Recombinant Transthyretin Purification and Competitive Binding with Organohalogen Compounds in Two Gull Species (Larus argentatus and Larus hyperboreus)

Article (PDF Available)inToxicological Sciences 107(2):440-50 · December 2008with54 Reads
DOI: 10.1093/toxsci/kfn240 · Source: PubMed
Abstract
Glaucous gulls (Larus hyperboreus) from Svalbard, Norway (marine), and herring gulls (Larus argentatus) from the Laurentian Great Lakes (freshwater) of North America are differentially exposed to persistent and bioaccumulative anthropogenic contaminants, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ether (PBDE) flame retardants and metabolic products. Such compounds can potentially perturb hormone transport via binding interactions with proteins such as transthyretin (TTR, prealbumin). In this present study, we isolated, cloned and sequenced TTR cDNA from the brain and liver of two species (herring and glaucous gull), which, to our knowledge, is the first report describing the TTR nucleic acid and amino acid sequences from any gull species. Identical TTR nucleotide and amino acid sequences were obtained from both gull species (liver and brain). Recombinant TTR (rTTR) was expressed and purified, and determined as a monomer of 18 kDa and homodimer of 36 kDa that putatively is comprised of the two protein monomers. Concentration dependent, competitive TTR-binding curves with each of the natural TTR ligands 3,5,3'-triiodothyronine (T(3)) and thyroxine (T(4)) were generated as well as by treatment with a range of concentrations (10(-3)-10(5)nM) of 2,2',3,4',5,5',6-heptaCB (CB187), 2,2',4,4'-tetrabromoDE (BDE47), and hydroxyl- (OH) and methoxyl (MeO)-containing analogs (i.e., 4-OH-CB187, 6-OH-BDE47, 4'-OH-BDE49, 4-MeO-CB187, and 6-MeO-BDE47). Relative to the nonsubstituted BDE47 and CB187 and their MeO-substituted analogs, the OH-substituted analogs all had lower K(i) and K(d) values, indicating greater affinity and more potent competitive binding to both T(3) and T(4). The OH-substitution position and/or the diphenyl ether substitution of the four bromine atoms resulted in more potent, greater affinity, and greater relative potency for 4'-OH-BDE49 relative to 6-OH-BDE47. CB187 was more comparable in binding potency and affinity to 4-OH-CB187, then was 6-OH-BDE47 and 4'-OH-BDE49 relative to BDE47 where the binding potency and affinity was several orders of magnitude greater for 6-OH-BDE47 and 4'-OH-BDE49. This indicated that the combination of the more thyroid hormone-like brominated diphenyl ether backbone (relative to the chlorinated biphenyl backbone), and in combination of having an OH-group, results in a more effective competitive ligand on gull TTR relative to both T(3) and T(4). Known circulating levels of 4-OH-CB187, 6-OH-BDE47, and 4'-OH-BDE49 in the plasma of free-ranging Svalbard glaucous gulls were comparable to the concentration of in vitro competitive potency of T(3) and T(4) with gull TTR. These results suggest that environmentally relevant and selected OH-containing PCB, and to a lesser extent PBDE congeners have the potential to be physiologically effective in these gull species via perturbation of T(4) and T(3) transport.
TOXICOLOGICAL SCIENCES 107(2), 440–450 (2009)
doi:10.1093/toxsci/kfn240
Advance Access publication November 25, 2008
Recombinant Transthyretin Purification and Competitive Binding
with Organohalogen Compounds in Two Gull Species
(Larus argentatus and Larus hyperboreus)
Francisco Uca´n-Marı´n,*
,
Augustine Arukwe, Anne Mortensen, Geir W. Gabrielsen,§ Glen A. Fox,*
and Robert J. Letcher*
,
,1
*National Wildlife Research Centre, Carleton University, Ottawa, Ontario, K1A 0H3, Canada; Department of Chemistry, Carleton University, Ottawa, Ontario
K1S 5B6, Canada; Department of Biology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway; and §Norwegian Polar
Institute, Tromsø NO-9296, Norway
Received July 11, 2008; accepted November 14, 2008
Glaucous gulls (Larus hyperboreus) from Svalbard, Norway
(marine), and herring gulls (Larus argentatus) from the Lau-
rentian Great Lakes (freshwater) of North America are differen-
tially exposed to persistent and bioaccumulative anthropogenic
contaminants, such as polychlorinated biphenyls (PCBs) and
polybrominated diphenyl ether (PBDE) flame retardants and
metabolic products. Such compounds can potentially perturb
hormone transport via binding interactions with proteins such as
transthyretin (TTR, prealbumin). In this present study, we
isolated, cloned and sequenced TTR cDNA from the brain and
liver of two species (herring and glaucous gull), which, to our
knowledge, is the first report describing the TTR nucleic acid and
amino acid sequences from any gull species. Identical TTR
nucleotide and amino acid sequences were obtained from both
gull species (liver and brain). Recombinant TTR (rTTR) was
expressed and purified, and determined as a monomer of 18 kDa
and homodimer of 36 kDa that putatively is comprised of the two
protein monomers. Concentration dependent, competitive TTR-
binding curves with each of the natural TTR ligands 3,5,3#-
triiodothyronine (T
3
) and thyroxine (T
4
) were generated as well
as by treatment with a range of concentrations (10
23
–10
5
nM) of
2,2#,3,4#,5,5#,6-heptaCB (CB187), 2,2#,4,4#-tetrabromoDE (BDE47),
and hydroxyl- (OH) and methox yl (MeO)-containing analogs (i.e.,
4-OH-CB187, 6-OH-BDE47, 4#-OH-BDE49, 4-MeO-CB187, and
6-MeO-BDE47). Relative to the nonsubstituted BDE47 and CB187
and their MeO-substituted analogs, the OH-substituted analogs all
had lower K
i
and K
d
values, indicating greater affinity and more
potent competitive binding to both T
3
and T
4
. The OH-substitution
position and/or the diphenyl ether substitution of the four bromine
atoms resulted in more potent, greater affinity, and greater relative
potency for 4#-OH-BDE49 relative to 6-OH-BDE47. CB187 was more
comparable in binding potency and affinity to 4-OH-CB187, then was
6-OH-BDE47 and 4#-OH-BDE49 relative to BDE47 where the
binding potency and affinity was several orders of magnitude greater
for 6-OH-BDE47 and 4#-OH-BDE49. This indicated that the
combination of the more thyroid hormone–like brominated diphenyl
ether backbone (relative to the chlorinated biphenyl backbone), and in
combination of having an OH-group, results in a more effective
competitive ligand on gull TTR relative to both T
3
and T
4
.Known
circulating levels of 4-OH-CB187, 6-OH-BDE47, and 4#-OH-BDE49
in the plasma of free-ranging Svalbard glaucous gulls were
comparable to the concentration of in vitro competitive potency of
T
3
and T
4
with gull TTR. These results suggest that environmentally
relevant and selected OH-containing PCB, and to a lesser extent
PBDE congeners have the potential to be physiologically effective in
these gull species via perturbation of T
4
and T
3
transport.
Key Words: thyroid hormones; Larid birds; transthyretin cloning
and expression; competitive binding; PCBs; PBDEs and
metabolites.
