Search

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., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

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 54,900ng/g and from 0 to 261,400ng/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., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

We previously demonstrated that in ovo exposure to the flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCPP) decreased plasma thyroxine levels, reduced growth parameters, and decreased gallbladder size in chicken embryos. In the current study DNA microarrays were used to evaluate global mRNA expression in liver tissue of male chicken embryos that exhibited the above mentioned effects. Injected doses were dimethyl sulfoxide vehicle control, 7.6 or 45 μg TDCPP/g egg. TDCPP caused significant changes in the expression of five genes at the low dose and 47 genes at the high dose (False Discovery Rate p ≤ 0.1, fold change ≥ 1.5). The gene expression analysis suggested a compromised immune function, a state of cholestatic liver/biliary fibrosis, and disrupted lipid and steroid metabolism. Circulating bile acid levels were elevated, which is an indication of liver dysfunction, and plasma cholesterol levels were reduced; however, hepatic bile acid and cholesterol levels were unaltered. Interactome analyses identified apolipoprotein E, hepatocyte nuclear factor 4 alpha, and peroxisome proliferator-activated receptor alpha as key regulatory molecules involved in the effects of TDCPP. Our results demonstrate a targeted effect of TDCPP toxicity on lipid metabolism, including cholesterol, that helps explain the aforementioned phenotypic effects, as chicken embryos are highly dependent on yolk lipids for growth and maintenance throughout development. Finally, our results are in concordance with the literature that describes TDCPP as a cancer-causing agent, since the majority of dysregulated genes were involved in cancer pathways., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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, p<0.0001). 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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., (Copyright 2001 Academic Press.)

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 studies 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 (ECTCDD 10%), and (3) a slope ratio method. For each method, the rank order in potency was TCDD > or = 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 > or = duck > or = 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.