Chick Embryo Hepatocytes Research Articles

Hypertonic stress regulates amino acid transport and cell cycle proteins in chick embryo hepatocytes

Hyperosmotic stress affects cell growth, decreasing cell volume and increasing the uptake of organic osmolytes. However, the sensitivity of embryonic cells to osmotic treatment remains to be established. We have analysed some aspects of cell-cycle control and amino-acid transport in hypertonic conditions during prenatal life. The effects of hyperosmotic stress on amino-acid uptake mediated by system A, (3)H-thymidine incorporation, and regulation of cell-cycle proteins were analysed in chick embryo hepatocytes. Hypertonic stress increased system A activity and caused cell-cycle delay. Effects on amino-acid transport involved p38 kinase activation and new carrier synthesis. Cyclin D1, cdk4 (cyclin-dependent kinase 4) and PCNA (proliferating-cell nuclear antigen) levels decreased, whereas cyclin E, p21 and p53 levels were unchanged. Incorporation of (3)H-leucine indicated decreased synthesis of cyclin D1. In contrast, analysis of mRNA by qRT-PCR (quantitative real-time PCR) showed a net increase of cyclin D1 transcripts, suggesting post-transcriptional regulation. The data show that chick embryo hepatocytes respond to hyperosmotic conditions by arresting cell growth to prevent DNA damage and increasing osmolyte uptake to regulate cell volume, indicating that the adaptive response to environmental stress exists during prenatal life.

Induction of cytochrome P4501A by highly purified hexachlorobenzene in primary cultures of ring-necked pheasant and Japanese quail embryo hepatocytes

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.

Leptin stimulates hepatic activation of thyroid hormones and promotes early posthatch growth in the chicken

Hepatic iodothyronine deiodinases (Ds) are involved in the conversion of thyroid hormones (THs) which interacts with growth hormone (GH) to regulate posthatch growth in the chicken. Previous studies suggest that leptin-like immunoreactive substance deposited in the egg may serve as a maternal signal to program posthatch growth. To test the hypothesis that maternal leptin may affect early posthatch growth through modifying hepatic activation of THs, we injected 5.0μg of recombinant murine leptin into the albumen of breeder eggs before incubation. Furthermore, chicken embryo hepatocytes (CEHs) were treated with leptin in vitro to reveal the direct effect of leptin on expression and activity of Ds. In ovo leptin administration markedly accelerated early posthatch growth, elevated serum levels of total and free triiodothyronine (tT3 and fT3), while that of total thyroxin (tT4) remained unchanged. Hepatic mRNA expression and activity of D1 which converts T4 to T3 or rT3 to T2, were significantly increased in leptin-treated chickens, while those of D3 which converts T3 to T2 or T4 to rT3, were significantly decreased. Moreover, hepatic expression of GHR and IGF-I mRNA was all up-regulated in leptin-treated chickens. Males demonstrated more pronounced responses. A direct effect of leptin on Ds was shown in CEHs cultured in vitro. Expression and activity of D1 were increased, whereas those of D3 were decreased, in leptin-treated cells. These data suggest that in ovo leptin administration improves early posthatch growth, in a gender-specific fashion, probably through improving hepatic activation of THs and up-regulating hepatic expression of GHR and IGF-I.

Highly purified hexachlorobenzene induces cytochrome P4501A in primary cultures of chicken embryo hepatocytes

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.

Effects of 18 Perfluoroalkyl Compounds on mRNA Expression in Chicken Embryo Hepatocyte Cultures

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.

QSAR study on the non-monotonic dose-response curve of PCBs in chicken embryo hepatocyte bioassay

Endocrine disrupting chemicals (EDCs) in the natural environment exhibit a unique non-monotonic dose-response curve and it is impossible to select one simple index to characterize the bilogogical activity of these compounds. Quantitative structure-activity relationship (QSAR) study on non-monotonic dose-response curve has become a real challenge presently. In order to explore the possible mechanism for the non-monotonic dose-response curve of polychlorinated biphenyls congeners (PCBs) in chicken embryo hepatocyte bioassay, AM1 method of ChemOffice was adopted to calculate necessary structure descriptors for PCBs, while the interactions between PCBs and simulated AhR ligand binding domain (LBD) were analyzed by using FlexX in SYBYL7.0. Different binding modes for PCBs have been distinguished not only from aligned conformation but also from free binding energy. Some QSAR models were established separately for both low and high doses ranges, revealing that receptor binding may predominate in the interference of the physiological function of cytochrome P4501A-P4501A in the low doses range. But with the higher doses range, the EROD suppression might be related to acute toxicity owing to molecular polarity or distribution of charges and consequently damage structure and function of chicken embryo hepatocyte.