Transthyretin (TTR), albumin (ALB), and thyroid binding
globulin (TBG) are the major hormone transport proteins of
thyroid hormones (THs) in all vertebrates (McKinnon et al.,
2005). These transport proteins bind to THs, and specifically
3,5,3-triidothyronine (T
3
) and thyroxine (T
4
), and circulate in
the blood. In vertebrates, the three TH carrier proteins are
synthesized by the liver, but only TTR is synthesized in the
brain (Dickson et al., 1987). Among vertebrate species there
are differences in the relative importance in terms of levels of
circulating levels of TTR, ALB, and TBG. In avian species, the
study of TTR has been mainly in the context of comparative
evolution relative to other taxa (Power et al., 2002; Richardson
et al., 1994). In contrast to humans, it has been shown that
TTRs from teleost fish (Yamauchi et al., 1999), amphibians
(Prapunpoj et al., 2000; Yamauchi et al., 1998), reptiles
(Prapunpoj et al., 2002), and birds (Chang et al., 1999) bind T
3
with higher affinity than T
4
.
In birds, two main groups of hormones are involved in the
growth of birds, growth hormones (GHs) together with the
associated insulin-like growth factors, and T
4
and T
3
. Normal
growth of posthatching birds requires GHs, T
4
, and T
3
(Decuypere et al., 2005). T
4
and especially active T
3
are also
1
To whom correspondence should be addressed at Department of
Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada. Fax:
(613) 998-0458. E-mail: robert.letcher@ec.gc.ca.
The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For permissions, please email: journals.permissions@oxfordjournals.org
critical in the regulation of metabolic functions and thermo-
genesis in birds, especially basal metabolic rate and cold-
induced thermogenesis (Silva, 1995). Therefore, circulating TH
levels can be perturbed by temperature stress. For example,
Ku¨hn and Nouwen (1978) reported that in domestic fowl
(Rhode Island Red strain), a gradual decline in ambient
temperature from 26.5 to 17.5C elevated serum T
3
in 40-day-
old chicks. Increases in T
4
occurred when the temperature was
lowered further to 12.5C. Physiological processes perturb
circulating TH levels and TH-dependent processes. THs are
very lipophilic molecules, and in the absence of TH distributor
proteins, they interact as carriers from serum into lipid
membranes (Ekins, 1990). The effect of THs on protein and
lipid metabolism is a biphasic nature: in low physiological
concentrations they are anabolic, whereas at higher concen-
trations they are catabolic (Decuypere et al., 2005).
A major concern is that TH-dependent processes, such as TH
transport, are susceptible to chemical stress and can be
disrupted by thyroidogenic, xenobiotic compounds accumu-
lated in an organism (Ishihara et al., 2003a, b). Such chemical
stressors include polychlorinated biphenyl (PCB) and poly-
brominated diphenyl ether (PBDE) flame retardant congeners as
well as hydroxylated (OH) analogs such as OH-PCBs and OH-
PBDEs. In vitro and in vivo studies with nonavian species have
reported that congeners of these contaminants can have effects
on TH-dependent processes (Hakk and Letcher, 2003; Legler
and Brouwer, 2003; Letcher et al., 2000; Meerts et al., 2000).
The metabolism of PCBs and particularly PBDE flame
retardants are not well understood in wildlife and particularly in
birds (Hakk and Letcher, 2003; Letcher et al., 2000). However,
putative OH-PCB and OH-PBDE metabolites have been
reported in the tissues of certain avian species. Several OH-
PCB and OH-PBDE congeners, and to a much lesser extent
methoxylated (MeO)-PBDEs, were recently quantified in the
plasma of adult glaucous gulls (Larus hyperboreus) from the
Norwegian Arctic (Verreault et al., 2005a, b), and the plasma
of bald eaglets (Haliaeetus leucocephalus) from the west coast
of North America (McKinney et al., 2006). In the plasma of
Norwegian glaucous gulls, 2,2#,4,4#-tetrabromo diphenyl ether
(BDE-47), 6-OH-BDE47, 4#-OH-2,2#,4#,5-tetrabromo diphenyl
ether (4#-OH-BDE49), 3#-MeO-BDE47, 4#-MeO-BDE49, and
most important among the OH-PCBs, 4-OH-2,2#,3,4#,5,5#,6-
heptachloro biphenyl (4-OH-CB187), tend to dominate. The
presence of OH-PCBs in the plasma of birds and other wildlife is
more than likely due to oxidative cytochrome P450 (CYP)–
mediated PCB biotransformation. However, both MeO-PBDEs
and some OH-PBDE congeners can also bioaccumulate in
aquatic food webs as natural products produced by marine
organisms such as sponges and algae (Malmva¨rn et al.,2005).
Thyroid system disrupting chemicals may target any of the
multiple pathways in a chemical-dependent manner, including
TH production, receptor binding, metabolism, and interaction
with transport proteins such as TTR (Ulrich, 2003). Thyroi-
dogenic activity via interaction with human TTR has been
reported for such organohalogen contaminants as congeners of
PCBs, PBDE flame retardants, OH-PCBs, and/or OH-PBDEs.
Several PCB, PBDE, OH-PCB, and/or OH-PBDE congeners
have been shown to competitively displace T
4
from human
TTR, which consequently can result in the release of (free) T
4
,
which can enhance T
4
metabolism and excretion (Brouwer
et al., 1998; Meerts et al., 2000; Purkey et al., 2004).
Neurological effects of PCBs and PBDEs have been reported in
mammals, and may be partly explained by the ability of these
compounds (or their metabolites) to decrease TH levels during
a sensitive time periods, for example, of neurodevelopment
(Costa and Giordano, 2007; Zoeller et al., 2002). Regardless of
origin from natural source accumulation of via metabolism of
PBDEs, thyroidogenic and estrogenic dysfunction have been
reported in laboratory rats exposed to OH-PBDEs (Meerts
et al., 2000, 2001).
To our knowledge, the binding affinity of natural T
4
and T
3
ligands to avian TTR transport protein has not been reported,
and especially the affinity to anthropogenic contaminants and/
or metabolite ligands such as OH-PCBs and OH-PBDEs
(McNabb, 2005). In the present study, brain and liver tissues of
two gull species from the genus Larus , glaucous gull from the
Norwegian Arctic and herring gull from the Laurentian Great
Lakes of North America were used to isolate TTR cDNA.
Isolated TTR cDNA were cloned and sequenced. The
recombinant TTR (rTTR) were expressed and purified, then
the rTTR protein was expressed and the product used in
a competitive binding determination assays. Comparisons were
performed on T
3
and T
4
with selected PCB, PBDE, OH-PCB,
OH-PBDE, and analogous MeO-containing congeners pre-
viously reported and known for their environmental importance
in the plasma of birds.
MATERIALS AND METHODS
Gull liver and brain tissue samples. Liver and brain samples were
collected in 2003 from herring gulls from Lake Ontario from colonies at
Hamilton Harbour and Scotch Bonnet Island. Tissue samples were collected in
cryogenic vials and immediately frozen and liquid nitrogen (LN2) and stored in
LN2 at Environment Canada’s National Wildlife Specimen Bank in Ottawa
(Carleton University, Canada). Since the early 1970#s, herring gulls from the
Laurentian Great Lakes have been used to monitor trends in levels and effects
of organochlorine contaminants (Mineau et al., 1984). The present herring gull
liver and brain samples were collected as part of the Great Lakes Herring Gull
Monitoring Program (GLHGMP) administered by the Canadian Wildlife
Service (Environment Canada). All aspects of the sample collections for the
GLHGMP have been approved by Environment Canada, and conform to all
animal handling guidelines. Liver and brain samples from glaucous gulls,
collected in 2002 and 2004 at Bear Island (7422#N1905#E; Norwegian
Arctic) (Verreault et al., 2004), were immediately frozen in LN2, and stored in
an ultra deep-freezer (80C) until analysis. All field methods employed in this
study were approved by the Governor of Svalbard (2002/00483-2 a. 512/2) and
the Norwegian National Animal Research Authority (S1030/02). The capture
and handling methods of glaucous gulls were approved by the Norwegian
National Animal Research Authority (P.O. Box 8147 Dep., NO-0033 Oslo,
Norway) and the Governor of Svalbard (Box 633, NO-9171 Longyearbyen,
Norway).
