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Research has demonstrated that the sensitivity of avian species to the embyrotoxic effects of dioxin-like compounds can be predicted by the amino acid identities at two key sites within the ligand-binding domain of the aryl hydrocarbon receptor 1 (AhR1). The domestic chicken (Gallus gallus domesticus) has been established as a highly sensitive species to the toxic effects of dioxin-like compounds. Results from genotyping and in vitro assays predict that the European starling (Sturnus vulgaris) is also highly sensitive to dioxin-like compound toxicity. The objective of the present study was to test that prediction in vivo. To do this, we used egg injections in field nesting starlings with 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), a dioxin-like polychlorinated biphenyl. Eggs were dosed with either the vehicle control or 1 of 5 doses (1.4, 7.1, 15.9, 32.1, and 52.9 ng PCB-126/g egg). A dose-dependent increase in embryo mortality occurred, and the median lethal dose (LD50; 95% confidence interval [CI]) was 5.61 (2.33–9.08) ng/g. Hepatic CYP1A4/5 messenger RNA (mRNA) expression in hatchlings also increased in a dose-dependent manner, with CYP1A4 being more induced than CYP1A5. No effect of dose on morphological measures was seen, and we did not observe any overt malformations. These results indicate that, other than the chicken, the European starling is the most sensitive species to the effects of PCB-126 on avian embryo mortality reported to date, which supports the prediction of relative sensitivity to dioxin-like compounds based on amino acid sequence of the AhR1. Environ Toxicol Chem 2014;33:2753–2758. © 2014 SETAC

The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highest doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism.

1,2-Dibromo-4-(1,2-dibromoethyl)-cyclohexane (DBE-DBCH; formerly abbreviated as TBECH) and tris(methylphenyl) phosphate (TMPP; formerly abbreviated as TCP) are additive flame retardants that are detected in the environment and biota. A recent avian in vitro screening study of 16 flame retardants identified DBE-DBCH and TMPP as important chemicals for follow-up in ovo evaluation based on their effects on cytotoxicity and mRNA expression in avian hepatocytes. In this study, technical mixtures of DBE-DBCH and TMPP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 54,900 ng/g and from 0 to 261,400 ng/g, respectively, to determine effects on pipping success, development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations. Both compounds were detectable in embryos at pipping and the β-DBE-DBCH isomer was depleted more rapidly than the α-isomer in tissue samples. DBE-DBCH had limited effects on the endpoints measured, with the exception of the up-regulation of two phase I metabolizing enzymes, CYP3A37 and CYP2H1. TMPP exposure caused embryonic deformities, altered growth, increased liver somatic index (LSI) and plasma bile acid concentrations, and altered mRNA expression levels of genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Overall, TMPP elicited more adverse molecular and phenotypic effects than DBE-DBCH albeit at concentrations several orders of magnitude greater than those detected in the environment. The increase in plasma bile acid concentrations was a useful phenotypic anchor as it was associated with a concomitant increase in LSI, discoloration of the liver tissue, and modulation of hepatic genes involved with xenobiotic and lipid metabolism.

Results of recent studies showed that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are equipotent in domestic chicken (Gallus gallus domesticus) while PeCDF is more potent than TCDD in ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica). To elucidate the mechanism(s) underlying these differences in relative potency of PeCDF among avian species, we tested the hypothesis that this is due to species-specific differential binding affinity of PeCDF to the aryl hydrocarbon receptor 1 (AHR1). Here, we modified a cell-based binding assay that allowed us to measure the binding affinity of dioxin-like compounds (DLCs) to avian AHR1 expressed in COS-7 (fibroblast-like cells). The results of the binding assay show that PeCDF and TCDD bind with equal affinity to chicken AHR1, but PeCDF binds with greater affinity than TCDD to pheasant (3-fold) and Japanese quail (5-fold) AHR1. The current report introduces a COS-7 whole-cell binding assay and provides a mechanistic explanation for differential relative potencies of PeCDF among species of birds.

World Health Organization (WHO) toxic equivalency factors are used to calculate toxic equivalent (TEQ) concentrations of complex mixtures of dioxin-like compounds (DLCs), such as polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls (PCBs), for mammals, fish and birds. The TEQ concept assumes that all species of a taxa respond with similar sensitivity to individual DLCs, but several reports do not support this assumption for birds. Our laboratory is conducting research to attempt to uncover the fundamental mechanism(s) underlying the reasons why avian species differ in sensitivity to DLCs. The present study determined concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) on ethoxyresorufin-O-deethylase (EROD) activity in primary cultures of northern bobwhite quail (Colinus virginianus) hepatocytes. Bobwhite quail were studied because (1) this species is used in the laboratory for toxicity testing and (2) the amino acids at all locations within the ligand binding domain (LBD) of aryl hydrocarbon receptor 1 (AHR1) in bobwhite quail and ring necked pheasant (Phasianus colchicus) are identical. Because earlier work indicated the importance of the identity of amino acids at key sites within the AHR1 LBD, we hypothesized that bobwhite quail and ring necked pheasant hepatocytes should have similar sensitivity to EROD induction by DLCs. ECthreshold-based relative sensitivity of the bobwhite quail compared to chicken for TCDD, PeCDF and PCB 126 was 0.11, 0.17 and 0.02, respectively. The rank order of potency was PeCDF > TCDD > PCB 126. The results confirm that bobwhite quail and ring-necked pheasant hepatocytes have similar sensitivity to EROD induction by TCDD, PeCDF and PCB 126.

Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are organic flame retardants detected in the environment and biota for which avian toxicological data are limited. In this study, domestic chicken eggs were injected with TCPP or TDCPP (maximum dose = 51,600 and 45,000ng/g egg, respectively) to determine dose-dependent effects on pipping success, development, hepatic messenger RNA (mRNA) expression levels of genes associated with xenobiotic metabolism and the thyroid hormone (TH) pathway, and TH levels following 20-22 days of incubation. Neither compound reduced pipping success; however, TCPP significantly delayed pipping at 9240 and 51,600ng/g and reduced tarsus length at 51,600ng/g. TDCPP exposure resulted in significant decreases in head plus bill length, embryo mass, and gallbladder size at 45,000ng/g and reduced plasma free T4 levels at 7640ng/g. Type I deiodinase, liver fatty acid-binding protein, and cytochrome P450 (CYP) 3A37 mRNA levels were significantly induced by TCPP, whereas TDCPP induced CYP3A37 and CYP2H1. Chemical analysis of egg contents at incubation days 0, 5, 11, 18, and 19 revealed that > 92% of the injected TCPP or TDCPP concentration was detectable up to day 5; however, < 1% was detected by day 19. The observed phenotypic responses to TCPP and TDCPP exposure may be associated with disruption of the TH axis, which is critical for normal growth and development in birds. The effects of TDCPP on the gallbladder indicate that the disturbance of lipid metabolism is a likely mechanism of toxicity.

