Recombinant transthyretin purification and competitive binding with organohalogen compounds in two gull species (Larus argentatus and Larus hyperboreus).

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.

Health of herring gulls (Larus argentatus) in relation to breeding location in the early 1990s. III. Effects on the bone tissue.

Health effects associated with the Great Lakes environment were assessed in adult herring gulls (Larus argentatus) in the early 1990s, including the size and quality of their bones. Femurs were excised from 140 individuals from 10 colonies distributed throughout the Great Lakes and 2 reference colonies in Lake Winnipeg (freshwater) and the Bay of Fundy (marine). Femurs of gulls from the Great Lakes differed from the freshwater or marine reference for 9 of 12 variables of size, composition, and strength assessed using peripheral quantitative computed tomography (pQCT) and biomechanical testing. Femurs of Great Lakes gulls were significantly smaller in length (-2.9%), periosteal circumference (-2.4%), and cross-sectional area (-5.4%) than freshwater reference birds. Femurs of the Great Lakes gulls had a lower significant cortical bone mineral content (-8.1%) and density (-2%) than the marine reference. A significant increase in the amount the bone could bend before it broke (+34%) and the energy required to break it (+44%) and a significant decrease (-16.3%) in stiffness during three-point biomechanical bending test were also detected in Great Lakes versus the freshwater gulls. These differences are indicative of impaired mineralization. When divided into high and low 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity equivalent (TCDD-TEQ) colonies, the amount the bone could bend before it broke and the energy required to break it were significantly higher in the high TEQ colonies, but not high polychlorinated biphenyl (PCB) colonies. Breeding location and dietary choices of Great Lakes herring gulls in the early 1990s resulted in modulations of physiological processes that affected the size, mineralization, and biomechanical properties of bone.

Health of herring gulls (Larus argentatus) in relation to breeding location in the early 1990s. I. Biochemical measures.

Tissues of 156 adult herring gulls (Larus argentatus) were sampled in the early 1990s from 11 colonies throughout the Laurentian Great Lakes and 2 reference colonies in Lake Winnipeg and the Bay of Fundy. Gulls from 1 or more Great Lakes differed from Lake Winnipeg or the Bay of Fundy for 17 of 19 clinical biochemical measures, whereas the freshwater and marine reference sites differed in only 3. Three differed with sex. There was little evidence to suggest that these differences reflect genotypic differences. Plasma thyroxine, albumin, calcium, magnesium, inorganic phosphorus, triglyceride, bile acids, total protein, uric acid, and urea nitrogen concentrations and aspartate aminotransferase (AST) activity were lower in gulls from one or more Great Lakes than for gulls from one or both reference sites, while those for globulins and glucose were higher. Highly carboxylated porphyrins accumulated in the livers of Great Lakes gulls and ethoxyresorufin O-deethylase (EROD) activity was induced. There was resistance to PCB/TCDD-induced EROD induction in the Lake Erie colonies. Gulls from five colonies were unable to obtain adequate food to maintain average body condition. Body condition was associated with seven biochemical measures. Colonies in designated Areas of Concern as well as those with high liver polychlorinated biphenyl (PCB) concentrations both differed for 50% of the biochemical measures. Associations between biochemical measures and delta15N-derived trophic position and/or contaminant levels in tissues suggest the effects may be toxicopathic responses. Associations were most frequently with PCBs and dioxin-like contaminants. The health of adult herring gulls varied with breeding location and "lifestyle" in the early 1990s, and Great Lakes gulls suffered from chemical and nutritional stressors that modulated physiological processes and endocrine function.

Health of herring gulls (Larus argentatus) in relation to breeding location in the early 1990s. II. Cellular and histopathological measures.