GULL TRANSTHYRETIN AND ORGANOHALOGEN BINDING
441
Chemicals and reagents. Trizol reagent for RNA purification, TA-cloning
kit with pCR2.1 vector and InVision His-tag In-gel staining were purchased from
Invitrogen (Carlsbad, CA). IScript cDNA synthesis kit and iTaq DNA
polymerase were purchased from Bio-Rad Laboratories (Hercules, CA). The
pET28a expression vector was obtained from Novagen (Madison, WI) and BL21-
RIPL Escherichia coli cells from Stratagene (La Jolla, CA). His-Trap-FF crude
Kit containing 1 ml columns were from GE Healthcare (Chalfort St. Giles, UK).
Human TTR (human pre-ALB, 98% pure) was purchased from Sigma-Aldrich
(Mississauga, ON, Canada).
The PCB, PBDE, OH-PCB, and OH-PBDE substrates used in the
competitive TTR–binding studies with T
4
and T
3
were those found to dominate
in the plasma of Norwegian glaucous gull (Verreault et al., 2005a, b). These
substrates were CB-187 from AccuStandard Chemical Reference Standards
(> 99% purity); and BDE-47 and the structurally analogous 4-OH-CB187,
6-OH-BDE47, 4#-OH-BDE49, 4-MeO-CB187, and 6-MeO-BDE47, which
were purchased from Cambridge Isotope Laboratories (Cambridge, MA, purity
99% in nonane). All chemicals used were of high purity, high-performance
liquid chromatography grade.
RNA isolation, cDNA synthesis, and PCR. The PCR primers were iden-
tified previously as an amino acid sequence set using the BLAST National
Center for Biotechnology Information (NCBI) using traces of chicken (Gallus
gallus) and using Primer3 (v. 0.4.0; http://mit.edu/), and the primer sequence
was purchased from Custom Oligonucleotide Synthesis (Biosearch Technologies,
Inc., Novato, CA). Total RNA was purified using frozen (80C preserved) brain
and liver tissues from herring gull and glaucous gull homogenized in Trizol
reagent according to the manufacturer’s protocol. The quality of the RNA was
determined by agarose-formamide gel electrophoresis, and RNA concentrations
were determined using a NanoDrop Spectrophotometer (NanoDrop Technologies,
Wilmington, DE). Total cDNA for the PCR were generated from 1 lg total RNA
using a combination of random and poly-T primers from a iScript cDNA Synthesis
Kit as described by the manufacturer (Bio-Rad Laboratories, Hercules, CA). PCR
was used to generate a 924-bp long product of TTR 3#-end. The 50 llofDNA
amplification reaction contained 0.25 ll of iTaq DNA polymerase, 5 llof
PCR buffer, 1.5 ll of MgCl (20mM), 1 ll of cDNA, and 200nM of each TTR
forward primer (5#-CTCCCATGGCTCTGTTGATT-3#) and reverse primer (5#-
TTGTCTGAATTTTTGCCAGGT-3#). The three-step PCR program included an
enzyme activation step at 95C (5 min) and 40 cycles of 95C (1 min), 55C
(1 min), and 72C (1 min).
One PCR product of 924-bp long representing TTR mRNA (pTTR2 plasmid)
for each of brain and liver of herring and glaucous gulls was cloned into pCR2.1
vector and transformed to INVaFinanE. coli (Invitrogen) bacteria culture. Each
plasmid was sequenced using an ABI-prism 3100 Genetic Analyzer (Applied
Biosystems, Foster City, CA). The amino acid sequences for each of brain and
liver of herring and glaucous gulls were confirmed and compared using NCBI
nucleotide BLAST online software (Genbank accession number bankit1047289-
EU352211) (http://www.ncbi.nlm.nih.gov/BLAST). The amino acid sequences
of herring and glaucous gull TTR were aligned using ClustalW analysis, and
Bootstrap values were obtained after 100 samplings. Positions with gaps were
excluded and corrections were made for multiple substitutions.
Sequence analysis. The 924-bp long PCR products representing TTR
mRNA from brain and liver of herring and glaucous gulls was cloned into
pCR2.1 vector in an E. coli INVaF strain (Invitrogen). TTR containing
plasmids were sequenced in both directions using an ABI-prism 3100 Genetic
Analyzer (Applied Biosystems). The generated nucleotide sequences were
confirmed using NCBIs Basic Local Alignment Search Tool, BLASTx (http://
www.ncbi.nlm.nih.gov/BLAST) and translated into amino acid sequences by
the aid of Expasy translation tool (http://us.expasy.org/tools/#translate).
Multiple sequences were aligned for TTR cDNAs from several vertebrates
(Crocodile (CAA11129), frog (NP001081349), zebrafish (AAH81488), human
(NP000362), rat (NP036813), chicken (NP990666), duck (ABC65926), and
gull (EU352211). Phylogenetic analysis of the TTRs was performed using
alignment and the neighbor-joining method (Saitou and Nei, 1987) option of
the Phylip program (Galtier et al., 1996) with 100 bootstrap replicates. For the
construction of the phylogenetic tree, the TTR-like protein sequence of
Campylobacter coli (Accession No. EAL57513) was used as an out-group.
Expression and purification of rTTR. The cloned TTR gene product was
transferred from the pCR2.1 vector into the pET28a E. coli expression vector
that generates a six times histidine tag (His-tag) positioned at the N-terminus of
the expressed proteins. An E. coli BL21 (RIPL) codon plus strain was used as
the expression host. E. coli was grown overnight (ON) in 3 ml of Lauria Bertani
medium (LB-medium; 5 g/l NaCl, 5 g/l yeast extract, and 10 g/l trypton)
containing 50 lg/ml kanamycin and 34 lg/ml chloramphenicol. Thereafter, the
synthesis of rTTR was accomplished by inoculating 250 ml of LB-medium
without antibiotics with 2.5 ml of ON culture. Cells were grown for 2 h at 37C
before the temperature was lowered to 30C and recombinant protein expression
was induced by addition of isopropyl-b-1-thio-galactopyranoside to a final
concentration of 0.5mM. After 4 h, cells were harvested by centrifugation (4200
3 g, 20 min) before storage at 80C. Cell pellets were resuspended in column
binding buffer (20mM sodium phosphate, 500mM NaCl, 5mM imidazole, pH
7.4) and lysozyme was added to a final concentration of 0.2 mg/ml. After 30-min
incubation on ice, Triton-X100 (1% vol/vol) was added to the lysis solution and
DNAse and RNAse (10 mg/ml each) were added for 30 min to reduce the sample
viscosity. Finally, the insoluble fraction of the samples was removed by
centrifugation (20 min at 20,000 3 g).