Primary cultures of ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica) embryo hepatocytes were used to compare the potencies of highly purified hexachlorobenzne (HCB-P), reagent-grade HCB (RG-HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. HCB-P, RG-HCB and TCDD all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNA. Induction was not caused by contamination of HCB with polychlorinated dibenzo-p-dioxins, dibenzofurans or biphenyls. Based upon a comparison of the EC50 and ECthreshold values for EROD and CYP1A4/5 concentration–response curves, the potency of HCB relative to TCDD was 0.001 in ring-necked pheasant and 0.01 in Japanese quail embryo hepatocytes. Differences in species sensitivity to HCB were found to be mainly dictated by differences in species sensitivity to TCDD rather than differences in the absolute potency of HCB. Consequently, ring-necked pheasant and Japanese quail embryo hepatocytes were found to be equally sensitive to HCB exposure. Species sensitivity comparisons were also made with chicken (Gallus gallus domesticus) and revealed that chicken embryo hepatocytes were less responsive to EROD induction (lower maximal response) by HCB compared to the embryo hepatocytes of pheasant and quail.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and tris(1-chloropropyl) phosphate (TCPP) belong to a group of chemicals collectively known as triester organophosphate flame retardants (OPFRs). OPFRs are used in a wide range of consumer products and have been detected in biota, including free-living avian species; however, data on toxicological and molecular effects of exposure are limited. An in vitro screening approach was used to compare concentration-dependent effects of TDCPP and TCPP on cytotoxicity and messenger RNA (mRNA) expression in cultured hepatocytes and neuronal cells derived from embryonic chickens. TDCPP was toxic to hepatocytes (LC₅₀ = 60.3 ± 45.8μM) and neuronal cells (LC₅₀ = 28.7 ± 19.1μM), whereas TCPP did not affect viability in either cell type up to the highest concentration administered, 300μM. Real-time reverse transcription-PCR revealed alterations in mRNA abundance of genes associated with phase I and II metabolism, the thyroid hormone (TH) pathway, lipid regulation, and growth in hepatocytes. None of the transcripts measured in neuronal cells (D2, D3, RC3, and Oct-1) varied in response to TDCPP or TCPP exposure. Exposure to ≥ 10μM TDCPP and TCPP resulted in significant upregulation of CYP2H1 (4- to 8-fold), CYP3A37 (13- to 127-fold), and UGT1A9 (3.5- to 7-fold) mRNA levels. Transthyretin was significantly downregulated more than twofold by TCPP at 100μM; however, TDCPP did not alter its expression. Liver fatty acid-binding protein, TH-responsive spot 14-α, and insulin-like growth factor-1 were all downregulated (up to 10-fold) in hepatocytes exposed to ≥ 0.01μM TDCPP and TCPP. Taken together, our results indicate that genes associated with xenobiotic metabolism, the TH pathway, lipid regulation, and growth are vulnerable to TDCPP and TCPP administration in cultured avian hepatocytes. The mRNA expression data were similar to those from a previous study with hexabromocyclododecane.

There is growing interest in assessing the neurotoxic and endocrine disrupting potential of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, prenatal and postnatal development and behavior. PFC exposure altered hormone levels and the expression of hormone-responsive genes in mammalian and aquatic species. This study is the first to assess the effects of PFCs on messenger RNA (mRNA) expression in primary cultures of neuronal cells in two avian species: the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following thyroid hormone (TH)–responsive genes were examined using real-time reverse transcription-PCR: type II iodothyronine 5′-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif–binding factor (Oct-1), and myelin basic protein. Several PFCs altered the mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. Short-chained PFCs (less than eight carbons) altered the expression of TH-responsive genes (D2, D3, TTR, and RC3) in chicken embryonic neuronal cells to a greater extent than long-chained PFCs (more than or equal to eight carbons). Variable transcriptional changes were observed in herring gull embryonic neuronal cells exposed to short-chained PFCs; mRNA levels of Oct-1 and RC3 were upregulated. This is the first study to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and may provide insight into the possible mechanisms of action of PFCs in the avian brain.

Some uncertainty exists regarding the purity of hexachlorobenzene (HCB) used in past toxicity studies. It has been suggested that reported toxic and biochemical effects initially attributed to HCB exposure may have actually been elicited by contamination of HCB by polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Herein, primary cultures of chicken embryo hepatocytes (CEH) were used to compare the potencies of two lots of reagent-grade hexachlorobenzene (HCB-old [HCB-O] and HCB-new [HCB-N]), highly purified HCB (HCB-P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A4 (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. The study also compared the EROD- and CYP1A4/5 mRNA-inducing potencies of HCB to the potencies of two mono-ortho substituted polychlorinated biphenyls (PCBs), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105) and 2,3'4,4',5-pentachlorobiphenyl (PCB 118). HCB-O, HCB-N and HCB-P all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNAs. Induction was not caused by contamination of HCB with PCDDs or PCDFs. Based upon a comparison of the EC(50) and EC(threshold) values for EROD and CYP1A4/5 mRNA concentration-response curves, the potency of HCB relative to the potency of TCDD was 0.0001, and was similar to that of PCB 105 and PCB 118. The maximal EROD activity and CYP1A4/5 mRNA expression differed greatly between HCB and TCDD, and may contribute to an overestimation of the ReP value calculated for highly purified HCB.

Egg injection studies were performed to confirm a proposed model of relative sensitivity of birds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this model, species are classified as belonging to one of three categories of sensitivity based on amino acid substitutions in the ligand-binding domain of the aryl hydrocarbon receptor. Embryo lethality and relative potencies of 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) were compared with TCDD for Japanese quail (Coturnix japonica; least sensitive), Common pheasant (Phasianus colchicus; moderately sensitive), and White Leghorn chicken (Gallus gallus domesticus; most sensitive). Doses ranging from 0.044 to 37 pmol/g egg (0.015-12 ng/g egg) were injected into the air cell of eggs prior to incubation. LD(50) (95% confidence intervals) values, based on rate of hatching for TCDD, PeCDF, and TCDF, were 30 (25-36), 4.9 (2.3-9.2), and 15 (11-24) pmol/g egg for the quail, 3.5 (2.3-6.3), 0.61 (0.28-1.2), and 1.2 (0.62-2.2) pmol/g egg for pheasant, and 0.66 (0.47-0.90), 0.75 (0.64-0.87), and 0.33 (0.23-0.45) pmol/g egg for chicken, respectively. LD(50)-based relative potencies of PeCDF and TCDF were 6.1 and 2.0 for quail, 5.7 and 2.9 for pheasant, and 0.88 and 2.0 for chicken, respectively. TCDD was not the most potent compound among the species tested, with PeCDF and TCDF being more potent than TCDD in the quail and pheasant. TCDF was the most potent in chicken. Species sensitivity was as expected for TCDD and TCDF, whereas for PeCDF, the chicken and pheasant were similar in sensitivity and both were more sensitive than the quail. Results from companion in vitro studies are generally similar to those reported here with a few exceptions.

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.

