Characterization of the avian aryl hydrocarbon receptor 1 from blood using non-lethal sampling methods.

The amino acid sequence of the aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) is an important determinant of sensitivity to dioxin-like compounds in avian species. We are interested in surveying AHR1 LBD sequences in a large number of birds as a means of identifying species that are particularly sensitive to dioxin-like compounds. Our original method for determining AHR1 LBD genotype used liver tissue and required lethal sampling. Here we present two alternate methods for determining AHR1 LBD genotype which use non-lethal sampling and are more appropriate for ecologically sensitive species. First, we establish that AHR1 LBD mRNA is expressed in avian blood and test a variety of blood collection and handling protocols in order to establish a method that is convenient for field collections. Our findings also identify which types of archival blood samples might be appropriate for AHR1 LBD sequence determination. Second, we present a method for obtaining AHR1 LBD coding sequences from DNA. A DNA-based method is advantageous because DNA can be isolated from many tissue types, is more stable than RNA, and requires less specific sample handling and preservation. This work extends applicability of a genetic screen for dioxin sensitivity to a larger number of species and sample types including endangered species and potentially museum specimens.

3,3',4,4',5-Pentachlorobiphenyl (PCB 126) impacts hepatic lipid peroxidation, membrane fluidity and beta-adrenoceptor kinetics in chick embryos.

Polychlorinated biphenyls (PCB) and other aryl hydrocarbon receptor (AHR) agonists induce oxidative stress and alter membrane lipid peroxidation and fluidity. This study tested the hypothesis that PCB-induced changes in membrane properties impact membrane beta-adrenoceptor (beta-AR) affinity and capacity in chick embryo hepatocytes. Embryos were injected into the air cell with 1.6 microg 3,3',4,4',5-pentachlorobiphenyl (PCB 126)/kg egg at day 0, and incubated to day 19 when livers were removed. This dose resulted in hepatic PCB 126 levels of 0.67 ng/g liver or 10.2 ng/g liver lipid; levels in untreated embryos were non-detectable. Hepatic microsomal EROD activity was elevated by approximately 12-fold and embryo mortality was significantly increased compared with the untreated group. Hepatic lipid peroxidation increased and membrane order (steady-state fluorescence anisotropy values) decreased with in ovo PCB 126 exposure. Consistent with changes in membrane structure, hepatic beta-AR affinity for CGP 12177 significantly decreased (Kd increased) without changes in receptor numbers. This study demonstrates that in ovo exposure to PCB 126 in chick eggs significantly impacted embryo survival, and this was correlated with altered hepatic membrane structure and ultimately membrane function.

Assessment of biological effects of chlorinated hydrocarbons in osprey chicks.

Osprey (Pandion haliaetus) eggs were collected during 1995 and 1996 at seven sites along the Fraser and Columbia River systems of British Columbia, Canada, and Washington and Oregon, USA. Fifty-four eggs were placed into a laboratory incubator. Thirty-eight of the hatched chicks were sacrificed within 24 h. Hatching success did not differ among sites and therefore between treatment and reference areas. Residual yolk sacs of eggs collected downstream of the large bleached-kraft pulp mill at Castlegar contained greater mean concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 2,930 ng/kg lipid) compared with reference sites such as the Nechako River, an upper tributary of the Fraser system (33.7 ng/kg). Total polychlorinated biphenyls (PCBs) in yolk sacs were also higher at Castlegar and in samples from the Columbia River downstream of Portland, Oregon, compared with those from the Nechako River. Concentrations of measured chemicals, including TCDD toxic equivalents (TEQs), total PCBs, p,p'-dichlorodiphenylethylene (p,p'-DDE), and other organochlorines were not different in eggs that failed to hatch compared with calculated whole-egg values for hatched eggs. There were significant biochemical responses; a hepatic cytochrome P4501A (CYP1A) cross-reactive protein was detected in all samples tested and correlated positively with ethoxyresorufin o-deethylase (EROD) activity and yolk sac concentrations of TEQs and total PCBs. Tissue concentrations of vitamin A compounds varied among sites and correlated positively with yolk sac concentrations of TEQs and PCBs. Morphological, histological, and other physiological parameters, including chick growth, edema, deformities, and hepatic and renal porphyrin concentrations, neither varied among sites nor showed concentration-related effects.

Role of oxidative stress and antioxidant defense in 3,3',4,4',5-pentachlorobiphenyl-induced toxicity and species-differential sensitivity in chicken and duck embryos.