Before purifying rTTR by affinity chromatography, the sample was filtered
through a 0.2-lm filter (Sarstedt, Nu¨mbrecht, Germany). Immobilized metal
(Ni
2þ
) affinity chromatography was conducted using His-Trap-FF crude Kit
(1-ml column; GE Healthcare) equilibrated with elution buffer (20mM sodium
phosphate, 500mM NaCl, 20mM imidazole, pH 7.4). Lysate with recombinant
protein was applied to the column and thereafter bound protein was eluted
using a stepwise imidazole gradient with 100, 200, 250, 300, and 500mM
imidazole. Aliquots of the eluates were collected and controlled by 12% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and InVision
His-tag In-gel staining (Invitrogen). For each of the liver and brain sourced
herring and glaucous gull rTTR, there were two bands (18 and 36 kDa), which
eluted at approximately 300mM imidazole. The protein concentration of each
purified rTTR was quantified using the Bradford method (Bradford, 1976).
Competitive rTTR binding. Competitive ligands, and T
4
and T
3
stock
solutions were prepared at concentrations ranging from 10
3
to 10
5
lM. A stock
solution of the purified rTTR protein (5nM) was prepared by dissolution in
equal parts of 0.1M Tris-HC1, 0.1mM NaC1, and 1mM EDTA buffer with
a pH 8. As a negative control solutions were prepared with dimethyl sulfoxide
(DMSO)/ethanol. For the assay volumes, the concentrations of rTTR and buffer
were proportionally adapted from previous methods with minor modifications
(Lans et al., 1993; Meerts et al., 2000). For the rTTR competitive binding
assays, a volume of 10 ll of stock concentrations (10
3
–10
5
lM) was added to
the assay incubation mixture, which had a final volume of 200 ll. For the
stocks of concentrations ranging from 10
3
to 10
1
M, a volume of 50 ll was
used for a final incubation volume of 1 ml.
The competitive binding assay was based on previous methods with minor
modifications (Lans et al., 1993; Meerts et al., 2000). For each of the
competitive ligand and DMSO control stock solutions, human TTR (30nM)
stock solution (using 3nM) or gull TTR (equilibrated to 5nM from an original
concentration of 0.77 mg/ml) was incubated with a mixture with each of
125
I-T
4
(5nM, 7000 cpm) and unlabeled T
4
(5nM) in Tris-HCl buffer. The treatment
was similar for
125
I-T
3
and unlabeled T
3
. The incubation mixtures were allowed
to reach binding equilibrium ON at 4C. After incubation, protein-bound and -
free
125
I-TH was separated by filtration. Protein-bound and -free
125
I-TH was
separated on a Biogel-P6DG column (bed volume: 1.2 ml; prepared in a 1-ml
disposable syringe) that was equilibrated with 300 ll of 10% (wt/vol) Tris-HC1
buffer, and centrifuged for 20 min at 4200 3 g at room temperature. The columns
were spin-forced (Jouan C412 centrifuge) again after an additional 200 llof
Tris-HC1 buffer was added. These first two eluant fractions, containing the
protein-bound
125
I-TH fraction were combined, and the total radioactivity
was counted and compared with the control incubations. Protein-bound
125
I-TH was quantified in the fractions. Total
125
I radioactivity per assay
(0.75 kBq per assay) was measured using a gamma counter (Cobra II Auto
442 UCA
´
N-MARI
´
N ET AL.
gamma, Perkin-Elmer, Montre´al, Canada). Protein-free
125
I-TH remained bound
to the Biogel P6DG column (BioRad, Richmond, CA), and therefore was not
present in the first two eluant fractions.
Data analysis. All TTR competitive binding assays were carried out in
triplicate, and the triplicate assay set was repeated on separate day, and showed
that relative competitive binding results were reproducible. Mean relative
competitive binding values were based on n ¼ 6 replicates (combined
triplicates on two different days). Competitive binding curves for the each
ligand were made by plotting the relative
125
I-T
4
or
125
I-T
3
protein binding (%
of control) against the natural logarithm of the competitor concentration.
Competitive binding curves were described by the sigmoidal function y ¼ a0 þ
a1/(1 þ exp ((a2 þ x)/a3)) (SlideWrite Plus 4.0, Advanced Graphics Software,
Carlsbad, CA). The relative potency of the individual competitors was
evaluated retrospectively. Using the T
3
(or T
4
) as Relative Potency 1, the value
was compared with the competitor ligands with T
3
(or T
4
). Where X (dose) and
Y (binding) are independent, the variance of Y/X is V(Y/X) ¼ E[Y^2] V (1/X) þ
V(Y) E [1/X]^2(V ¼ variance, E ¼ expected value). A one-way ANOVA with
a Studentized Newman-Keuls test was used to assess the statistical significance
(p < 0.05) of the differences among competitive binding assays to validate the
triplicate response of binding. Competitive binding constants were calculated
according to Cheng and Prusoff (1973), where the affinity constant K
i
¼ K
d
(1 þ T
0
/K
d
*) or K
i
¼ IC
50
/(1 þ ([radioligand]/K
d
)). The IC
50
is the concentration
of inhibitor resulting in 50% inhibition, the K
d
is the dissociation constant of
inhibitor-binder reaction, K
d
* is the dissociation constant of the tracer-binder
reaction, and T
0
is the total added concentration of the tracer.
RESULTS
Cloning and Characterization of Gull rTTR
The isolated TTR cDNA from brain and liver tissues of
herring and glaucous gull were sequenced in both directions
and sequence analysis showed identical nucleotide sequences
between the species and between liver and brain. The achieved
nucleotide sequence was translated and the deduced gull rTTR
(where the common ‘gull’ rTTR from liver and brain is here
on in referred to as gull TTR) translation showed 126 amino
acid residues with a calculated molecular mass of 13.8 kDa
(mass without his-tag labeling) and a theoretical isoelectric
point (pI) of 5.1. A multiple alignment analysis was performed
using the gull, duck, chicken, crocodile, human, rat, frog and
zebrafish TTR variant (Fig. 1) (Schuler et al., 1991). The
alignment showed that TTR is highly conserved among
vertebrate species, but the N-terminal region presented a low
sequence homology. To assess the relationship of gull TTR to
TTRs from other vertebrate species, a phylogenetic tree was
constructed using the neighbor-joining method and boot-
strapped 100 times (Fig. 2). This tree clearly grouped the gull
TTR with avian (chicken and duck) TTR variants. Avian TTR
is more similar to crocodile TTR than those from human, rat,
frog or zebrafish.
After transfection of the E. coli expression host, gull TTR
was expressed and purified using affinity column chromatog-
raphy. SDS-PAGE analysis was performed to determine the
expression of gull TTR in the E. coli expression host (Fig. 3).
Gel analysis of the eluates showed a stepwise imidazole
gradient with 100, 200, 250, 300, and 500mM and the his-tag
labeled gull TTR protein was observed as a protein monomer of
18 kDa and homodimer of 36 kDa that putatively comprises of
the two protein monomers (Fig. 3). Eluants containing > 250mM
imidazole contained gull TTR of high purity, which was
subsequently isolated.
FIG. 1. A multiple alignment of the predicted gull rTTR translation with TTRs from chicken, duck, crocodile, zebrafish, frog, human, and rat was generated
using MACAW. Identical and similar residues are indicated, darker shade corresponds to the most similar and no shade denotes no sequence homology. Accession
numbers are: crocodile (CAA11129), frog (NP001081349), zebrafish (AAH81488), human (NP000362), rat (NP036813), chicken (NP990666), duck (ABC65926),
and gull (sequence reported herein; EU352211).