Relative potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7, 8-tetrachlorodibenzofuran (TCDF) were determined in vitro in primary hepatocyte cultures of chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus), and Japanese quail (Coturnix japonica) embryos. Concentration-dependent effects on ethoxyresorufin O-deethylase (EROD) activity and expression of cytochrome P4501A4 and cytochrome P4501A5 (CYP1A4 and CYP1A5) messenger RNA (mRNA) were determined in hepatocytes exposed to serial dilutions of TCDD, PeCDF, or TCDF for 24 h. In chicken hepatocytes, the three compounds were equipotent inducers of EROD activity and CYP1A4/CYP1A5 mRNA expression. However, in ring-necked pheasant and Japanese quail hepatocytes, PeCDF was more potent than TCDD (3- to 5-fold in ring-necked pheasant and 13- to 30-fold in Japanese quail). Among species, the rank order of sensitivity (most to least) to EROD and CYP1A4/CYP1A5 mRNA induction for TCDD and TCDF was chicken > ring-necked pheasant > Japanese quail. In contrast, the three species were approximately equisensitive to EROD and CYP1A4/CYP1A5 mRNA induction by PeCDF. It has generally been assumed that TCDD is the most potent "dioxin-like compound" (DLC) and that the chicken is the most sensitive avian species to CYP1A induction by all DLCs. This study indicates that PeCDF is more potent than TCDD in ring-necked pheasant and Japanese quail hepatocytes and that ring-necked pheasant, Japanese quail, and chicken hepatocytes are equally sensitive to CYP1A induction by PeCDF.

We describe a statistically significant correlation between two well-characterized responses to dioxin-like compounds in birds; induction of 7-ethoxyresorufin-O-de- ethylase (EROD) activity in cultured hepatocytes, and embryo mortality. Data were obtained from a review of the literature. EROD EC50 values for 2,3,7,8-tetrachlo- rodibenzo-p-dioxin (TCDD) and 6 polychlorinated biphe- nyls (PCBs) were strongly correlated with LD50 values in chicken embryos (r 2 = 0.93, P \ 0.005). Similarly, EROD EC50 values for TCDD and a potent dioxin-like compound, PCB 126, were correlated with embryonic LD50 values in different species of birds (chicken, ring-necked pheasant, turkey, double-crested cormorant, and common tern) (r 2 = 0.92, P \ 0.005). Our findings contribute to a devel- oping understanding of the molecular basis for differential dioxin sensitivity in birds, and validate the EROD bioassay as a useful predictive tool for ecological risk assessment.

Many studies have characterized the effects of perfluoroalkyl compounds (PFCs) in mammalian species, but limited information exists on the effects of PFCs in birds. PFCs have been detected in serum and liver of avian wildlife worldwide. While the molecular mechanisms have yet to be elucidated in detail, PFCs alter lipid metabolism through peroxisome proliferation, xenobiotic metabolism by activating the cytochrome P450 (CYP) system, and serum cholesterol levels by inducing or repressing key genes. Here, we employed a simple messenger RNA (mRNA) screening method using quantitative PCR to assess the effects of PFCs on mRNA expression in chicken embryo hepatocytes (CEH). CEH cultures were treated with perfluoroalkyl sulfonates and perfluoroalkyl carboxylates of varying chain lengths and linear or technical grade potassium perfluoro-1-octane sulfonate (L-PFOS and T-PFOS). T-PFOS comprised 80% perfluorooctane sulfonate isomers (62% linear) and various PFCs and inorganic salts. Relative mRNA expression levels of the following genes were examined: acyl-CoA oxidase (ACOX), liver fatty acid-binding protein (L-FABP), CYP1A4/1A5 and CYP4B1, 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase, and sterol regulatory element-binding protein 2 (SREBP2). Compared to L-PFOS, T-PFOS altered the mRNA expression level of more genes and produced greater fold changes. L-FABP was upregulated by PFCs greater than or equal to eight carbons, while CYPs were upregulated by PFCs less than or equal to eight carbons. ACOX, HMG-CoA, and SREBP2 showed little to no change following PFC exposure. This study is the first to expose CEH cultures to multiple PFCs in vitro and demonstrates that exposure to PFC solutions of different isomeric content or chain length causes variable transcriptional responses.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and other halogenated aromatic hydrocarbons (HAHs) elicit a variety of adverse biological effects on the cardiovascular systems of mammalian, piscine and avian species. Many of the cardiotoxic effects of HAHs are mediated by the aryl hydrocarbon receptor (AHR). Induction of cytochrome P4501A (CYP1A) is a well-known AHR-dependent response to HAHs in the liver, but there are a limited number of studies on CYP1A induction by these compounds in the heart. We used an in vitro approach to examine effects of TCDD and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) on CYP1A in the avian heart. The responses of primary cultures of chicken embryo cardiomyocytes (CEC) and chicken embryo hepatocytes (CEH) to TCDD and PCB 77 were compared using immunofluorescence staining for CYP1A, the ethoxyresorufin-O-deethylase (EROD) assay, and real-time RT-PCR analysis of CYP1A4 mRNA and CYP1A5 mRNA. Immunofluorescent detection of CYP1A indicated that induction of CYP1A by TCDD was localized within the cytoplasm of CEC cells. EROD activity and CYP1A4/5 mRNA levels were strongly induced in CEC and CEH cultures by TCDD and PCB 77, and the shapes of the concentration-response curves in CEC and CEH cultures were similar. The studies provide clear evidence that the AHR signaling pathway is induced by TCDD and PCB 77 in CEC, and establish a new in vitro approach for studying the effects of HAHs in the avian heart. Induction of CYP1A5 by TCDD in avian cardiomyocytes is a novel finding, and might help direct future studies on mechanisms of action of HAHs in the heart.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in a wide range of consumer products. Previous studies have suggested that PBDEs can disrupt thyroid hormone homeostasis and the developing central nervous system in rodents, but few studies have determined whether PBDEs cause similar effects in birds. An in vitro method was used to determine effects of a commercial PBDE flame retardant (DE-71) on mRNA expression in primary chicken neuronal cells derived from the cerebral hemisphere. Real-time RT-PCR assays were developed to quantify changes in mRNA abundance of genes associated with the thyroid hormone pathway; thyroid hormone receptors (TRalpha and TRbeta) and transthyretin (TTR). We also used a new differential display PCR methodology, fluorescent RNA arbitrarily primed PCR (FRAP-PCR), to determine additional effects of DE-71 on mRNA expression. Neither of the TRs responded to DE-71 exposure, but TTR mRNA decreased approximately 2-fold following exposure to 0.1, 1 and 3 microM DE-71. Candidate transcripts associated with signal transduction, neurosteroidogenesis, and neurite and axonal growth were up-regulated by DE-71 exposure. Taken together, the findings from this study indicate that this in vitro cell culture method can be used to characterize the effects of PBDEs in the avian brain.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces cytochrome P4501A (CYP1A) catalyzed ethoxyresorufin-O-deethylase (EROD) activity in chickens and other avian species. To investigate mechanisms underlying the effectiveness of EROD activity as a biomarker for exposure to dioxin-like compounds in avian models, we characterized inter-species differences in isoform-specific CYP1A mRNA expression, induction, and stability in chickens (Gallus gallus domesticus) and herring gulls (Larus argentatus). Exposure to 100 nM TCDD significantly increased CYP1A4 and CYP1A5 mRNA expression in chicken and herring gull embryo hepatocyte cultures. Chicken CYP1A4 and CYP1A5 were induced 61-fold and 25-fold respectively. The herring gull isoforms were induced 2.2- and 4.3-fold respectively. In both species, the isoform that was preferentially induced exhibited lower constitutive expression. Half-lives of chicken CYP1A4, chicken CYP1A5, and herring gull CYP1A5 mRNA ranged from 5.0 to 7.0 h in cultured hepatocytes. The half-life of herring gull CYP1A4 mRNA was 2.5 h. Our findings indicate that expression, induction, and stability of CYP1A4 and CYP1A5 mRNA are differentially regulated in chickens and herring gulls. In particular, CYP1A4 is preferentially induced in chickens, while CYP1A5 is preferentially induced in herring gulls. We propose that CYP1A5 mRNA expression may be a sensitive biomarker of exposure to dioxin-like compounds in some avian species.

