Sean W. Kennedy

Impact of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls on human and environmental health, with special emphasis on application of the toxic equivalency factor concept

A scientific evaluation was made of the mechanisms of action of polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls. Distinction is made between the aryl-hydrocarbon (Ah) receptor-mediated and non-Ah receptor-mediated toxic responses. Special attention is paid to the applicability of the toxic equivalency factor (TEF) concept.

In ovo effects of two organophosphate flame retardants, TCPP and TDCPP, on pipping success, development, mRNA expression and thyroid hormone levels in chicken embryos

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.

Effects of Tris(1,3-dichloro-2-propyl) phosphate and Tris(1-chloropropyl) phosphate on Cytotoxicity and mRNA Expression in Primary Cultures of Avian Hepatocytes and Neuronal Cells

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.

Amino Acid Sequence of the Ligand-Binding Domain of the Aryl Hydrocarbon Receptor 1 Predicts Sensitivity of Wild Birds to Effects of Dioxin-Like Compounds

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, p < 0.0001) 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.

Rapid in vitro metabolism of the flame retardant triphenyl phosphate and effects on cytotoxicity and mRNA expression in chicken embryonic hepatocytes.

The organophosphate flame retardant, triphenyl phosphate (TPHP), has been detected with increasing frequency in environmental samples and its primary metabolite is considered to be diphenyl phosphate (DPHP). Information on the adverse effects of these compounds in avian species is limited. Here, we investigate the effects of TPHP and DPHP on cytotoxicity and mRNA expression, as well as in vitro metabolism of TPHP, by use of a chicken embryonic hepatocyte (CEH) screening assay. After 36 h of exposure, CEH cytotoxicity was observed following exposure to >10 μM TPHP (LC50 = 47 ± 8 μM), whereas no significant cytotoxic effects were observed for DPHP concentrations up to 1000 μM. Using a custom chicken ToxChip polymerase chain reaction (PCR) array, the number of genes altered by 10 μM DPHP (9 out of 27) was greater than that by 10 μM TPHP (4 out of 27). Importantly, 4 of 6 genes associated with lipid/cholesterol metabolism were significantly dysregulated by DPHP, suggesting a potential pathway of importance for DPHP toxicity. Rapid degradation of TPHP was observed in CEH exposed to 10 μM, but the resulting concentration of DPHP accounted for only 17% of the initial TPHP dosing concentration. Monohydroxylated-TPHP (OH-TPHP) and two (OH)2-TPHP isomers were identified in TPHP-exposed CEH, and concentrations of these metabolites increased over 0 to 36 h. Overall, this is the first reported evidence that across 27 toxicologically relevant genes, DPHP altered more transcripts than its precursor, and that TPHP is also metabolized via a hydroxylation pathway in CEH.

Effects of Hexabromocyclododecane and Polybrominated Diphenyl Ethers on mRNA Expression in Chicken (Gallus domesticus) Hepatocytes

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 to >or= 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 at >or= 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 to >or= 1 microM HBCD-alpha and HBCD-TM. DE-71 also downregulated these three target genes twofold to fivefold at concentrations >or= 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.

Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins

There are large differences in sensitivity to the toxic and biochemical effects of dioxins and dioxin-like compounds (DLCs) among vertebrates. Previously, we demonstrated that the difference in sensitivity between domestic chicken (Gallus gallus domesticus) and common tern (Sterna hirundo) to aryl hydrocarbon receptor 1 (AHR1)-dependent changes in gene expression following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is based upon the identities of the amino acids at two sites within the ligand binding domain of AHR1 (chicken--highly sensitive; Ile324_Ser380 vs common tern--250-fold less sensitive than chicken; Val325_Ala381). Here, we tested the hypotheses that (i) the sensitivity of other avian species to TCDD, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) is also determined by the amino acids at sites that are equivalent to sites 324 and 380 in chicken, and (ii) Ile324_Ala380 and Val324_Ser380 genotypes confer intermediate sensitivity to DLCs in birds. We compared ligand-induced transactivation function of full-length AHR1s from chicken, common tern, ring-necked pheasant (Phasianus colchicus; Ile324_Ala380) and Japanese quail (Coturnix japonica; Val324_Ala380), and three Japanese quail AHR1 mutants. The results support our hypothesis that avian species can be grouped into three general classes of sensitivity to DLCs. Both AHR1 genotype and in vitro transactivation assays predict in vivo sensitivity. Contrary to the assumption that TCDD is the most potent DLC, PeCDF was more potent than TCDD at activating Japanese quail (13- to 26-fold) and common tern (23- to 30-fold) AHR1. Our results support and expand previous in vitro and in vivo work that demonstrated ligand-dependent species differences in AHR1 affinity. The findings and methods will be of use for DLC risk assessments.

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

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

Tris(1,3-dichloro-2-propyl) phosphate perturbs the expression of genes involved in immune response and lipid and steroid metabolism in chicken embryos

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.

Sensitivity of Japanese Quail (Coturnix japonica), Common Pheasant (Phasianus colchicus), and White Leghorn Chicken (Gallus gallus domesticus) Embryos to In Ovo Exposure to TCDD, PeCDF, and TCDF

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.