GULL TRANSTHYRETIN AND ORGANOHALOGEN BINDING
443
Competitive TTR–Binding Assays
Competitive T
3
and T
4
binding assays with were performed
and concentration-dependent binding curves and constants
generated with commercially available human and/or purified
gull TTR (Fig. 4, Table 1), which showed that both T
3
and T
4
more competitively bind with higher affinity for gull TTR
relative to human TTR. Also, gull TTR is more effective at
binding T
3
relative to T
4
, whereas for human TTR T
4
more
effectively binds relative to T
3
. Concentration-dependent
competitive binding curves and constants for gull TTR with
T
3
and T
4
were all also generated for the exogenous ligands
under study (Figs. 5 and 6). As shown in Figure 5 and Table 1,
CB187 < 4-MeO-CB187 < 4-OH-CB187 with respect to
competitive binding potency in the displacement of both T
3
and T
4
. Similarly as shown in Figure 6 and Table 2, BDE47 < 6-
MeO-BDE47 < 6-OH-BDE47 and 4#-OH-BDE49 with respect
to competitive displacement potency for both T
3
and T
4
.
DISCUSSION
Herring gulls from Lake Ontario (Laurentian Great Lakes of
North America) and glaucous gull from Bear Island, in the
Norwegian Arctic are top avian predators in their respective
aquatic ecosystems. Herring gulls inhabits a freshwater system
(Lake Ontario), and are distributed within highly urbanized
areas such as the Greater Toronto Area, whereas the Bear
Island glaucous gulls inhabit a marine system, and inhabit
a more remote Arctic location where contaminant exposure is
a manifestation of contaminant input through atmospheric
transport and subsequent accumulation into the marine food
web. As these are different Larus species and populations and
are exposed to different organohalogen contaminant profiles,
they may possess differing TTR protein structure and ligand
interactions and differences with respect to the homeostasis of
circulating TH levels (McNabb, 2003, 2005). In the present
study, we compared the binding parameters of commercial
human TTR and recombinant gull TTR. The recombinant gull
FIG. 2. Phylogenetic analysis of the amino acid sequences of TTR from several vertebrate species and Campylobacter coli. The tree was constructed using the
neighbor-joining method and bootstrap values from 100 replicates. The sequence accession numbers of analyzed genes are: crocodile (CAA11129), frog
(NP001081349), zebrafish (AAH81488), human (NP000362), rat (NP036813), chicken (NP990666), duck (ABC65926) and gull (EU352211).
FIG. 3. Purification of gull rTTR expressed in E. coli BL21-RIPL. The
electrophoretic gel shows eluants from a stepwise imidazole gradient with
(from right to left) 250, 300, and 500mM imidazole concentrations. For
example, at the far right of the figure is the fifth eluant aliquot for the elution
with 250mM of imidazole. The his-tag labeled gull rTTR protein is observed as
a monomer of 18 kDa and homodimer of 36 kDa that is comprised of the two
monomer forms.
444 UCA
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TTR protein resembles the human TTR; however, there is
a truncation of approximately 26 amino acids in the N-terminal
end of gull TTR. In addition, the rTTR contains six histidine-
tag residues. In the present attempt to further understand the
similarities or differences of the present gull TTR with human
TTR, one confounding factor to consider is that minor
differences may have occurred in the post-translational modi-
fication of the recombinant gull TTR.
TTR and Phylogenetic Analysis with Other Vertebrates
To our knowledge, this is the first report describing the TTR
nucleic acid and amino acid sequences as well as the
expression and purification of rTTR from any gull species.
Previously in birds, the synthesis, expression and secretion of
TTR in the liver and brain (choroid plexus) has been
demonstrated in chicken, quail and pigeon (Zanotti et al.,
2001). For this reason, it was presently decided to evaluate the
expression of TTR mRNA in brain and liver of both gull
species to identify and potential differences or similarities. The
TTR sequences in the liver and brain of both herring and
glaucous gull were found to be identical, and thus it was
possible to use one tissue source of TTR protein that was rep-
resentative of Larus genus and for use in competitive binding
assays. TTR is a major, circulating TH-binding protein in birds,
herbivorous marsupials and small eutherians (Richardson et al.,
1996). TTR is considered the only TH-binding protein that is
synthesized in the cells of the blood-cerebroespinal fluid
barrier, in addition to its synthesis in the liver (Schreiber and
Richardson, 1997).
Comparison of the gull TTR sequence with other vertebrate
species showed the evolutionary conservation of TTR
nucleotide and amino acid sequences (Figs. 1 and 2). The
deduced TTR amino acid sequences indicated that the protein
is highly conserved among avian species. However, it was
presently observed that the N-terminal region of TTR was less
homologous among species. This is not surprising because the
noninvolvement of this region in and/or influencing TH binding
was previously proposed by Chang et al. (1999). Overall, when
grouped based on similarities in the alignment of genome
sequences, the amino acids in the N-terminal regions of the
TTRs in marsupials, birds, reptiles, amphibians and fish, differ
from that of the order insectivora (Schreiber and Richardson,
1997). One explanation for this is that the basic structure of TTR
(four identical subunits, central channel, thyroxine-binding sites
in mammals) evolved while this protein was an extra-cellular
brain protein, long before the initiation of its synthesis in the
liver (Hamilton and Benson, 2001). However, although the TH
binding region of TTR is highly conserved among vertebrate
species including birds, other TH binding factors can affect the
binding affinity among species and in different stages of
development (Richardson et al., 1994).
Natural T
3
and T
4
Ligand Binding to gull TTR versus Human
TTR
The competitive binding curves and constants (K
i
and K
d
)
demonstrated that gull TTR is more effective at binding T
3
TABLE 1
Competitive Binding Parameters for Gull TTR Protein and T
3
or T
4
in the Presence of Model PCB and Substituted Structural
Analog Ligands, and Human TTR Values for T
3
and T
4
as
Reference
Compound K
i
(nM)
a
Relative
potency
b
K
d
c
Maximum %
competition
d
T
3
4.48 1 13.1 87 ± 4.3
CB187 771 6.02 3 10
3
± 2.9 3 10
4
195 89 ± 3.7
4-MeO-CB187 62.67 0.08 ± 4.5 3 10
3
23.5 90 ± 2.6
4-OH-CB187 1.1 3.77 ± 0.55 11.1 91 ± 2.7
T
4
10.31 1 27.5 91 ± 3.7
CB187 71.3 0.139 ± 0.01 36.04 76 ± 5.8
4-MeO-CB187 941 0.010 ± 1.35 3 10
3
432 86 ± 2.6
4-OH-CB187 12.5 0.8 ± 0.05 10.8 90 ± 1.8
Human TTR
T
4
50.89 1 14.9 98 ± 2.1
T
3
214 0.19 ± 0.02 60.1 77.3 ± 3.7
Note. Results presented as the mean of individual measurements, and based
on n ¼ 6 replicates (combined triplicates on two different days). The ± SD for
relative potency and maximum % competition is based on the standard
deviation of the two values comprising the ratio. See Figure 5A for competitive
binding curves.
a
K
i
¼ K
d
(1 þ T
0
/K
d
*) or K
i
¼ IC
50
/(1 þ ([radioligand]/K
d
) (Cheng and
Prusoff, 1973). See ‘‘Materials and Methods’’ section for further details.
b
The relative potency is a ratio of K
i
values and the variance of the ratio is
estimated by the equation V(Y/X) ¼ E[Y^2] V (1/X) þ V (Y ) E [1/X]^2(V ¼
variance, E ¼ expected value), where X (dose) and Y (binding) are independent.
c
The K
d
values (mean ± SD) were determined from the slope of the linear
regression line of Scatchard plots.
d
Percentage of competition reached at highest tested concentration (1 3 10
5
nM).