Comparison of Genetic and Physiological Properties ofSalmonella entericaIsolates from Chickens Reveals One Major Difference Between Serovar Kentucky and Other Serovars: Response to Acid

For unknown reasons, Salmonella enterica Kentucky has become the serovar most frequently isolated from chickens and chicken carcasses in the United States. In an attempt to identify traits that may underlie this phenomenon, genetic and physiological features of 30 serovar Kentucky chicken isolates were compared with those of chicken isolates belonging to a range of other S. enterica serovars. Most of the well-known Salmonella virulence genes were detected in the serovar Kentucky isolates by PCR, but the cdtB, spvB, spvC, and pefA genes were not found. The serovar Kentucky isolates were as invasive as the non-Kentucky isolates in in vitro assays involving chicken embryo hepatocytes, but were less invasive than the Enteritidis, Mbandaki, and Typhimurium isolates when incubated with human HCT-8 cells. Statistical comparison of growth, biofilm formation, and stress survival data from the serovar Kentucky and those from the serovar Enteritidis, Hadar, Mbandaka, Senftenberg, Typhimurium, and Worthington isolates demonstrated either no differences or differences with only a few of the serovars; however, three data sets were different. These data sets included the OD(600) values of cultures grown in tryptic soy broth (TSB) adjusted to pH 5.5 with acetic acid and survival counts of cells grown in either TSB pH 7 or TSB adjusted to pH 5.5 with acetic acid and then transferred into TSB adjusted to pH 2.5 with HCl. Most notable was the log(10) reduction for acetic acid pre-exposed Kentucky isolates of 3.1 versus <1 log(10) for the other isolates upon transfer to pH 2.5. The connection, if any, between this acid response phenotype and the prevalence of the serovar Kentucky in poultry remains to be elucidated, but it is possible that slightly better growth in the presence of acetic acid in conjunction with not mounting a strong, energy-consuming acetic acid-induced adaptive acid response provides a small competitive advantage to this serovar in low acid environments such as the cecum where the pH is around 5.5.

Chicken Embryo Cardiomyocyte Cultures—A New Approach for Studying Effects of Halogenated Aromatic Hydrocarbons in the Avian Heart

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.

Effects of co-exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin and perfluorooctane sulfonate or perfluorooctanoic acid on expression of cytochrome P450 isoforms in chicken ( Gallus gallus) embryo hepatocyte cultures

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 µM and 60 µM with or without co-exposure to TCDD (0.3 nM at maximum). PFOA did not decrease cell viability even at maximum concentrations of 60 µM. 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 µM PFOS plus TCDD (0.3 nM) induced CYP4V2 mRNA compared to TCDD alone ( P < 0.05). This trend was similar to that observed with co-exposure to TCDD plus PFOA, suggesting that PFOA alone did not induce CYP4V2 mRNA, whereas co-exposure to TCDD plus PFOA induced the expression levels. PFOS alone decreased CYP3A37 mRNA by a maximum of 45%, however after co-exposure to TCDD, recovery of mRNA expression to levels measured in DMSO-treated cells was observed. Our data suggest a complex gene response to mixtures of dioxin-like and perfluorinated compounds.

Perfluorooctane sulfonate (PFOS) toxicity in domestic chicken ( Gallus gallus domesticus) embryos in the absence of effects on peroxisome proliferator activated receptor alpha (PPARα)-regulated genes

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 (PPARα)-regulated genes in cultured primary chicken embryo hepatocytes. The present study examined whether PPARα-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 μg PFOS/g egg into the air cell prior to incubation. Embryos were incubated until pipping, after which the expression of PPARα-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 μg/g (3.54 μg/g–672,910 μg/g, 95% confidence interval). Hepatic PFOS concentrations increased concomitantly with dose. The PPARα-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 PPARα-regulated genes at any of the doses examined in day 21 chicken embryos.