Recent years have witnessed an explosion of different genomic approaches which, through a combination of advanced biological, instrumental and bioinformatic techniques, can yield a previously unparalleled amount of data concerning the molecular and biochemical status of organisms. Fueled partially by large, well-publicized efforts such as the human genome project, genomic research has become a rapidly growing topical area in multiple biological disciplines. Since 1999 when the term toxicogenomics was coined to describe the application of genomics to toxicology, a citation analysis reveals a rapid increase in publications dealing with the topic. The potential utility of toxicogenomics in toxicological research and regulatory activities has been the subject of scientific discussions and, as with any new technology, there is a wide range of opinion. The purpose of this feature article is to consider roles of toxicogenomics in the field of regulatory ecotoxicology, explore current limitations in the science and practice of genomics, and propose possible avenues to approach and resolve some of the major challenges. A significant amount of input to our analysis came from a workshop sponsored by the Society of Environmental Toxicology and Chemistry in Pellston, MI in September, 2005.

Common terns (Sterna hirundo) and roseate terns (Sterna dougallii) breed on Bird Island, Massachusetts, USA, near a Superfund site highly contaminated with polychlorinated biphenyls (PCBs). Observations of skewed sex ratios and female–female pairings among endangered roseate terns (Nisbet and Hatch (1999) Ibis 141, 307) suggested the possibility of contaminant-related endocrine disruption in these birds and prompted investigation of common terns as a surrogate species. In 1993 and 1994, 60–90% of pipping male common tern embryos sampled exhibited ovarian cortical tissue in their testes (ovotestes) (Nisbet et al. (1996) Bull. Environ. Contam. Toxicol. 57, 895; Hart et al. (1998) Mar. Environ. Res. 46, 174). To examine the possible impact of ovotestes on the reproductive capabilities of common terns, we examined gonadal histology in common tern prefledglings (approximately 21 days old) collected from Bird Island in 1995. As a measure of embryonic contaminant exposure, contaminants were measured in a subset of eggs collected from the same nests as the prefledglings. Concentrations of total PCBs in these eggs ranged from 14.4 to 546 µg/g lipid. No evidence of ovotesticular development was observed in any of the 19 male prefledglings examined. Some gonadal irregularities were observed, including small nodules of testicular tissue within the epithelial capsule of the testes, but these were judged not likely to affect testicular function. There was no relationship between any observed irregularities and levels of contaminants present in the matched eggs. The results suggest that the ovotestes that occur in 60–90% of pipping common tern embryos from this site become fully regressed by approximately 21 days posthatch. Our data from this and previous studies are consistent with the idea that ovotestes occur naturally in some individual common terns at hatching, although the frequency of occurrence may be enhanced by exposure to chlorinated organic contaminants such as PCBs. In either case, we suggest that the presence of ovotestes in common tern embryos from PCB-contaminated sites such as Bird Island does not lead to permanent alterations in gonadal histology that would be expected to impair reproductive function.

Graded doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were added to primary hepatocyte cultures of bald eagle (Haliaeetus leucocephalus) embryos to determine their sensitivity to induction of cytochrome P4501A (CYP1A) and porphyrin accumulation. No porphyrin accumulation was observed, but both CYP1A catalytic activity (using the ethoxyresorufin-O-deethylase (EROD) assay) and immunodetectable CYP1A were induced by relatively high concentrations of TCDD. Bald eagle hepatocytes were less sensitive to CYP1A induction than hepatocytes from any other avian species that we have studied to date. These in vitro results are in general agreement with recent assessments of field data, which indicate that bald eagles are relatively insensitive to some of the effects of TCDD and related compounds. Preparation of bald eagle hepatocytes was challenging because existing methods did not yield monolayers of cells. Here we describe details of a new method that was successful for bald eagle hepatocytes. This new method is used routinely in our laboratory to prepare hepatocyte cultures from birds for examination of various biochemical responses to environmental contaminants.

Dioxins and dioxin-like chemicals (DLCs) cause a suite of adverse effects in terrestrial species. Most of the adverse effects occur subsequent to binding to the aryl hydrocarbon receptor. Avian species vary in their sensitivity to the effects of DLCs and current research indicates that this is mediated by variations in the amino acid sequence within the ligand binding domain (LBD) of the aryl hydrocarbon receptor 1 (AHR1). Eighty-eight avian species have been classified into three broad categories of sensitivity, based on the amino acid variations within the AHR1 LBD: sensitive type 1 (Ile324_Ser380), moderately sensitive type 2 (Ile324_Ala380), and relatively insensitive type 3 (Val324_Ala380). Risk assessment of avian species can be complicated due to the variability in sensitivity among species. A predictive tool for selecting the priority species at a given site would have broad implications for the risk assessment community. We present a method for AHR1 genotyping using plucked feathers as a source of RNA. The method is extremely robust, requires minimal sample processing and handling, and eliminates the need for blood sampling or tissue collection from the species of interest. Using this method we were able to determine the amino acid sequence of the AHR LBD of three avian species: the chicken, the herring gull, and the zebra finch, and to categorize them based on the identity of amino acids at key sites within the LBD.

Avian species differ in sensitivity to the toxic effects of dioxin-like compounds (DLCs) and recent reports have provided insight into the molecular mechanisms underlying this variability. The sensitivity of avian species to DLCs is associated with the identity of amino acids at positions 324 and 380 within the ligand-binding domain (LBD) of the aryl hydrocarbon receptor 1 (AHR1). 6-formylindolo [3,2-b] carbazole (FICZ), a naturally produced photo-oxidation product of tryptophan, is a highly potent AHR ligand. Few studies have attempted to determine if there are species differences in AHR activation by FICZ in a systematic manner. Here we describe results from an in vitro assay that measures AHR1-mediated luciferase reporter gene activity to determine concentration-dependent effects of FICZ and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in COS-7 cells transfected with AHR1 constructs from chicken (Gallus gallus domesticus), ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica) and common tern (Sterna hirundo), and three mutant AHR1 constructs. Data were used to (a) compare the potency of FICZ and TCDD for each AHR1 construct (relative potency; ReP) and (b) the sensitivity of each construct to AHR1 activation by FICZ and TCDD (relative sensitivity; ReS). The results show that (1) FICZ was considerably more potent than TCDD in cells transfected with chicken AHR1 (RePavg=41), ring-necked pheasant AHR1 (RePavg=93), Japanese quail AHR1 (RePavg=1392) and common tern AHR1 (RePavg=1534), (2) there were no significant differences in sensitivity to FICZ in cells expressing chicken, pheasant, quail and tern AHR1, but there were significant differences in sensitivity to TCDD, (3) alteration of amino acids at positions 324 and 380 had no effect on avian AHR1 activity in response to FICZ, (4) there was no time-dependent change in the relative potency of FICZ in COS-7 cells, and (5) neither FICZ nor TCDD induced ethoxyresorufin O-deethylase (EROD activity) in COS-7 cells. Our results suggest that FICZ and TCDD activate avian AHR1 by different modes of interaction with AHR1.

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 μg 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.

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.