The role of oxidative stress and antioxidant defense in 3,3',4,4',5-pentachlorobiphenyl (PCB 126)-induced toxicity and species-specific sensitivity was examined in White Leghorn chicken (Gallus domesticus) and Pekin duck (Anas platyrhynchos) embryos. Eggs were injected into the air cell with 0.4-1.6 microgram PCB 126/kg egg in corn oil prior to incubation. Lipid peroxidation measured by thiobarbituric acid reactive substances (TBARS), the GSSG:GSH ratio, and glutathione peroxidase (GPox) activities were determined in liver and adipose tissue of day 19 chicken and day 26 duck embryos. In chicken embryos, PCB 126 increased mortality and the incidence of edema and liver lesions, decreased embryo size, increased eye and head malformations, and markedly reduced fat storage. In contrast, no effects on the endpoints were observed in duck embryos even at the highest dose used in chicken embryos. PCB 126 increased hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity in a dose-dependent manner in chicken but not duck embryos. PCB 126 significantly increased TBARS levels in liver and to a greater degree in adipose tissue of chicken embryos, indicating that adipose tissue is a sensitive target for this compound. Increases in lipid peroxidation by PCB 126 were associated with significant decreases in GPox activity in these tissues. These biochemical changes support oxidative stress playing a role in PCB 126-induced embryo toxicity while antioxidant defenses provided protection against oxidative damage induced by this compound. Ducks, the less-sensitive species, showed higher basal levels of hepatic GPox than chickens, suggesting that this antioxidant enzyme may contribute to the differences in sensitivity to this compound between the two species.

Towards molecular understanding of species differences in dioxin sensitivity: initial characterization of Ah receptor cDNAs in birds and an amphibian.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to most vertebrate animals, but there are dramatic species differences in sensitivity, both within and among vertebrate classes. For example, studies in cultured avian hepatocytes have revealed differential sensitivity of birds to PHAHs [Kennedy et al. (1996). Toxicol. Appl. Pharmacol., 141, 214-230]. Differences in the characteristics or expression of the aryl hydrocarbon receptor (AHR) could contribute to these species differences in PHAH responsiveness. To investigate the molecular mechanism of differential PHAH sensitivity, we have begun to characterize the AHR in white leghorn chicken (Gallus gallus), Pekin duck (Anas platyrhynchos), and common tern (Sterna hirundo), as well as an amphibian, mudpuppy (Necturus maculosus). Partial AHR cDNAs encompassing the helix-loop-helix and PAS domains were cloned and sequenced. Comparison of amino acid sequences in this region indicated a high degree of sequence conservation among the bird species (97% amino acid identity). The percent identity between bird sequences and either mouse or mudpuppy was lower (79%); the mudpuppy AHR was 74% identical to the mouse AHR. Phylogenetic analysis of these and other AHR amino acid sequences showed that the bird and mudpuppy AHRs were more closely related to mammalian and fish AHR1 forms than to fish AHR2. Future studies include the in vitro expression and functional characterization of AHRs from these and other non-mammalian vertebrates.

Relationships between environmental organochlorine contaminant residues, plasma corticosterone concentrations, and intermediary metabolic enzyme activities in Great Lakes herring gull embryos.

Experiments were conducted to survey and detect differences in plasma corticosterone concentrations and intermediary metabolic enzyme activities in herring gull (Larus argentatus) embryos environmentally exposed to organochlorine contaminants in ovo. Unincubated fertile herring gull eggs were collected from an Atlantic coast control site and various Great Lakes sites in 1997 and artificially incubated in the laboratory. Liver and/or kidney tissues from approximately half of the late-stage embryos were analyzed for the activities of various intermediary metabolic enzymes known to be regulated, at least in part, by corticosteroids. Basal plasma corticosterone concentrations were determined for the remaining embryos. Yolk sacs were collected from each embryo and a subset was analyzed for organochlorine contaminants. Regression analysis of individual yolk sac organochlorine residue concentrations, or 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs), with individual basal plasma corticosterone concentrations indicated statistically significant inverse relationships for polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDDs/PCDFs), total polychlorinated biphenyls (PCBs), non-ortho PCBs, and TEQs. Similarly, inverse relationships were observed for the activities of two intermediary metabolic enzymes (phosphoenolpyruvate carboxykinase and malic enzyme) when regressed against PCDDs/PCDFs. Overall, these data suggest that current levels of organochlorine contamination may be affecting the hypothalamo-pituitary-adrenal axis and associated intermediary metabolic pathways in environmentally exposed herring gull embryos in the Great Lakes.

Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife.