Cell-specific signal transduction pathways regulating Na+-K+-ATPase. Focus on “Short-term effects of thyroid hormones on the Na+-K+-ATPase activity of chick embryo hepatocytes during development: focus on signal transduction”

the na+-k+-atpase is a complex integral membrane protein that carries out active transport of sodium and potassium across the cell plasma membrane, maintaining the ionic gradients. Each subunit has multiple isozymes that are expressed in tissue and developmental specific fashion. In addition to

Short-term effects of thyroid hormones on Na+-K+-ATPase activity of chick embryo hepatocytes during development: focus on signal transduction

Nongenomic effects of thyroid hormones on Na+-K+-ATPase activity were studied in chick embryo hepatocytes at two different developmental stages, 14 and 19 days of embryonal age, and the signal transduction pathways involved were characterized. Our data showed the following. 1) 3,5,3'-Triiodo-L-thyronine (T3) and 3,5-diiodo-L-thyronine (3,5-T2) rapidly induced a transient inhibitory effect on the Na+-K+-ATPase; the extent and duration depended on the developmental age of the cells. 2) 3,5-T2 behaved as a true hormone and fully mimicked the effect of T3. 3) Thyroxine had no effect at any of the developmental stages. 4) The inhibition of Na+-K+-ATPase was mediated by activation of protein kinase A, protein kinase C, and phosphoinositide 3-kinase, suggesting several modes of modulation of ATPase activity through phosphorylation at different sites. 5) The MAPK pathway did not seem to be involved in the early phase of hormone treatment. 6) The nonpermeant analog T3-agarose inhibited Na+-K+-ATPase activity in the same way as T3, confirming that hormone signaling initiated at a receptor on the plasma membrane. From these results, it can be concluded that the cell response mechanisms change rapidly and drastically within the early phase of embryo growth. The differences found at the two stages probably reflect the different roles of thyroid hormones during development and differentiation.

Exposure to perfluorooctane sulfonate or fenofibrate causes PPAR-α dependent transcriptional responses in chicken embryo hepatocytes

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-α). Activated PPAR-α up-regulates PPAR-α 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-α, we exposed chicken embryo primary hepatocyte cultures ( N = 3 independent cell cultures) to PFOS or fenofibrate, a mammalian PPAR-α agonist, and examined the expression of PPAR-α and PPAR-α 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-α mRNA expression was not altered by PFOS or fenofibrate. This study provides the first evidence that PFOS can induce PPAR-α-dependent transcriptional responses in an avian species and provides the first characterization of fenofibrate induced transcriptional responses in chicken embryo hepatocyte cultures.

Chenodeoxycholic acid suppresses the activation of acetyl-coenzyme A carboxylase-α gene transcription by the liver X receptor agonist T0-901317

The therapeutic utility of liver X receptor (LXR) agonists in treating atherosclerosis is limited by an undesired accumulation of triglycerides in the blood and liver. This effect is caused by an increase in the transcription of genes involved in fatty acid synthesis. Here, we show that the primary bile acid, chenodeoxycholic acid (CDCA), antagonizes the stimulatory effect of the synthetic LXR agonist, T0-901317, on the expression of acetyl-coenzyme A carboxylase-alpha (ACCalpha) and other lipogenic enzymes in chick embryo hepatocyte cultures. CDCA inhibits T0-901317-induced ACCalpha transcription by suppressing the enhancer activity of a LXR response unit (-101 to -71 bp) that binds LXR and sterol-regulatory element binding protein-1 (SREBP-1). We also demonstrate that CDCA decreases the expression of SREBP-1 in the nucleus and the acetylation of histone H3 and H4 at the ACCalpha LXR response unit. The CDCA-mediated reduction in ACCalpha expression is associated with a decrease in the expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) and small heterodimer partner and an increase in the expression of fibroblast growth factor-19 (FGF-19). Ectopic expression of FGF-19 decreases T0-901317-induced ACCalpha expression. Inhibition of p38 mitogen-activated protein kinase (MAPK) and/or extracellular signal-regulated kinase (ERK) suppresses the effects of CDCA on the expression of ACCalpha, SREBP-1, PGC-1alpha, and FGF-19. These results demonstrate that CDCA inhibits T0-901317-induced ACCalpha transcription by suppressing the activity of LXR and SREBP-1. We postulate that p38 MAPK, ERK, PGC-1alpha, and FGF-19 are components of the signaling pathway(s) mediating the regulation of ACCalpha gene transcription by CDCA.

