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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.

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.

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.

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.