The World Health Organization toxic equivalency factors (WHO-TEFs) for birds were developed to simplify risk assessments of environmental mixtures of dioxin-like compounds (DLCs). Under this framework, toxic equivalents (TEQs) are used to represent the toxic potency of DLC mixtures as an equivalent concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Recently, a luciferase reporter gene (LRG) assay, measuring aryl hydrocarbon receptor 1 (AHR1)-mediated gene expression, accurately predicted the relative potency of individual polychlorinated biphenyl (PCB) congeners in different avian species. The study presented here used the LRG assay to predict the relative potency of Aroclors 1016, 1221, 1242, 1248, 1254, and 1260 on induction of LRG activity in cells transfected with chicken, ring-necked pheasant, or Japanese quail AHR1 constructs. LRG assay results were compared to (1) results of ethoxyresorufin-O-deethylase (EROD) assays conducted in chicken hepatocytes and (2) calculated TEQs from the literature. The relative potencies of Aroclors were similar between the LRG and EROD assays, and bioassay-derived TEQs for the chicken closely resembled calculated TEQs. However, LRG assay-derived TEQs for the Japanese quail construct were 1-2 orders of magnitude higher than calculated TEQs for Aroclors 1254 and 1016. These results suggest that the WHO-TEFs are not representative of relative PCB potency for all avian species.

Novel methods that predict the sensitivity of avian embryos to the toxic effects of dioxin-like compounds (DLCs) using either (1) knowledge of the identity of amino acids at key sites within the ligand binding domain of aryl hydrocarbon receptor 1 (AHR1) or (2) a luciferase reporter gene assay that measures AHR1 activation were recently reported. Results from both methods predict that European starling (Sturnus vulgaris) and domestic chicken (Gallus gallus domesticus) embryos have similar sensitivity to the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (TCDF). Chicken embryos are highly sensitive to DLC toxicity, and the prediction that starlings are equally sensitive is surprising given their widespread distribution and large population size. In an attempt to learn more about starling sensitivity to DLCs, we determined concentration-dependent effects of TCDD, PeCDF and TCDF on cytochrome P4501A4 and 1A5 (CYP1A4 and 1A5) mRNA levels in primary cultures of hepatocytes prepared from embryonic European starlings. It has been demonstrated that the sensitivity of avian hepatocytes to CYP1A4/5 induction is well correlated with LD50 values of DLCs for several avian species. The results of the present study indicate that European starling hepatocytes are indeed as sensitive as chicken hepatocytes to CYP1A4/5 induction after exposure to TCDD. However, starling hepatocytes are less sensitive than chicken hepatocytes to CYP1A4/5 induction by PeCDF and TCDF.

Concentration-dependent effects of halogenated aromatic hydrocarbons(HAHs) on cytochrome P4501A (CYP1A) induction in primary hepatocyte cultures prepared from embryos of chickens (four breeds), pheasants, turkeys, ducks (three breeds), and herring gulls were determined. CYP1A activity was estimated by measuring ethoxyresorufin O -deethylase (EROD) activity and the concentration of immunodetectable CYP1A was estimated using mouse monoclonal antibody 1-12-3 that was prepared against scup ( Stenotomus chrysops ) CYP1A1. The HAHs studied were 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3′,4,4′-tetrachlorobiphenyl (PCB 77, IUPAC nomenclature), 3,4,4′,5-tetrachlorobiphenyl (PCB 81), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), 2,3,3′,4,4′-pentachlorobiphenyl (PCB 105), and 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118). Two general types of comparisons were made: (1) relative potencies of compounds within a species (expressed relative to TCDD as induction equivalency factors, IEFs) and (2) relative sensitivity of each species to EROD induction by each compound. Three methods for estimating potency were compared. These were: (1) the concentration of inducer that produced a half-maximal (EC50) EROD response, (2) the concentration producing a response equivalent to 10% of the maximal response produced by TCDD (ECTCDD10%), and (3) a slope ratio method. For each method, the rank order in potency was TCDD ⩾ TCDF > PCB 126 > PCB 81 > PCB 77 > PCB 169 in chicken, pheasant, and turkey hepatocytes. The rank order was similar in duck and herring gull hepatocytes with the following exceptions: TCDF was approximately 2- to 4-fold more potent than TCDD in duck hepatocytes; PCB 169 induced EROD in gulls, but PCB 77 had no measurable effect in this species. PCB 118 was a relatively weak EROD inducer in most species/breeds, but it did not induce EROD in Pekin ducks or gulls. PCB 105 was a weak inducer in White Leghorn chicken and turkey hepatocytes, but it did not induce EROD in other species. The EC50, ECTCDD10% and slope ratio methods for estimating potencies generally gave similar IEFs for compounds that produced a maximal response that was at least 60% of the maximal response produced by TCDD. For compounds that caused a response that was 50% or lower than that produced by TCDD, EC50-based IEFs were greater (10- to 100-fold) than ECTCDD10%-based IEFs or slope-ratio-based IEFs. Among species, the rank order in sensitivity to EROD induction was chicken > pheasant > turkey ⩾ duck ⩾ herring gull. The relative sensitivity of avian hepatocyte cultures to EROD induction by PCB 77 was similar to the relative sensitivity of these species (reported elsewhere) to lethality after in ovo injection of PCB 77. Chicken hepatocyte cultures were 5–10 times more sensitive to EROD induction by TCDD than were pheasant hepatocyte cultures, which is identical to the difference in sensitivity of these species to the lethal effect of TCDD after in ovo injection. Measuring the sensitivity of hepatocyte cultures to EROD induction might be useful for estimating the sensitivity of avian species (including rare or endangered species, where it is impossible to conduct in vivo studies) to the embryotoxic effects of TCDD, non- ortho substituted PCBs, and other aryl hydrocarbon receptor agonists.

During the1992 breeding season, eggs of bald eagles (Haliaeetus leucocephalus) were collected within a gradient of exposure to chlorinated hydrocarbon pollutants, particularly from pulp mill point sources, on the southern coast of British Columbia, Canada. Twenty-five eggs were placed in a laboratory incubator, of which 18 hatched; chicks were sacrificed within 24 h. Hatching success was not significantly different between eggs taken from pulp mill sites and reference sites. A hepatic cytochrome P4501A (CYP1A) cross-reactive protein was induced nearly sixfold in chicks from near a pulp mill at Powell River compared to those from a reference site (p < 0.05). Hepatic ethoxyresorufin-O-deethylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were also significantly elevated in chicks from nests located near pulp mills compared to reference sites (p < 0.0005 and p < 0.02, respectively). A hepatic CYP2B cross-reactive protein was threefold higher in chicks from pulp mill versus reference sites, but the difference was not significant. Residual yolk sacs of eggs collected near pulp mill sites contained greater concentrations of 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) compared to reference areas. No significant differences in concentrations of polychlorinated biphenyls (PCBs), non-ortho congeners, and organochlorine pesticides occurred among sites. Regressions showed that the hepatic CYP1A cross-reactive protein and EROD and BROD activities were positively correlated with 2,3,7,8-TCDD, 2,3,7,8-TCDF, and toxic equivalents (TEQsWHO—Wrld Health Organization toxic equivalence factors) in yolk sacs. No significant concentration-related effects were found for morphological, physiological, or histological parameters, such as chick growth, edema, or density of thymic lymphocytes. Using hepatic CYP1A induction as a biomarker, a no-observed-effect-level (NOEL) of 100 ng/kg and a lowest-observed-effect-level (LOEL) of 210 ng/kg TEQsWHO on a whole egg (wet weight basis) are suggested for bald eagle chicks.