An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in vivo evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties.

Subchronic toxicity of PCB 105 (2,3,3',4,4'-pentachlorobiphenyl) in rats.

The toxicity of 2,3,3',4,4'-pentachlorobiphenyl (PCB 105) was investigated in Sprague-Dawley rats following dietary exposure to this substance at levels of 0, 0.05, 0.5, 5 or 50 ppm for 13 weeks. Growth rate and food consumption were not affected and no clinical signs of toxicity were observed. Increased incidences of enlarged, fatty liver and decreased thymic weight were observed in the highest-dose groups of both genders; these groups also had elevated hepatic microsomal ethoxyresorufin deethylase activity and uroporphyrin. Significant increases in serum cholesterol and hepatic pentoxyresorufin dealkylase activity were observed in the highest-dose males and two highest-dose females. By contrast, liver UDP-glucuronosyl transferase activity was elevated in the two highest-dose males and the highest-dose females. Urinary ascorbic acid excretion was increased in the highest-dose males. While the amount of vitamin A was decreased dose-dependently, starting at 0.5 ppm in the liver of both sexes and in the lung of the females, the level in the kidney of the highest-dose group was increased. Administration of PCB 105 resulted in decreased dopamine in the caudate nucleus region of the brain in males and homovanillic acid in caudate nucleus and nucleus accumbens of females. Increased 5-hydroxytryptamine and 5-hydroxyindoleacetic acid were observed in the substantia nigra region of both sexes, with most of the increases being seen in highest-dose females. Anemia, characterized by decreased hemoglobin, hematocrit and red cell indices, occurred in the highest-dose group, as did eosinophilia. Treatment with PCB 105 caused dose-dependent histopathological changes in the liver and thyroid. Thymic changes were observed in the highest-dose males and two highest-dose females. Tissue residue data showed a dose-dependent accumulation of this congener in fat, liver and spleen, kidney and brain. Based on these data the no-observable-effect level of PCB 105 was judged to be 0.05 ppm or 3.9 microg kg(-1) body wt. day(-1) in males and 4.2 microg kg(-1) body wt. day(-1) in females.

Environmental contamination and developmental abnormalities in eggs and hatchlings of the common snapping turtle (Chelydra serpentina serpentina) from the Great Lakes-St Lawrence River basin (1989-1991).

During 1989-1991, we assessed developmental abnormalities in embryos and hatchlings from eggs of the common snapping turtle (Chelydra serpentina serpentina). Eggs were collected and artificially incubated from eight sites in Ontario, Canada and Akwesasne/New York, USA. In eggs from the same clutches we measured 20 organochlorine pesticides, 48 polychlorinated biphenyl (PCBs) congeners including 6 non-ortho PCBs, 8 polychlorinated dibenzodioxins (PCDDs), 14 polychlorinated dibenzofurans (PCDFs) and total mercury. We found a significant increase in abnormal development with increasing polychlorinated aromatic hydrocarbon exposure in eggs, particularly PCDD and PCDF concentrations. In contrast, the risk of abnormality was not significantly higher as toxic equivalent concentrations increased in eggs. We also found significant 7-ethoxyresorufin O-deethylase and Cytochrome P4501A responses in livers of hatchling turtles from Lake Ontario relative to hatchlings from a clean, inland site whereas we did not find any evidence of porphyria in the hatchlings from either site.

Subchronic toxicity of 2,2',3,3',4,4'-hexachlorobiphenyl in rats.

The subchronic toxicity of 2,2',3,3',4,4'-hexachlorobiphenyl (PCB 128) was investigated in rats following dietary exposure at 0, 0.05, 0.5, 5, or 50 ppm for 13 wk. The growth rate was not affected by treatment and no apparent clinical signs of toxicity were observed. There was a significant increase in liver weight in the 50 ppm females. The liver ethoxyresorufin deethylase (EROD) activity was increased by five- and fourfold in the highest dose males and females, respectively, while aminopyrine demethylase (ADPM) activity was significantly increased only in the highest dose females. Liver vitamin A was significantly reduced in the highest dose females. No other biochemical or hematological effects were observed. Treatment-related histopathological changes were seen in the thyroid and liver, and to a lesser extent in the bone marrow and thymus. Residue data showed a dose-dependent accumulation of PCB 128 in the following tissues: fat, liver, kidney, brain, spleen, and serum, with the highest concentration being found in fat followed by liver and kidney. Based on these data, the no-observable-adverse-effect level of PCB 128 was judged to be 0.5 ppm in diet or 42 micrograms/kg body weight.