Organosomatic indices, hematological indicators of stress, and histopathological lesions were quantified for over 150 incubating herring gulls (Larus argentatus) sampled in 11 colonies throughout the Great Lakes and reference colonies in Lake Winnipeg and the Bay of Fundy. Of 21 parameters assessed, significantly more differed between Great Lakes colonies and reference colonies than between the two reference colonies. Relative adrenal, kidney, and liver masses of gulls from some Great Lakes colonies were reduced and thyroid masses increased relative to gulls from reference colonies. Foci of cellular atypia were observed in the hepatocytes of two Great Lakes gulls. Chronic periportal hepatitis, lipogranulomas and vacuolation of hepatocytes, and chronic granulomatous interstitial nephritis were more prevalent or severe in gulls from Great Lakes colonies and were associated with contaminants. The kidneys of gulls from the three most contaminated locations were damaged and functionally compromised. Interstitial nephritis was likely the most functionally significant histopathological lesion. Portal-tract fibrosis, granulomatous hepatitis, and kidney tubule dilation/obstruction and splenic enlargement were more prevalent or severe at reference sites and were associated with blood-borne parasites. Amyloid deposits were observed in the spleen, kidneys, or liver of nearly half of the gulls. Associations between the prevalence or severity of lesions and contaminant levels in gull tissues or the trophic level of their diet suggest some lesions are toxicopathic. Associations were most frequently found with planar halogenated aromatic hydrocarbons and lead. The stress response, as measured by the heterophil/lymphocyte ratio, was attenuated in gulls from Areas of Concern and associated with decreased adrenal mass. Our findings suggest that adult Great Lakes gulls suffered from chronic exposure to chemical stressors in the early 1990s sufficient to modulate endocrine function and physiological processes and induce structural changes in tissues.

DNA strand length and EROD activity in relation to two screening measures of genotoxic exposure in Great Lakes herring gulls.

We collected tissues from herring gulls (Larus argentatus) nesting within and outside of the Great Lakes basin. Genotoxin exposure was assessed as fluorescent aromatic compounds (FACs) in bile and SOS Chromotest-inducing activity in muscle extracts. We determined whether these exposures were associated with decreased erythrocyte DNA strand length and/or induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity. FACs were detected in all bile samples. Most muscle extracts produced a positive or marginal SOS response in the presence of S9. SOS induction potentials were strongly associated with dietary trophic level. The median molecular length of DNA isolated from erythrocytes for 14 of 17 adult and 10 of 11 prefledgling collections was reduced compared to the modal class for their respective age group suggesting widespread DNA damage. DNA damage was greatest in gulls from Saginaw Bay, Lake Huron. Median EROD activity in both adults and prefledglings from remote locations was significantly lower than that of gulls from the lower Great Lakes and was not associated with concentrations of benzo[a]pyrene (B[a]P)-like FACs. Our results indicate Great Lakes herring gulls were exposed to genotoxins and Ah-receptor activating agents in biologically significant concentrations in the early 1990s. These agents appear to be persistent bioaccumulative compounds and/or their metabolites.

Development and validation of a herring gull embryo toxicokinetic model for PCBs.

A toxicokinetic model was developed to describe polychlorinated biphenyl (PCB) accumulation by herring gull (Larus argentatus) embryos during development. The model consists of two components, a bioenergetics model that predicts the lipid mass balance of embryo and yolk compartments with time and an empirical toxicokinetic model that describes PCB partitioning between lipid compartments in the egg. The model was calibrated using data on PCB and lipid partitioning between embryo and yolk + albumen at four time points during incubation in herring gull eggs injected with a PCB mixture, combined with data sets on herring gull embryo growth rates and bioenergetic demands with time. The model was validated using independent data consisting of maternally exposed, field-incubated Lake Superior herring gull eggs that varied in incubation ages over the range of 8.5 d to pipping age (26-28 days). PCB concentrations in 6-9 d embryos were nearly an order of magnitude less than predicted by equilibrium lipid partitioning between the embryo and yolk + albumen compartments of the eggs. PCB concentrations in embryos were adequately predicted by equilibrium partitioning, however, for eggs incubated for 23-28 d. An empirical relationship was developed to account for the apparent nonequilibrium behaviour of PCBs during early development. The model was sensitive to the mass of yolk lipids and the mass of PCBs deposited to fresh eggs and much of the variability in embryo PCB concentrations could by explained by accounting for variability in these input parameters. Consistent with experimental data for other avian species, the model predicts that the highest PCB concentrations in the embryo/chick occur during pipping or soon after when yolk lipids have been completely resorbed by the embryo.