A bioassay that uses chicken embryo hepatocyte (CEH) primary cultures for measuring 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (TCDD-EQ) concentrations in extracts prepared from wild bird eggs ...

The toxicity of 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and 2,3',4,4',5-pentachlorobiphenyl (PCB 118) was investigated in rats following subchronic dietary exposure. Groups of 10 male and 10 female weanling Sprague-Dawley rats were administered PCB 77 in the diet at 0, 10, 100, 1000, or 10,000 ppb for 13 weeks. PCB 118 was administered to males in the diet at 0, 10, 100, 1000, and 10,000 ppb, while the female groups received 0, 2, 20, 200, or 2000 ppb of the congener for 13 weeks. Growth rate and food consumption were not affected by treatment. No clinical signs of toxicity were observed. Increased spleen weight occurred in male rats fed 1000 or 10,000 ppb PCB 77. Male rats receiving 10,000 ppb PCB 118 had increased liver weight and hepatic ethoxyresorufin O-deethylase (EROD) activity. Increased hepatic EROD activity but not liver weight was observed in female rats given the 2000-ppb PCB 118 diet. Increased EROD activity was also noted in male rats given 10,000 ppb and in female groups receiving 1000 or 10,000 ppb PCB 77. Male rats exposed to 10,000 ppb PCB 77 had decreased vitamin A in the liver and lung and elevated levels in the kidney. Liver vitamin A of both 1000- and 10,000-ppb PCB 77 female groups was decreased. PCB 118 had no effects on tissue vitamin A at the levels studied. No hematological changes or serum biochemical changes were seen in any of PCB 118- and PCB 77-treated groups, nor were liver uroporphyrin levels altered. A reduction in dopamine and homovanillinic acid in substantia nigra region of the brain was observed in female rats fed 2000 ppb PCB 118, while 10,000 ppb PCB 77 was associated with an elevation in 3,4-dihydroxyphenylacetic acid in the nucleus accumbens region of male rat brains. Mild to moderate changes were observed in the liver and thyroid of rats given PCB 77 or PCB 118. PCB 118 accumulated in a dose-dependent manner in fat and to a much lesser extent in liver. In contrast, very low levels of PCB 77 residue were found in the tissues examined. Based on the above data it was concluded that NOAEL of PCB 77 is 100 ppb in diet or 8.7 micrograms/kg and that of PCB 118 is 200 ppb in diet or 17 micrograms/kg body wt/day.

Perfluoroundecanoic acid (PFUdA) is one of the most highly detected perfluoroalkyl compounds in wild bird tissues and eggs. Although PFUdA does not affect hatching success, many PFCs are known to impair post-hatch development and survival. Here we use microarrays to survey the transcriptional response of cultured chicken embryonic hepatocytes (CEH) to PFUdA for potential targets of PFUdA action that could lead to developmental deficiencies in exposed birds. At 1 μM and 10 μM PFUdA significantly altered the expression of 346 and 676 transcripts, respectively (fold-change>1.5, p

The sensitivity of avian species to the toxic effects of dioxin-like compounds (DLCs) varies up to 1000-fold among species, and this variability has been associated with interspecies differences in aryl hydrocarbon receptor 1 ligand-binding domain (AHR1 LBD) sequence. We previously showed that LD(50) values, based on in ovo exposures to DLCs, were significantly correlated with in vitro EC(50) values obtained with a luciferase reporter gene (LRG) assay that measures AHR1-mediated induction of cytochrome P4501A in COS-7 cells transfected with avian AHR1 constructs. Those findings suggest that the AHR1 LBD sequence and the LRG assay can be used to predict avian species sensitivity to DLCs. In the present study, the AHR1 LBD sequences of 86 avian species were studied, and differences at amino acid sites 256, 257, 297, 324, 337, and 380 were identified. Site-directed mutagenesis, the LRG assay, and homology modeling highlighted the importance of each amino acid site in AHR1 sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin and other DLCs. The results of the study revealed that (1) only amino acids at sites 324 and 380 affect the sensitivity of AHR1 expression constructs of the 86 avian species to DLCs and (2) in vitro luciferase activity of AHR1 constructs containing only the LBD of the species of interest is significantly correlated (r (2) = 0.93, p0.0001) with in ovo toxicity data for those species. These results indicate promise for the use of AHR1 LBD amino acid sequences independently, or combined with the LRG assay, to predict avian species sensitivity to DLCs.

Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species.

In a recent egg injection study, we showed that in ovo exposure to perfluorohexane sulfonate (PFHxS) affects the pipping success of developing chicken (Gallus gallus domesticus) embryos. We also found evidence of thyroid hormone (TH) pathway interference at multiple levels of biological organization (i.e., somatic growth, messenger RNA expression, and circulating free thyroxine levels). Based on these findings, we hypothesize that PFHxS exposure interferes with TH-dependent neurodevelopmental pathways. This study investigates global transcriptional profiles in cerebral hemispheres of chicken embryos following exposure to a solvent control, 890 or 38,000 ng PFHxS/g egg (n = 4-5 per group); doses that lead to the adverse effects indicated above. PFHxS significantly alters the expression (≥ 1.5-fold, p ≤ 0.001) of 11 transcripts at the low dose (890 ng/g) and 101 transcripts at the high dose (38,000 ng/g). Functional enrichment analysis shows that PFHxS affects genes involved in tissue development and morphology, cellular assembly and organization, and cell-to-cell signaling. Pathway and interactome analyses suggest that genes may be affected through several potential regulatory molecules, including integrin receptors, myelocytomatosis viral oncogene, and CCAAT/enhancer-binding protein. This study identifies key functional and regulatory modes of PFHxS action involving TH-dependent and -independent neurodevelopmental pathways. Some of these TH-dependent mechanisms that occur during embryonic development include tight junction formation, signal transduction, and integrin signaling, whereas TH-independent mechanisms include gap junction intercellular communication.

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R(2)≥0.87, p0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals.

Some currently used brominated flame retardants (BFRs), such as hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO), bis(2-ethylhexyl)tetrabromophthalate (BEHTBP), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) and decabromodiphenylethane (DBDPE), are persistent organic contaminants detected in various environmental matrices, including wild birds. Data on potential toxicological and molecular responses to exposure of these BFRs are lacking for avian species. A combined in vitro/in ovo approach was used to determine the concentration-dependent effects of these BFRs on overt toxicity and hepatic messenger RNA (mRNA) expression levels of 11 transcripts in (1) primary cultures of chicken embryonic hepatocytes (CEH; all four BFRs) and (2) chicken embryos (HCDBCO and BTBPE only). Neither hepatocyte viability nor embryonic pipping success were affected by the BFRs at any of the administered concentrations (CEH: 0.001-30 μM, egg injection: 0.1-10 μg/g nominal dose). In CEH, 10 μM HCDBCO induced cytochrome P450 2H1 (CYP2H1) and CYP3A37, while CYP1A4/5 were down-regulated at all tested concentrations. In contrast, only transthyretin was down-regulated by HCDBCO in embryonic liver. There was concordance between the BTBPE-induced transcriptional responses in vitro and in ovo for CYP1A4/5 (up-regulated) and type III iodothyronine 5'-deiodinase (DIO3; down-regulated). DBDPE induced CYP1A4/5 29- and 59-fold at 0.2 μM in CEH and increased DIO1. None of the gene targets were responsive to BEHTBP exposure in CEH. The multi-tiered in vitro/in ovo screening approach was effective for assessing toxicological and molecular biological effects of these BFRs in an avian species.

