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Exposure to dioxin-like compounds is consistently associated with concentration-dependent induction of cytochrome P4501A (CYP1A) enzymes in primary cultures of avian hepatocytes. We have previously demonstrated that the median effective concentration (EC50) for induction of this response is predictive of in vivo sensitivity to dioxin-like compounds in birds. We investigated sources of interindividual variation in the CYP1A response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild herring gulls and considered how this variation may complicate dioxin sensitivity estimates based on the CYP1A bioassay. Concentration-dependent effects of TCDD on CYP1A mRNA expression were characterized in 55 hepatocyte cultures prepared from individual herring gull embryos. A large degree of variability was observed among the hepatocyte culture preparations. For example, 1) basal CYP1A4 and CYP1A5 mRNA expression varied by 20- and 126-fold, respectively, among individuals, and 2) exposure to TCDD induced CYP1A4 mRNA expression by 57-fold in the most responsive sample but did not significantly induce CYP1A4 mRNA expression above baseline values in 42% of hepatocyte culture preparations. Environmental and genetic factors contributing to the observed variability are discussed. Despite the large amount of interindividual variation, we conclude that reproducible EC50-based estimates of species sensitivity can be obtained from the CYP1A cell culture bioassay when samples are collected from relatively uncontaminated colonies. Environ Toxicol Chem 2019;38:660-670. © 2019 SETAC., (© 2019 SETAC.)

The liver is a key metabolic organ contributing significantly to both lipid and cholesterol homeostasis in vertebrates. This study examines whether the human pharmaceutical atorvastatin (ATV), which is designed to lower cholesterol biosynthesis, could disrupt lipid dynamics in fish. The study investigates the effects of ATV at a physiologically relevant exposure regimen (concentration and duration) on gene transcripts and the biosynthesis of cholesterol and other lipid and non-lipid molecules in primary rainbow trout hepatocytes. Trout hepatocytes exposed to ATV increased the transcript abundance of genes involved in lipid metabolism (HMGCR1, LDLR, PPARα, PPARγ, and SREBP1) and xenobiotic metabolism (CYP3A27), and reduced cholesterol synthesis. This study demonstrates that lipid metabolism in trout hepatocytes is sensitive to the effects of ATV, and changes in gene expression occur within 3-6h after exposure., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tetradecabromo-1,4-diphenoxybenzene (TeDB-DiPhOBz) and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (BDE-209) are flame retardant chemicals that can undergo photolytic degradation. The present study compared the time-dependent photolyic degradation of TeDB-DiPhOBz and BDE-209, and dioxin-like product formation as a result of (UV) irradiation (I; irradiation time periods of 0, 1, 4, 15 and 40 days). Photo-degraded product fractions of UV-I-TeDB-DiPhOBz (nominal concentration: 1.9 μM) were administered to chicken embryonic hepatocytes (CEH), and significant induction of CYP1A4/5 mRNA expression was observed for fractions collected at the day 15 and 40 time points (fold change of 7.3/3.6 and 9.1/4.7, respectively). For the UV-I-BDE-209 fractions (nominal concentration: 10 μM), significant CYP1A4/5 up-regulation occurred at all time points, and the fraction collected on day 1 induced the greatest fold change of 510/86, followed by 410/68 (day 4) and 110/26 (day 15), respectively. For the UV-I-BDE-209 fraction collected at day 40, significant CEH cytotoxicity was observed. As a result, CYP1A4/5 expression was determined at a nominal concentration of 1 μM instead of 10 μM and CYP1A4/5 fold changes of 11/8.2 (day 40) were observed. Fractions eliciting the greatest CYP1A4/5 mRNA upregulation were further screened for transcriptomic effects using a PCR array comprising 27 dioxin-responsive genes. A total of 6 and 16 of the 27 target genes were up or down-regulated following UV-I-TeDB-DiPhOBz and UV-I-BDE-209 exposure, respectively. Overall, and regardless of the formation rate, these results raise concerns regarding the potential formation of dioxin-like compounds from flame retardants in products and materials such as plastics, and in natural sunlight irradiation situations in the environment (e.g. in landfill sites or electronic waste facilities)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We report on two highly brominated polyphenyl ether flame retardants, tetradecabromo-1,4- diphenoxybenzene (TeDB-DiPhOBz) and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (BDE-209), that formed photolytic degradation products in tetrahydrofuran (THF)/hexane solvent after 21 days of natural sunlight irradiation (SI). These degradation products of SI-TeDB-DiPhOBz and SI-BDE-209 included the numerous polybrominated homologue groups of polybenzofurans and dibenzofurans, respectively. Formation of similar polybenzofuran and dibenzofuran products was also observed following a 3 month exposure of the solid powder forms of TeDB-DiPhOBz and BDE-209 to natural SI. These resulting degradation product mixtures were administered to chicken embryonic hepatocytes (CEH) to determine effects on mRNA expression levels of 27 dioxin-responsive genes. For the solvent-based SI study, equivalent concentrations of 1 or 25 μM of SI-TeDB-DiPhOBz or 1 or 10 μM of SI-BDE-209 resulted in gene expression profiles that were similar to those of the most potent dioxin-like compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin. In addition, a concentration-dependent induction of CYP1A4 and CYP1A5 mRNA was observed following exposure to SI-TeDB-DiPhOBz and SI-BDE-209. Based on ECthreshold values for CYP1A4/5 mRNA expression, relative potency (ReP) values were 1 × 10(-6) and 1 × 10(-5) for SI-TeDB-DiPhOBz and SI-BDE-209, respectively. The SI TeDB-DiPhOBz and BDE-209 powder degradation product mixture also significantly induced CYP1A4 mRNA levels in CEH. Our findings clearly show that the environmental stability of TeDB-DiPhOBz and BDE-209, and possibly other highly brominated polyphenyl ethers, is of great concern from a dioxin-like degradation products and toxicity perspective.