Hepatic EROD activity is not a useful biomarker of polychlorinated biphenyl exposure in the adult herring gull (Larus argentatus).

Liver concentrations of polychlorinated biphenyls (PCBs) and chicken embryo hepatocyte (CEH) bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQs) were measured in livers of adult herring gulls (Larus argentatus) collected from several locations on the Great Lakes and two reference sites. Total PCB concentrations (sum of 42 congeners) and TCDD-EQ concentrations were compared with hepatic ethoxyresorufin-O-deethylase (EROD) activity, methoxyresorufin-O-deethylase (MROD) activity and immunodetectable cytochrome P4501A (CYP1A) protein concentration. EROD and MROD activity were not significantly correlated with total PCB concentration or TCDD-EQ concentration in liver tissue. CYP1A protein concentration was significantly correlated with total PCB concentration, but the linear relationship had little predictive power. We conclude that EROD is not a useful biomarker of PCB exposure in the adult herring gull.

Biological effects of marine PCB contamination on black guillemot nestlings at Saglek, Labrador: liver biomarkers.

Black guillemots (Cepphus grylle) in Saglek Bay, Labrador have elevated polychlorinated biphenyl (PCB) concentrations due to marine sediment contamination around a former military site. We measured liver biomarkers and sigmaPCB concentrations in 31 nestlings from three PCB-exposure groups: Reference group (range: 15-46 ng/g liver, wet wt.), moderately exposed Islands group (24-150 ng/g), and highly exposed Beach group (170-6200 ng/g). Biomarker responses were dose-dependent and in some cases sex-dependent. Livers of female Beach nestlings were enlarged 36% relative to Reference females. In both sexes, Beach nestlings had liver ethoxyresorufin-O-deethylase (EROD) activities elevated 79% and liver retinol concentrations reduced 47%. Retinyl palmitate concentrations were reduced 50% but only among female nestlings. Island nestlings also exhibited EROD induction (57%) and reductions in retinol and retinyl palmitate concentrations (28 and 58%, respectively). Liver lipid content increased with sigmaPCBs in both sexes, and correlated with liver mass in males. Malic enzyme activity and porphyrin concentrations showed little association with sigmaPCBs. Although similar associations between liver biomarkers and organochlorine exposure in fish-eating birds are well documented, typically exposures involve multiple contaminants and there is uncertainty about specific PCB effects. Our findings indicate that liver biomarkers respond to relatively low PCB exposures (approximately 73 ng/g liver) in guillemots.

Avian thyroid development in chemically contaminated environments: is there evidence of alterations in thyroid function and development?

Poor reproductive success, developmental abnormalities, and behavioral alterations in fish-eating birds in some Great Lakes areas have led to more than 35 years of toxicological studies and residue monitoring of herring gull (Larus argentatus) populations. Polyhalogenated aromatic hydrocarbons (PHAHs), especially polychlorinated biphenyls (PCBs), are widespread contaminants in the Great Lakes ecosystem. The introduction of regulations and elimination of point sources since the 1970s have resulted in decreased PHAHs in fish-eating bird eggs and tissues. PCB exposure is associated with thyroid disruption (hypothyroidism) in mammals, but much less is known of PCB effects on avian thyroid function. Our 1998-2000 studies of herring gulls from the Great Lakes show that both pipping embryos and prefledglings from highly contaminated sites have marked depletion of thyroid gland hormone stores compared with similarly aged gulls at the reference sites. However, organismal hypothyroidism was not apparent in many embryo and chick collections where severe depletion of thyroid gland hormone was observed. Adults, sampled at two high PCB sites and a low PCB site in the Great Lakes and the maritime reference colony in 2001, showed no differences in organismal thyroid status across sites, but gulls from the high sites had enlarged thyroid glands and depressed thyroid gland hormone stores. Here we discuss the evidence that ecological exposure to PHAHs are responsible for thyroid deficiencies in gulls and that during development these deficiencies lead to developmental abnormalities in young gulls from highly contaminated Great Lakes sites.