Dechlorane Plus (DP) is an additive chlorinated flame retardant comprising two major isomers, syn- and anti-DP, that is used in a variety of commercial/industrial products. It has been detected in biotic and abiotic matrices including the eggs of herring gulls collected from the Laurentian Great Lakes. However, data on potential toxicological and molecular responses to exposure are lacking, especially for avian species. A combined in vitro/in ovo approach was used to determine concentration-dependent effects of DP in chicken embryonic hepatocytes (CEH) and chicken embryos following injection of DP into the air cell of eggs prior to incubation. Overt toxicity (i.e. cytotoxicity and pipping success) and mRNA expression levels of transcripts previously determined to be responsive to a brominated flame retardant were assessed in CEH and hepatic tissue. DP was not cytotoxic up to a maximum concentration of 3 μM in CEH, and no effects on pipping success were observed up to the highest nominal dose group of 500 ng/g egg. A significant shift in isomeric content of syn- and anti-DP was detected between stock solutions of the commercial mixture and hepatic tissue; the proportion of the syn-DP isomer increased from 0.34 to 0.65 with a concomitant decrease of anti-DP from 0.66 to 0.35. None of the mRNA transcripts changed as a result of in vitro or in ovo exposure to DP indicating that, although there was concordance between the two approaches, DP may evoke its toxicity through other modes of action. At current environmental exposure levels, no adverse effects of DP on embryonic viability or pathways associated with the genes assessed are predicted.

Primary cultures of chick embryo hepatocytes were maintained in Waymouth's MD 705/1 medium that was unexposed or had been exposed for 4 hr to 366 nm ultraviolet (UV) light. The cultures were assayed for 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB)-mediated porphyrin accumulation and ethoxyresorufin-O-deethylase (EROD) induction. A decrease in 3,3',4,4'-TCB-mediated porphyrin accumulation, due in part to tryptophan depletion after exposure of the cell culture medium to UV light, was observed. In contrast, EROD activity of cells maintained in irradiated medium was shown to increase over basal levels, possibly due to the presence of photoproducts of medium components. Further studies indicated that photoproducts obtained after irradiation of a concentrated aqueous solution of tryptophan were inducers of EROD activity in primary chick embryo hepatocytes.

Concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) on cytochrome P4501A (CYP1A) induction were determined in primary cultures of embryonic herring gull (Larus argentatus) hepatocytes exposed for 24 h. Based on the concentration that induced 50% of the maximal response (EC50), the relative potencies of TCDD and TCDF did not differ by more than 3.5-fold. However, also based on the EC50, PeCDF was 40-fold, 21-fold, and 9.8-fold more potent for inducing ethoxyresorufin-O-deethylase (EROD) activity, CYP1A4 mRNA expression, and CYP1A5 mRNA expression than TCDD, respectively. The relative CYP1A-inducing potencies of PeCDF and of other dioxin-like chemicals (DLCs) in herring gull hepatocytes (HEH RePs), along with data on concentrations of DLCs in Great Lakes herring gull eggs, were used to calculate World Health Organization toxic equivalent (WHO-TEQ) concentrations and herring gull embryonic hepatocyte toxic equivalent (HEH-TEQ) concentrations. The analysis indicated that, when using avian toxic equivalency factors (TEFs) recommended by the WHO, the relative contribution of TCDD (1.1-10.2%) to total WHO-TEQ concentration was higher than that of PeCDF (1.7-2.9%). These results differ from the relative contribution of TCDD and PeCDF when HEH RePs were used; PeCDF was a major contributor (36.5-52.9%) to total HEH-TEQ concentrations, whereas the contribution by TCDD (1.2-10.3%) was less than that of PeCDF. The WHO TEFs for avian species were largely derived from studies with the domestic chicken (Gallus gallus domesticus). The findings of the present study suggest that it is necessary to determine the relative potencies of DLCs in wild birds and to re-evaluate their relative contributions to the biochemical and toxic effects previously reported in herring gulls and other avian species.

Hexabromocyclododecane (HBCD) is an additive flame retardant used primarily in polystyrene foams. HBCD is a persistent contaminant that has been detected in abiotic and biotic matrices, including wild avian species. The toxicological implications of exposure are not well characterized. We recently identified molecular end points responsive to HBCD exposure in chicken embryonic hepatocytes (CEHs) including genes involved in xenobiotic metabolism, thyroid hormone transport, and lipid metabolism. In the current study, a technical mixture of HBCD (HBCD-TM), comprising 12% alpha-, 11% beta-, and 77% gamma-stereoisomers, was injected into the air cell of chicken eggs prior to incubation. Embryonic viability to pipping, isomer-specific HBCD accumulation, and hepatic mRNA expression of the genes identified in the in vitro study were determined. Concentrations of 100 and 10,000 ng/g decreased pipping success while 50, 300, and 1000 ng/g had no effect. In contrast to HBCD-TM, the isomeric composition in liver tissue was significantly different for alpha- (31%) and gamma-HBCD (61%) demonstrating that isomer-specific processes were occurring in the egg and/or developing embryo. Exposure to 1000 ng/g HBCD-TM significantly upregulated cytochrome P450 (CYP) 2H1, CYP3A37, uridine 5'-diphospho-glucuronosyltransferase, and deiodinase 2, while liver fatty acid-binding protein and insulin-growth factor 1 expression were significantly decreased at 100 and 10,000 ng/g, respectively. The alterations in hepatic mRNA levels were in concordance with those observed in CEH highlighting the utility of both approaches for identifying molecular mechanisms of action. Research on the effects of HBCD in wild avian species is warranted.

Ethoxyresorufin O-deethylase (EROD) activity was measured in primary cultures of ring-necked pheasant (Phasianuscolchicus) and Japanese quail (Coturnix japonica) embryonic hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) for 12, 24, 36 and 48 h. In ring-necked pheasant hepatocytes there was a significant time-dependent increase in the EROD-inducing potency of TCDD, PeCDF and TCDF (i.e. decrease of the EC50). In Japanese quail hepatocytes there was no time-dependent change in the EROD-inducing potency of TCDD, PeCDF and TCDF. There was no time-dependent change in the relative potency of PeCDF and TCDF (i.e. compared to the potency of TCDD) in ring-necked pheasant hepatocytes and of PeCDF in Japanese quail hepatocytes. The results indicate that the relative potencies of these compounds at 24h are representative of their relative potencies between 12 and 48 h. However, in Japanese quail hepatocytes, the relative potency of TCDF decreased in a time-dependent manner (up to 3.6-fold difference). These results suggest that the effect of time on the EROD-inducing potency of TCDD, PeCDF and TCDF in ring-necked pheasant and Japanese quail hepatocytes is compound- and species-specific, but experimental conditions could also be involved in the differences observed.