FIG. 4. Concentration-dependent, competitive binding curves of gull TTR
(gTTR) and commercially available human TTR (hTTR) with T
3
or T
4
. The
error bars denote the standard deviation of n ¼ 6 replicated (two n ¼ 3 replicate
sets performed on different days).
GULL TRANSTHYRETIN AND ORGANOHALOGEN BINDING
445
relative to T
4
, whereas for human TTR T
4
more effectively
binds relative to T
3
. The contrasting T
3
and T
4
binding affinity
for human versus gull TTR may be partially explained from an
evolutionary perspective, where different taxon levels have
differing specificity with respect to transport proteins and the
hormones transported. For the present gull TTR, where T
3
is
more effectively binds relative to T
4
, there is a consistency with
other reports in the literature, where it has been reported that
chicken TTR has about twice the affinity for T
3
relative to T
4
(Schreiber, 2002). TTR in birds, reptiles, amphibians, and
teleost fish have generally been found to preferentially bind T
3
over T
4
(Kawakami et al., 2006). The preference of avian TTR
for T
3
relative to T
4
could be related to the N-terminus of the
TTR subunits that are more than merely changes in the primary
structure, but are manifested in the relative affinity of the TTR
homotetramer for T
4
and T
3
. Prapunpoj et al. (2006) recently
demonstrated in a reptile model the causal relationship between
the N-terminal region of the TTR subunits and the affinity of
the homotetramer for THs. They showed that removing the
N-terminus of the Crocodylus porosus TTR subunit or replacing
it with the N-terminus of the human subunit resulted in a
homotetramer with increased affinity for T
4
. The present human
TTR–binding results are also consistent with that for humans,
where mammalian TTR preferentially binds T
4
relative to T
3
.
Also in humans, TTR has higher affinity for THs than ALB, and
TBG has higher affinity for THs than TTR. Schreiber and
Richardson (1997) suggested that the onset of hepatic TTR
synthesis correlated with the development of homeothermy and
the increase in lipid volume to body mass ratio.
The present results suggests that in gulls, T
3
binding relative
to that of T
4
would be less susceptible to competitive dis-
placement by exogenous ligands present (e.g., in the blood
stream), which suggests that T
4
delivery targeting the liver or
brain and likely other organs may be more perturbed relative to
T
3
. However, the thyroid gland synthesizes and releases into
the circulation primarily T
4
(95%, 5% T
3
), which is the less
active precursor that is subsequently deiodinated to T
3
at the
target tissues. Also, besides the differences among avian and
mammalian TTR binding T
3
and T
4
, in mammals about 12% of
total thyroid transport proteins is synthesized by the choroid
FIG. 5. Concentration-dependent, competitive binding curves of (A) T
4
and (B) T
3
displacement from gull TTR by 2,2#,3,4#,5,5#,6-heptaCB (CB-187),
4-hydroxy-CB187 (4-OH-CB187), or 4-methoxy-CB187 (4-MeO-CB187). The
competitive binding parameters are listed in Table 1. The error bars denote the
standard deviation of n ¼ 6 replicated (two n ¼ 3 replicate sets performed on
different days).
FIG. 6. Concentration-dependent, competitive binding curves of (A) T
4
and (B) T
3
displacement from gull TTR by 2,2#,4,4#-tetrabromoDE (BDE-47)
flame retardant and 6-hydroxy-BDE47 (6-OH-BDE47), 6-methoxy-BDE47 (6-
MeO-BDE47), or 4-OH-2,2#,4,5-tetrabromoDE (4-OH-BDE49). The compet-
itive binding parameters are listed in Table 2. The error bars denote the standard
deviation of n ¼ 6 replicated (two n ¼ 3 replicate sets performed on different
days).
446 UCA
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plexus, where about 50% of such proteins that are secreted is
TTR (Schreiber and Richardson, 1997). Because much more T
4
(relative to T
3
) is associated with circulating TTR, in gulls
(relative to humans) this would suggest that circulating and
target organ levels of T
4
would be less sensitive to exogenous
ligand competition.
Competitive Gull rTTR Binding of T
3
and T
4
with Exogenous
Contaminant Ligands
Like other brominated flame retardants, PBDEs have been
used in a wide array of products, including building materials,
electronics, furnishings, motor vehicles, plastics, polyurethane
foams, and textiles. As a consequence, PBDEs in the
environment and exposure to wildlife are a major contamina-
tion issue. Birds are no exception, and congeners such as
BDE47 are persistent and bioaccumulative in wildlife in-
cluding tissues and/or eggs of Great Lakes herring gulls and
Svalbard glaucous gulls (Gauthier et al., 2008; Verreault et al.,
2005b). Although there are no present reports for Great Lakes
herring gulls, several OH-PCB and OH-PBDE congeners, and
to a much lesser extent MeO-PBDEs, have been recently
quantified in glaucous gull adult plasma from the Norwegian
Arctic (Verreault et al., 2005a, b), where 6-OH-BDE47,
4#-OH-BDE49, 3#-MeO-BDE47, 4#-MeO-BDE49, and most
important among the OH-PCBs, 4-OH-CB187 was detected
and dominant. The persistence of OH-PCB and OH-PBDE
congeners in the blood of birds and other wildlife has been
postulated as being via competitive binding to TH transport
proteins and specifically TTR.
In the present study, the gull TTR–binding affinity and
potency was exceptionally low for BDE47 relative to T
4
or T
3
and as compared with the structurally analogous MeO-PBDE
and especially the OH-PBDE ligands. This is consistent with
a recent studies on the PBDE competitive binding with T
4
and
human TTR, where no substantial TTR binding was observed
for 17 (Meerts et al., 2001) and 19 (Hamers et al., 2006) PBDE
congeners, including BDE47, at maximum or higher concen-
trations than 250nM. This T
4
displacement potency for human
TTR is consistent with gull TTR where > 1000nM of BDE47 was
required (Fig. 6). Morgado et al. (2007) also reported a lack of
competitive PBDE (including BDE47) binding with sea bream
TTR, also at high nanomolar treatment concentrations.
OH-PCB and OH-PBDE congeners, and other brominated
phenolic ligands, have been shown to competitively displace
T
4
from human TTR (Brouwer et al., 1998; Legler and
Brouwer, 2003). Hamers et al. (2006) screened a test set of
twenty-seven individual BFRs, including 6-OH-BDE47, for
their relative potency to compete with T
4
for binding human
TTR. Meerts et al. (2001) also reported high human TTR
binding competitive with T
4
for 6-OH-BDE47, but was of
lesser potency than for other brominated phenolic substances
and used as flame retardants such as tetrabromobisphenyl
A (TBBPA) and 2,4,6-tribromophenol (2,4,6-TBP). The human
TTR–binding potencies of TBBPA and 2,4,6-TBP exceeded
that of T
4
. Similarly for selected OH-PCB congeners, Lans
et al. (1993) demonstrated high binding competitiveness with
T
4
and human TTR. The present study results were consistent,
that is, 6-OH-BDE47 and 4#-OH-BDE49 (Fig. 6, Table 2) and
4-OH-CB187 (Fig. 5, Table 1) had comparable or greater
binding affinity for gull TTR than T
4
(Fig. 6, Table 2). For both
OH-PBDEs and OH-PCBs, it has been proposed that for
optimal human TTR competitive binding (with T
4
), hydroxyl-
ation should exist at a para position (relative to the aromatic
ring linkage), and that there be one, but preferably two, halogen
substituents on carbons adjacent to the OH-group (Hamers
et al., 2006; Lans et al., 1993; Morgado et al., 2007). TTR is
a homotetramer consisting of a dimer of dimers. The binding
channel for TH is at an interaction site between the two dimers.