The potency of tetrachlorodibenzo-p-dioxin (TCDD) and 18 polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity was assessed in primary hepatocyte cultures prepared from chicken (Gallus domesticus), Pekin duck (Anas platyrhynchos domesticus), and greater scaup (Aythya marila). TCDD and 8 of the PAHs induced EROD activity in a concentration-dependent manner. Seven of these were previously shown to be acutely toxic to avian embryos, while the 10 congeners that did not produce an EROD response caused limited mortality. The rank order potency of the EROD-active congeners in all three species was as follows: TCDD>dibenz[ah]anthracene>benzo[k]fluoranthene>indeno[1,2,3-cd]pyrene>benzo[a]pyrene>chrysene≈benz[a]anthracene≈benz[ghi]perylene>benzo[b]naphtho[2,3-d]thiophene. Chicken hepatoctyes were more sensitive than duck hepatocytes to EROD induction by all test compounds, but the gap in species sensitivity was 100-fold for TCDD, and generally ≤10-fold for PAHs. This study is the first to use in vitro methods to rank the AHR-mediated potency of PAHs in birds. These data may be useful for assessing risks associated with exposure to PAHs in the environment.

Silver nanoparticles (AgNPs) are found in many consumer products yet their biological effects on non-target aquatic organisms are yet to be fully understood. This research aimed to investigate the effects of AgNPs on cell signaling in rainbow trout (Oncorhynchus mykiss) hepatocytes. We focused on the β-adrenoreceptor (AR), which mediates glycogenolysis, and the glucocorticoid receptor (GCR), which mediates gluconeogenesis. These two receptors have been extensively studied in trout hepatocytes due to their key roles during the stress response to increase glucose availability (among other things), allowing the organisms to cope with the stressor. We show for the first time that AgNPs at a concentration of 1 μg/mL did not interfere with the function of either the β-AR or the GCR systems in rainbow trout hepatocytes, but at the concentration of 10 μg/mL AgNPs stimulated glycogenolysis which was apparently receptor-independent. This study suggests that AgNPs could affect hormone-regulated cell signaling pathways at a concentration of 10 μg/mL., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Silver nanoparticles (AgNPs) are present in a multitude of consumer and medical products; however, the toxicity of AgNPs is not fully understood. This research aimed to elucidate the relationship between AgNP cytotoxicity and oxidative stress and damage in rainbow trout (Oncorhynchus mykiss) hepatocytes and erythrocytes in comparison to silver ions (Ag(+)). Generally the cytotoxicity of AgNPs and Ag(+) was similar, such that both silver types generated reactive oxygen species, decreased glutathione levels, and decreased activities of glutathione reductase and glutathione-S-transferase. Nonetheless, the two silver types had different cellular targets; AgNPs increased lipid peroxidation without apparent uptake into the cells whereas Ag(+) increased DNA damage. Furthermore, the toxicity of both silver types was generally decreased in cells treated with cysteine while treatment with buthionine sulfoximine increased the toxicity of both silver types., (© 2013.)