Role of the PI3-kinase/mTor pathway in the regulation of the stearoyl CoA desaturase (SCD1) gene expression by insulin in liver

The stearoyl-CoA desaturase 1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids. This enzyme is a critical control point regulating hepatic lipogenesis and lipid oxidation. Therefore SCD1 may be a potential therapeutic target in the treatment of obesity and metabolic syndrome. Regulation of SCD1 expression occurs primarily at the level of transcription. In the present study, we characterized the insulin response elements (IREs) and the insulin signaling pathway mediating the regulation of SCD1 gene transcription in liver. In chicken embryo hepatocytes (CEH) and HepG2 cells, insulin stimulates SCD1 promoter activity by 2.5 folds. This activation is mediated by two different IREs on the chicken promoter, one localized between -1,975 and -1,610 bp and one between -372 and -297 bp. The latter binds both NF-Y and SREBP-1 transcription factors in response to insulin. We also demonstrated that insulin induction of SCD1 gene expression and promoter activity is abolished by pre-incubation of cells with specific inhibitors of both PI3-kinase (LY294002) and mTor (Rapamycin) or by over-expression of a dominant negative mutant of PI3-kinase. The PI3-kinase and mTor pathway mediates the insulin response on both IREs. In summary, insulin activates SCD1 gene expression in liver via a signaling pathway that involves PI3-kinase and mTor and the downstream transcription factors NF-Y and SREBP-1. Sentence summary: Insulin regulates SCD1 gene expression via two different IREs. The most 3' IRE is localized between -372 and -297 bp and binds the NF-Y and SREBP-1 transcription factors in response to insulin. PI3-kinase and mTor mediate the action of insulin on both IREs.

Same-sample analysis of ethoxyresorufin- O-deethylase activity and cytochrome P4501A mRNA abundance in chicken embryo hepatocytes

Inducibility of the cytochrome P4501A4 (CYP1A4) enzyme, measured as ethoxyresorufin- O-deethylase (EROD) activity, has been used as a biomarker for sensitivity to the effects of dioxin-like compounds in avian species. Here, we present a quantitative reverse transcriptase-PCR (Q-PCR) method for assessing this biomarker response at the level of messenger RNA (mRNA) expression. The method was validated for use in fresh samples as well as samples that have been analyzed for EROD activity previously. Concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on CYP1A4 and CYP1A5 mRNA abundance were detected in fresh and post-EROD hepatocyte cultures. Although the quality of the RNA obtained from post-EROD samples was low, quantification of the CYP1A mRNA response to TCDD was not compromised. Several benefits of evaluating CYP1A mRNA expression in addition to EROD activity were noted. The CYP1A mRNA bioassay may provide more accurate estimates for the potency of environmental mixtures of contaminants and has a very low detection limit. When working with hepatocytes cultured from wild or endangered species, our approach can help to circumvent the problem of small sample size by maximizing the amount of data obtained from each sample.

The mechanism mediating the activation of acetyl-coenzyme A carboxylase-α gene transcription by the liver X receptor agonist T0-901317

In birds and mammals, agonists of the liver X receptor (LXR) increase the expression of enzymes that make up the fatty acid synthesis pathway. Here, we investigate the mechanism by which the synthetic LXR agonist, T0-901317, increases the transcription of the acetyl-coenzyme A carboxylase-alpha (ACC alpha) gene in chick embryo hepatocyte cultures. Transfection analyses demonstrate that activation of ACC alpha transcription by T0-901317 is mediated by a cis-acting regulatory unit (-101 to -71 bp) that is composed of a liver X receptor response element (LXRE) and a sterol-regulatory element (SRE). The SRE enhances the ability of the LXRE to activate ACC alpha transcription in the presence of T0-901317. Treating hepatocytes with T0-901317 increases the concentration of mature sterol-regulatory element binding protein-1 (SREBP-1) in the nucleus and the acetylation of histone H3 and histone H4 at the ACC alpha LXR response unit. These results indicate that T0-901317 increases hepatic ACC alpha transcription by directly activating LXR*retinoid X receptor (RXR) heterodimers and by increasing the activity of an accessory transcription factor (SREBP-1) that enhances ligand induced-LXR*RXR activity.