In this study, it is possible that the high differences found in the
K
d
and K
i
values comparing gull TTR with human TTR
binding can be attributed to the fact that we have studied
interaction of TH and organohalogen compounds with the
dimer form.
Better understanding OH-PCB and OH-PBDE binding with
TTR (and other TH transport proteins) is important with
respect to effects on the thyroid system and target organs, for
example, in the brain because the binding of OH-PCBs and
OH-PBDEs to TTR may be an avenue for these chemicals to
TABLE 2
Competitive Binding Parameters for Gull TTR Protein and T
3
or T
4
in the Presence of Model PBDE and Substituted Structural
Analog Ligands
Compound K
i
(nM)
a
Relative
potency
b
K
d
c
Maximum %
competition
d
T
3
4.48 1 13.1 87 ± 4.3
BDE47 671 6.69 ± 4.4 3 10
4
201 88 ± 3.8
6-OH-BDE47 78 0.07 ± 3.91 3 10
3
7.1 91 ± 2.7
4-OH-BDE49 57 0.81 ± 0.013 6.3 90 ± 2.2
6-MeO-BDE47 233.7 0.019 ± 2.3 3 10
3
29.6 90 ± 3.5
T
4
10.31 1 27.5 91 ± 3.7
BDE47 89.1 0.13 ± 0.02 213 94 ± 3.1
6-OH-BDE47 4.91 2.17 ± 0.173 14.9 96 ± 2.6
4-OH-BDE49 4.68 2.20 ± 0.161 13.8 95 ± 2.7
6-MeO-BDE47 54.32 0.189 ± 0.032 139.3 96 ± 2.8
Note. Results presented as the mean of individual measurements, and based
on n ¼ 6 replicates (combined triplicates on two different days). The ± SD for
relative potency and maximum % competition is based on the standard
deviation of the two values comprising the ratio. See Figure 5B for competitive
binding curves.
a
K
i
¼ K
d
(1 þ T
0
/K
d
*) or K
i
¼ IC
50
/(1 þ ([radioligand]/K
d
) (Cheng and
Prusoff, 1973). See ‘‘Materials and Methods’’ section for further details.
b
The relative potency is a ratio of K
i
values and the variance of the ratio is
estimated by the equation V(Y/X) ¼ E[Y^2] V (1/X) þ V(Y) E [1/X]^2(V ¼
variance, E ¼ expected value), where X (dose) and Y (binding) are independent.
c
The K
d
values (mean ± SD) were determined from the slope of the linear
regression line of Scatchard plots.
d
Percentage of competition reached at highest tested concentration (1 3 10
5
nM).
GULL TRANSTHYRETIN AND ORGANOHALOGEN BINDING
447
reach TH receptors in target organs. Thyroidogenic contami-
nants such as OH-PCBs and OH-PBDEs that can displace T
4
from TTR, would subsequently release free T
4
, which may
enhance T
4
metabolism and excretion (Brouwer et al., 1998).
A sufficiently increased excretion of T
4
could result in
a decrease in circulating T
4
, potentially leading to more serious
effects such as hypothyroidism.
The present gull TTR molecular characterization and
competitive binding studies also clearly showed that human
TTR cannot be used as a surrogate to assess the effects on
circulating THs in birds (or at least gull species). Profound
differences in the binding affinity of T
4
and T
3
and several
environmentally relevant PCB, PBDE, OH-PCB, OH-PBDE,
MeO-PCB, and MeO-PBDE congeners for both human and/or
gull TTR. The OH-containing forms of PCBs and PBDEs are
strong binding competitors for gullTTR. Depending on the
circulatory quantities of OH-PCBs and OH-PBDEs in the
blood, it is possible to influence and/or change on the
circulating THs, both levels and relative proportions of T
3
and T
4
. Such TTR interactions could affect the circulatory TH
homeostasis, and perhaps the TH-dependent function and
health of exposed birds. OH-PCB binding affinity to TTR in
mammals has been linked to alterations of TH and vitamin A
levels in OH-PCB exposed laboratory rats (Schuur et al.,
1998). OH-PCB congeners (e.g., 4-OH-CB106) have also been
shown to bind to the human TH receptor (TR) (You et al.,
2006). Kimura-Kuroda et al. (2005) reported that 4#-OH-
CB106 and 4#-OH-CB159 significantly inhibited T
3
-dependent
extension of Purkinje cell dendrites extracted from mouse
cerebellum in vitro.
To our knowledge there are no reports on circulating OH-
PCB, OH-PBDE, PBDE, or possibly MeO-PBDEs in herring
gulls from the Laurentian Great Lakes. Recently it was reported
that the mean concentrations of 6-OH-BDE47 and 4#-OH-
BDE49 in the plasma of male and female glaucous gulls from
the Norwegian Arctic was up to 0.32 ng/g (wet weight) or
~0.6nM (Verreault et al., 2005b). The competitive potency was
~10nM for both OH-PBDE congeners with T
3
and T
4
on gull
TTR (Fig. 6), and therefore in free-ranging Svalbard gulls OH-
PBDEs may have a profound effect on circulating T
3
and/or T
4
levels. The mean concentrations of 4-OH-CB187 in the plasma
of male and female glaucous gulls from the Norwegian Arctic
was recently reported to be up to 17.5 ng/g (wet weight) or
~40nM (Verreault et al., 2005a). The competitive potency was
5–10nM for 4-OH-CB187 with T
3
and T
4
on gull TTR (Fig. 5),
and thus 4-OH-CB187 levels in free-ranging Svalbard gulls are
even more likely (relative to OH-PBDEs) to effect circulating
T
3
and T
4
levels. However, in recent studies on Svalbard
glaucous gulls, neither T
4
nor T
3
levels were associated with
concentrations of the selected organohalogens including OH-
PCBs and OH-PBDEs (Verreault et al., 2007). It is probable
that the patterns and levels of exposure to these OH-containing
contaminants differ in Great Lakes herring gull blood, and thus
the potential of effecting circulating levels of T
3
and T
4
via
competitive interaction with TTR. The present results also
demonstrate that there may be potential physiological con-
sequences of the competitive binding of OH-containing
organohalogens and perturbation of T
4
and T
3
levels in blood
via interaction with gull TTR. One disruption outcome from
the displacement of T
4
from TTR could result in less T
4
at
target tissues, and thus decreased prohormone for deiodinase/
mediated (including the 5# deiodinases DI and DII), for T
4
conversion to active T
3
(Verhoelst et al., 2005).
Like other bird species, gulls not only have TTR but also
ALB, which also bind and are involved in the transport of THs.
In the context of overall TH binding in birds, TTR may be of
lesser importance because in birds the proportion of circulating
TH-binding transport proteins is low for TTR. According to
McNabb et al. (1984, 2003) in chicken the circulating T
4
is
bound 75% to ALB, 17% to TTR, and 7.5% to an a-globulin.
Research is presently underway in the molecular cloning,
expression and purification of ALB for these gull species,
which is being used to comparatively assess the binding
affinities and potencies of the various exogenous ligands and
byproducts that potentially could disrupt TH transport. Ligand
binding parameter assessments for TH transport protein in gull
species, and in wildlife in general, are necessary to more fully
understand the potential effects in reproductive, nutritional,
physiological and environmental (e.g., temperature) factors that
can influence circulating T
4
and T
3
and subsequently on TH-
dependant processes.