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<0.05, false discovery rate-corrected). Using functional, pathway and interactome analysis we identified several potentially important targets of PFUdA exposure, including the suppression of the acute-phase response (APR). We then measured the expression of five APR genes, fibrinogen alpha (fga), fibrinogen gamma (fgg), thrombin (f2), plasminogen (plg), and protein C (proC), in the liver of chicken embryos exposed in ovo to PFUdA. The expression of fga, f2, and proC were down-regulated in embryo livers (100 or 1000 ng/g, p<0.1) as predicted from microarray analysis, whereas fibrinogen gamma (fgg) was up-regulated and plg was not significantly affected. Our results demonstrate the utility of CEH coupled with transcriptome analysis as an in vitro screening tool for identifying novel effects of toxicant exposure. Additionally, we identified APR suppression as a potentially important and environmentally relevant target of PFUdA. These findings suggest in ovo exposure of birds to PFUdA may lead to post-hatch developmental deficiencies, such as impaired inflammatory response., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

The role of methionine supplementation in fish metabolism remains largely unexplored. This study investigates the effects of methionine deprivation (MD) on glucose uptake in rainbow trout (Oncorhynchus mykiss) hepatocytes. To this end, primary hepatocytes were incubated in the presence (+M) or the absence (-M) of methionine for 48h and glucose uptake was assessed using a novel non-radioactive, fluorescent-linked enzymatic assay. Evidence indicated that glucose uptake increased under methionine deprivation, primarily due to the increased abundance of membrane bound sodium-glucose transporter 2 (SGLT2), which was likely facilitated by the cellular reduction in [ATP] resulting from increased mitochondrial uncoupling, as supported by elevated transcript levels of uncoupling protein 2a (UCP2a). This study is the first to suggest that the mechanisms responsible for the rapid glucose uptake associated with MR are facilitated by the greater abundance of SGLT2 glucose transporter and mitochondrial uncoupling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na(+)/K(+)-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1μM βNF for 48h did not alter hepatocyte AEC, protein synthesis, or Na(+)/K(+)-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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.)

The effects of chronic social stress on hepatic glycogen metabolism were examined in rainbow trout Oncorhynchus mykiss by comparing hepatocyte glucose production, liver glycogen phosphorylase (GP) activity, and liver β-adrenergic receptors in dominant, subordinate, control, fasted, and cortisol-treated fish. Hepatocyte glucose production in subordinate fish was approximately half that of dominant fish, reflecting hepatocyte glycogen stores in subordinate trout that were just 16% of those in dominant fish. Fasting and/or chronic elevation of cortisol likely contributed to these differences based on similarities among subordinate, fasted, and cortisol-treated fish. However, calculation of the "glycogen gap"--the difference between glycogen stores used and glucose produced--suggested an enhanced gluconeogenic potential in subordinate fish that was not present in fasted or cortisol-treated trout. Subordinate, fasted, and cortisol-treated trout also exhibited similar GP activities (both total activity and that of the active or a form), and these activities were in all cases significantly lower than those in control trout, perhaps reflecting an attempt to protect liver glycogen stores or a modified capacity to activate GP. Dominant trout exhibited the lowest GP activities (20%-24% of the values in control trout). Low GP activities, presumably in conjunction with incoming energy from feeding, allowed dominant fish to achieve the highest liver glycogen concentrations (double the value in control trout). Liver membrane β-adrenoceptor numbers (assessed as the number of (3)H-CGP binding sites) were significantly lower in subordinate than in dominant trout, although this difference did not translate into attenuated adrenergic responsiveness in hepatocyte glucose production in vitro. Transcriptional regulation, likely as a result of fasting, was indicated by significantly lower β(2)-adrenoceptor relative mRNA levels in subordinate and fasted trout. Collectively, the data indicate that social status shapes liver metabolism and in particular glycogen metabolism, favoring accumulation of glycogen reserves from incoming energy in dominant fish and reliance on onboard fuels in subordinate fish.

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 EC(50) and EC(threshold) 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recently it was discovered that the perfluorooctane sulfonate (PFOS) detected in wildlife, such as fish-eating birds, had a greater proportion of linear PFOS (L-PFOS) than the manufactured technical product (T-PFOS), which contains linear and branched isomers. This suggests toxicological studies based on T-PFOS data may inaccurately assess exposure risk to wildlife. To determine whether PFOS effects were influenced by isomer content, we compared the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either L-PFOS or T-PFOS using Agilent microarrays. At equal concentrations (10 µM), T-PFOS altered the expression of more transcripts (340, >1.5-fold change, p < 0.05) compared with L-PFOS (130 transcripts). Higher concentrations of L-PFOS (40 µM) were also less transcriptionally disruptive (217 transcripts) than T-PFOS at 10 µM. Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, hepatic system development, and cellular growth and proliferation. Pathway and interactome analysis suggested that genes may be affected through the RXR receptor, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling, and PPARγ and SREBP receptors. In all functional categories and pathways examined, the response elicited by T-PFOS was greater than that of L-PFOS. These data show that T-PFOS elicits a greater transcriptional response in CEH than L-PFOS alone and demonstrates the importance of considering the isomer-specific toxicological properties of PFOS when assessing exposure risk., (Copyright © 2011 SETAC.)

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.)

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., (Copyright 2010 SETAC.)

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., (Copyright 2010 Elsevier Ltd. All rights reserved.)

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.

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.