Exposure to 3,3′,4,4′,5‐pentachlorobiphenyl during embryonic development has a minimal effect on the cytochrome P4501A response to 2,3,7,8‐tetrachlorodibenzo‐P‐dioxin in cultured chicken embryo hepatocytes

Concentrations of dioxin-like compounds in avian eggs can vary substantially between individuals, species, and collection sites. Although the inducibility of cytochrome P4501A (CYPIA) in hepatocyte cultures is commonly used to predict species sensitivity to environmental contaminants, it is not known how exposure to dioxin-like compounds during embryonic development might alter this biomarker response. To investigate this question, we injected vehicle or 0.4, 0.8, or 1.6 microg/kg of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) into the air cell of fertilized chicken eggs before incubation, and we measured CYP1A endpoints in cultured hepatocytes of day-19 embryos from each treatment group. The CYP1A response to PCB 126 also was assessed in whole liver tissue. Embryonic exposure to the most environmentally relevant treatment, 0.4 microg/kg of PCB 126, increased CYPIA4 mRNA expression 29-fold compared to control values in whole liver tissue but only twofold compared to control values in cultured hepatocytes. The CYPIA response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was not altered in hepatocytes cultured from embryos treated with 0.4 microg/kg of PCB 126, but at 0.8 and 1.6 microg/kg of PCB 126, several concentration-dependent effects were observed. For example, embryos exposed to higher concentrations of PCB 126 in ovo were more responsive to CYP1A mRNA induction by TCDD in vitro. These findings suggest that exposure to environmental levels of dioxin-like compounds during incubation is not likely to alter species-sensitivity estimates derived from in vitro CYP1A data.

Hypothyroidism Reduces Tricarboxylate Carrier Activity and Expression in Rat Liver Mitochondria by Reducing Nuclear Transcription Rate and Splicing Efficiency

The tricarboxylate carrier (TCC), also known as citrate carrier, is an integral protein of the mitochondrial inner membrane. It is an essential component of the shuttle system by which mitochondrial acetyl-CoA, primer for both fatty acid and cholesterol synthesis, is transported into the cytosol, where lipogenesis occurs. The effect of hypothyroidism on the activity and expression of the hepatic mitochondrial TCC was investigated in this study. TCC activity was significantly decreased in hypothyroid rats as compared with euthyroid animals. This hormone deficiency effect was due to a reduction in the amount of carrier protein, which resulted from a proportionate decrease of the specific mRNA. Hypothyroidism did not influence TCC mRNA stability. On the other hand, nuclear run-on assay revealed that the transcriptional rate of TCC mRNA decreased by approximately 40% in the nuclei from hypothyroid versus euthyroid rats. In addition, the ribonuclease protection assay showed that, in the nuclei of hypothyroid rats, the ratio of mature to precursor RNA decreased, indicating that the splicing of TCC RNA is affected. Furthermore, we found that the ratio of polyadenylated/unpolyadenylated TCC RNA as well as the length of the TCC RNA poly(A) tail were similar in both euthyroid and hypothyroid rats. Thus, the rate of formation of the TCC 3'-end is not altered in hypothyroidism. These results suggest that hypothyroidism affects TCC expression at both the transcriptional and post-transcriptional levels.

Nongenomic Actions of Thyroid Hormones: Focus on Membrane Transport Systems

Extranuclear or nongenomic effects of thyroid hormones are unaffected by inhibitors of protein synthesis, and their rapid time course cannot be explained by interaction of the hormone molecule with nuclear receptors. Their origin has been localized at the plasma membrane, but also at organelles such as the endoplasmatic reticulum and mitochondria. Thyroid hormone has been reported to activate, by both genomic and non genomic mechanisms, the Ca2+-ATPase that stores calcium from the cytosol in the sarcoplasmic reticulum; the decrease in intracellular Ca2+ leads to muscle relaxation. Considering the important effects on the cardiovascular system, T3 can actually be envisaged as a potent inotropic drug. T3 is also a major regulator of the plasma membrane Na+/K+-ATPase activity; T3 and its analog 3,5-diiodothyronine rapidly inhibits Na+/K+-ATPase in chick embryo hepatocytes, whereas the activity is up-regulated in alveolar epithelial cells. Also the ubiquitous plasma membrane Na+/H+ exchanger, that regulates cell volume and pH by exchanging extracellular Na+ with cytoplasmic H+ according to the concentration gradient, is activated by T3 via both genomic and nongenomic mechanisms. A growing number of natural and synthetic thyroid hormone analogs are available to study the physiological importance of extranuclear effects; this may lead to compounds that selectively target either genomic or nongenomic receptors. Such drugs may make it possible to activate separately only a part of the complex effects normally induced by thyroid hormones, this could be of clinical relevance for the cardiovascular system, bone tissue and the Central Nervous System.