FUNDING
Natural Science and Engineering Research Council
(NSERC) of Canada Discovery Grant to R.J.L.; Premier
Research Excellence Award to R.J.L.; Wildlife Toxicology and
Disease Program (Environment Canada); and the Norwegian
Research Council.
ACKNOWLEDGMENTS
Thanks goes to France Maisonneuve (NWRC, Environment
Canada) for training and assistance with the TTR competitive
binding assays, and the NWRC Specimen Bank and field staff
(herring gull liver and brain collections). Thanks also to
Jonathan Verreault, Hallvard Strøm, and Rosa A. Villa (the
Norwegian Polar Institute) for collection of glaucous gull
samples from Bear Island; thanks to Chriptopher Sørmoe for
guidance during rTTR synthesis (Norwegian University of
Science and Technology).
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    • "For example, OH-PBDEs could compete with thyroid hormones (TH) for binding to proteins such as transthyretin (TTR) and thyroxine binding globulin (TBG), and then exert thyroid hormone activity (Uc an-Marín et al., 2009). The binding affinity of OH-PBDEs with TTR is significantly greater than TH, parental PBDEs and their MeO-substituted analogs (Li et al., 2010; Uc an-Marín et al., 2009). In addition, OH-tetra-BDEs could disturb Ca 2þ homeostasis, neurotransmitter release and induce greater cytotoxicity and genotoxicity than tetra-BDEs (Dingemans et al., 2008; Ji et al., 2011; Li et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are of great concern due to their potential risk to animal and human health. The biotransformation potential of OH-PBDEs in organisms is important for the understanding of their health risk. In the present study, the biotransformation of 3′-OH-2,4-di-BDE (3′-OH-BDE-7), 4′-OH-2,2′,4-tri-BDE (4′-OH-BDE-17) and 3-OH-2,2′,4,4′-tetra-BDE (3-OH-BDE-47) by pig liver microsomes was studied. Compared with their precursor PBDEs, the three OH-PBDEs were more readily biotransformed by pig liver microsomes, and the biotransformation rate followed the order: 3′-OH-BDE-7 > 4′-OH-BDE-17 > 3-OH-BDE-47. These results revealed that the biotransformation rate of OH-PBDEs was decreased with an increase in the number of bromine substituents. Cleavage of the diphenyl ether bond was the dominant pathway for biotransformation of the three OH-PBDEs by pig liver microsomes, while debromination and hydroxylation were found to be of less importance. CYP3A4 was suggested to be the specific enzyme responsible for the biotransformation of OH-PBDEs via associated inhibition assay. These findings may enrich our understanding of health risk associated with OH-PBDEs in mammals and human beings.
    Full-text · Article · Apr 2016
    • "Another avenue of mechanistic studies involves thyroid hormone transport proteins in blood. PBDEs and/or PCBs as well as their OH-and MeO-containing metabolites were shown to displace thyroid hormones from plasma carrier proteins such as recombinant transthyretin (TTR) and albumin in adult herring gulls (Ucán-Marín et al., 2009; Ucán-Marin et al., 2010). HPT axis investigations at the genomic level may represent a relevant and sensitive tool to assess organohalogen-mediated effects on thyroid hormone homeostasis. "
    [Show abstract] [Hide abstract] ABSTRACT: A number of studies have reported altered circulating thyroid hormone levels in birds exposed either in controlled settings or in their natural habitat to ubiquitous organohalogen compounds including organochlorines (OCs) and polybrominated diphenyl ether (PBDE) flame retardants. However, limited attention has been paid to underlying homeostatic mechanisms in wild birds such as changes in the expression of genes in the hypothalamic-pituitary-thyroid (HPT) axis. The objective of the present study was to investigate the relationships between hepatic concentrations of major organohalogens (PBDEs and OCs), and circulating thyroid hormone (free and total thyroxine (T4) and triiodothyronine (T3)) levels and transcription of 14 thyroid-related genes in three tissues (thyroid, brain, and liver) of an urban-adapted bird exposed to high organohalogen concentrations in the Montreal area (QC, Canada), the ring-billed gull (Larus delawarensis). Positive correlations were found between liver concentrations of several polychlorinated biphenyls (PCBs), PBDEs as well as chlordanes and total plasma T4 levels. Hepatic concentrations of several PBDEs were negatively correlated with mRNA levels of deiodinase type 3, thyroid peroxidase, and thyroid hormone receptor β (TRβ) in the thyroid gland. Liver PCB (deca-CB) correlated positively with mRNA levels of sodium-iodide symporter and TRα. In brain, concentrations of most PBDEs were positively correlated with mRNA levels of organic anion transporter protein 1C1 and transthyretin, while PCBs positively correlated with expression of TRα and TRβ as well as deiodinase type 2. These multiple correlative linkages suggest that organohalogens operate through several mechanisms (direct or compensatory) involving gene transcription, thus potentially perturbing the HPT axis of this highly organohalogen-contaminated ring-billed gull population.
    Full-text · Article · Mar 2016
    • "hyperboreus) and herring gulls (L. argentatus) demonstrate that some hydroxylated PBDE metabolites can also bind to avian TTR with comparable or greater potency than thyroxine (T 4 ), but not triiodothyronine (T 3 ) [9], and to avian albumin (another thyroid transport protein in birds [10]) with greater potency than either T 3 or T 4 [11]. In whole animal studies, exposure to PBDEs has increased hepatic clearance of T 4 in rats [12, 13], reduced circulating thyroid hormones in fish [14, 15] Acc e p ted P r e p r i nt "
    [Show abstract] [Hide abstract] ABSTRACT: High concentrations of polybrominated diphenyl ethers (PBDEs) accumulate in predatory birds. Several PBDE congeners are considered thyroid disruptors however avian studies are limited. Here, we examined circulating thyroid hormones, thyroid gland function of nestling American kestrels (Falco sparverius) at 17-20 d of age, following embryonic exposure by maternal transfer only to environmentally relevant levels of PBDEs (DE-71 technical mixture). Nestlings were exposed to in ovo sum (Σ) PBDE concentrations of 11,301 ± 95 ng/g ww (high exposure), 289 ± 33 ng/g ww (low exposure), or 3.0 ± 0.5 ng/g ww (controls; background exposure). Statistical comparisons are made to controls of respective sexes and account for the relatedness of siblings within broods. Circulating concentrations of plasma total thyroxine (TT4 ) and triiodothyronine (TT3 ) in female nestlings were significantly influenced overall by the exposure to DE-71. Following the intra-muscular administration of thyroid stimulating hormone (TSH), the temporal response of the thyroid gland in producing and/or releasing TT4 was also significantly affected by the females' exposure to DE-71. The altered availability of T4 for conversion to T3 outside of the gland, and/or changes in thyroid-related enzymatic activity, may explain the lower TT3 concentrations (baseline, overall) and moderately altered temporal TT3 patterns (p = 0.06) of the treatment females. Controlling for the significant effect on TT3 levels of the delayed hatching of treatment females, baseline TT3 levels were significantly and positively correlated with body mass (10, 15, 20 d), with PBDE-exposed females generally being smaller and having lower TT3 concentrations. Given that exposure concentrations were environmentally relevant, similar thyroidal changes and associated thyroid-mediated processes relating to growth, may also occur in wild female nestlings. This article is protected by copyright. All rights reserved.
    Article · Jan 2016
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