. NA: no correlation analysis could be carried out because measurable concentration ratios were found only in less than five samples.
Simple regression analysis was conducted; liver/muscle concentration (lipid) ratios ¼ a 3 [relative expression levels of CYP1A5 mRNA or HMW CYP1Alike protein] þ b.
a
greater than those for lower chlorinated congeners, whereas no
elevation was found in the L/M ratios for CB-77 and monoortho Co-PCBs. These results suggest congener-specific hepatic sequestrations by the induced CYP1A(5) in JCs. In rats
and mice, dose-dependent increases in liver retention have
been reported for several congeners, including 2378substituted PCDDs/DFs, CB-126, and CB-169 (Chen et al.,
2001; De Vito et al., 1998; Körner et al., 2002; Van den Berg
et al., 1994). A previous study on the toxicokinetics of monoortho Co-PCBs in mice showed no dose-dependent hepatic
sequestration for several mono-ortho Co-PCBs (De Vito et al.,
1998). A study using CYP1A2 knockout mice provided direct
evidence that CYP1A2 is responsible for the sequestration of
2378-T4CDD and 23478-P5CDF in hepatic tissue (Diliberto
et al., 1999). A more recent study demonstrated no altered
2378-T4CDD accumulation in Cyp1a1(-/-) knockout mice liver
(Uno et al., 2004). These reports suggest that hepatic sequestration is caused by CYP1A2, and not by CYP1A1, in mammalian species. The present study indicated that CYP1A5
might be involved in hepatic sequestration of certain DRCs
in JC. Comparing the amino acid sequences of chicken
CYP1A4/5 isoforms with those of mammalian CYP1A isoforms, neither chicken CYP1A4 nor CYP1A5 appears to be
directly orthologous to CYP1A1 or CYP1A2 (Gilday et al.,
1996). On the other hand, the CYP1A5 is involved mainly in
arachidonic acid epoxygenation (Nakai et al., 1992; Rifkind
et al., 1994), which is preferentially catalyzed by mammalian
CYP1A2 (Jacobs et al., 1989; Lambrecht et al., 1992).
398
WATANABE ET AL.
Furthermore, Handly-Goldstone and Stegeman (in press)
suggested that avian and mammalian CYP1A paralog pairs
might have resulted from a single gene duplication event, and
that concerted evolution has obscured orthologous relationships. Since the phylogenetic analysis of substrate recognition
sites 2–4 of CYP1A revealed that there is no evidence that
CYP1As have undergone conversion, they proposed that
chicken CYP1A4 and CYP1A5 are orthologous to mammalian
CYP1A1s and CYP1A2s, respectively. Considering that avian
CYP1A4s and CYP1A5s may be orthologous to mammalian
CYP1A1s and CYP1A2s, respectively, it is consistent that JC
CYP1A5 is involved in hepatic sequestration of DRCs. In
addition, we could find significantly greater hepatic sequestrations of PCDD/DF and some Co-PCB congeners in JCs than
those in cormorant (Fig. 2). Further research is necessary to
characterize species- and isoform-specific function of avian
CYP1As, particularly in terms of metabolism and hepatic
sequestration toward DRCs.
ACKNOWLEDGMENTS
We thank Prof. An. Subramanian, Ehime University, for critical reading of
this manuscript. Financial assistance was provided by ‘‘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), and for
Exploratory Research (no. 13027101), and by ‘‘21st Century COE Program’’
from the Ministry of Education, Culture, Sports, Science and Technology,
Japan. Conflict of interest: none declared.
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