Several perfluoroalkyl compounds (PFCs) are ubiquitous environmental contaminants that can biomagnify in species at high trophic levels including wild birds. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been detected in wild birds and are known to reduce hatching success of laboratory-exposed chicken embryos at environmentally relevant concentrations. Limited toxicity data are available regarding avian exposure to PFCs of chain lengths greater than C(8), which are of increasing environmental relevance following the recent phase-out of PFOS and PFOA. In this study, linear PFOA, perfluoroundecanoic acid (PFUdA) and perfluorodecane sulfonate (PFDS) were injected into the air cell of white leghorn chicken eggs (Gallus gallus domesticus) prior to incubation to determine effects on embryo pipping success. Furthermore, mRNA expression of key genes involved in pathways implicated in PFC toxicity was monitored in liver tissue. PFOA, PFUdA or PFDS had no effect on embryonic pipping success at concentrations up to 10 microg/g. All PFCs accumulated in the liver to concentrations greater than the initial whole-egg concentration as determined by HPLC/MS/MS. Hepatic accumulation was highest for PFOA (4.5 times) compared to PFUdA and PFDS. Cytochrome P450 1A4 and liver fatty acid binding protein mRNA expression increased after exposure to PFUdA but was only statistically significant at 10 microg/g; several orders of magnitude higher than levels found in wild bird eggs. Based on the present results for white leghorn chickens, current environmental concentrations of PFOA, PFUdA and PFDS are unlikely to affect the hatching success of wild birds.

In this study we investigated the effect of a single-compound exposure or two compound co-exposure to tetrachlorodibenzo-p-dioxin (TCDD) plus perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA) on the mRNA expression of cytochromes P450 (CYP) 1A4, 4V2 and 3A37, ethoxyresorufin-O-deethylase (EROD) activity and cell viability in chicken (Gallus gallus domesticus) embryo primary hepatocyte cultures. Cell viability after 24 h of incubation was significantly decreased in cells exposed to PFOS at concentrations between 30 microM and 60 microM with or without co-exposure to TCDD (0.3 nM at maximum). PFOA did not decrease cell viability even at maximum concentrations of 60 microM. TCDD induced CYP1A4 mRNA and EROD activity substantially as reported previously. PFOS also increased CYP1A4 mRNA in a concentration-dependent manner. Co-exposure of cells to PFOS plus TCDD did not change CYP1A4 mRNA levels compared to cells treated with TCDD alone. PFOS alone did not induce CYP4V2 mRNA, however 40-50 microM PFOS plus TCDD (0.3 nM) induced CYP4V2 mRNA compared to TCDD alone (P

Hexabromocyclododecane (HBCD) and polybrominated diphenyl ethers (PBDEs) are additive flame retardants used in a wide range of consumer products. Both compounds have been detected in free-living avian species, but toxicological and molecular end points of exposure are limited. An in vitro approach was used to compare concentration-dependent effects of HBCD and the commercial penta-brominated diphenyl ether mixture DE-71 on cytotoxicity and mRNA expression in cultured hepatocytes derived from embryonic chickens. Neither HBCD-alpha, HBCD-technical mixture (TM), nor DE-71 effected hepatocyte viability at the highest concentrations assessed (30-100 microM). Real-time RT-PCR assays were developed to quantify changes in mRNA abundance of genes associated with chicken xenobiotic-sensing orphan nuclear receptor activation, the thyroid hormone (TH) pathway, and lipid regulation. Exposure toor= 1 microM HBCD-alpha and HBCD-TM resulted in significant upregulation of cytochrome P450 (CYP) 2H1 (fourfold to sevenfold) and CYP3A37 (5- to 30-fold) at 24 and 36 h. In contrast, 30 microM DE-71 caused a twofold increase of CYP2H1 only. UGT1A9 expression was only upregulated by HBCD-alpha to a maximum of fourfold ator= 1 microM. Transthyretin, thyroid hormone-responsive spot 14-alpha, and liver fatty acid-binding protein were all significantly downregulated (up to sevenfold) for cells exposed toor= 1 microM HBCD-alpha and HBCD-TM. DE-71 also downregulated these three target genes twofold to fivefold at concentrationsor= 3 microM. Taken together, our results indicate that xenobiotic-metabolizing enzymes and genes associated with the TH pathway and lipid regulation are vulnerable to HBCD and DE-71 administration in cultured avian hepatocytes and might be useful molecular markers of exposure.

Perfluorooctane sulfonate (PFOS) is a widely distributed industrial compound that has been detected in the eggs of various wild avian species. Laboratory studies have indicated that PFOS is embryotoxic to domestic chickens (Gallus gallus domesticus), but the mechanisms of toxicity in the developing avian embryo remain unknown. We recently demonstrated that PFOS acts as a peroxisome proliferator by causing increased expression of peroxisome proliferator activated receptor alpha (PPARalpha)-regulated genes in cultured primary chicken embryo hepatocytes. The present study examined whether PPARalpha-regulated genes were dose-dependently affected in chicken embryos exposed in ovo to PFOS. White leghorn chicken eggs were injected with 0.1, 5.0 or 100.0 microg PFOS/g egg into the air cell prior to incubation. Embryos were incubated until pipping, after which the expression of PPARalpha-regulated genes was measured in the liver tissue of surviving embryos using real-time reverse transcription polymerase chain reaction. A dose-dependent decrease in embryo pippability was observed with an LD50 of 93 microg/g (3.54 microg/g-672,910 microg/g, 95% confidence interval). Hepatic PFOS concentrations increased concomitantly with dose. The PPARalpha-regulated genes measured were peroxisomal acyl CoA oxidase, bifunctional enzyme, liver fatty acid binding protein and peroxisomal 3-ketoacyl thiolase. PFOS exposure via egg injection prior to incubation did not affect the transcriptional activity of any of the assayed PPARalpha-regulated genes at any of the doses examined in day 21 chicken embryos.

Perfluorooctane sulfonate (PFOS) is a globally distributed environmental contaminant that is detected in the serum and liver of numerous mammalian and avian species. PFOS acts as a peroxisome proliferator in rodents, which occurs subsequent to activation of the nuclear receptor peroxisome proliferator activated receptor-alpha (PPAR-alpha). Activated PPAR-alpha up-regulates PPAR-alpha target genes, most of which are involved in lipid metabolism. Although several studies have investigated the effects of PFOS exposure on mammalian gene expression, there are few studies in avian species. To determine if PFOS is capable of activating avian PPAR-alpha, we exposed chicken embryo primary hepatocyte cultures (N=3 independent cell cultures) to PFOS or fenofibrate, a mammalian PPAR-alpha agonist, and examined the expression of PPAR-alpha and PPAR-alpha target genes using quantitative real-time PCR. The target genes examined were peroxisomal acyl-CoA oxidase (ACOX), liver fatty acid binding protein (L-FABP), enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase bifunctional enzyme (BIEN), peroxisomal 3-ketoacyl thiolase (PKT), and malic enzyme (ME). All five target genes were induced in response to PFOS exposure and all of the target genes, except L-FABP, were induced in response to fenofibrate. PPAR-alpha mRNA expression was not altered by PFOS or fenofibrate. This study provides the first evidence that PFOS can induce PPAR-alpha-dependent transcriptional responses in an avian species and provides the first characterization of fenofibrate induced transcriptional responses in chicken embryo hepatocyte cultures.

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.