Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are known to elicit adverse health effects including skin toxicity and cancer to animals and humans. 4-Monochlorobiphenyl (PCB3), a low-chlorinated airborne PCB conger is present in human blood and the environment. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of PCB3, has been shown to induce oxidative stress and toxicity in human mammary and prostate epithelial cells. These studies were designed to investigate and characterize the cellular responses to 4-ClBQ in HaCaT human skin keratinocytes. We found that 4-ClBQ treatment increased cellular reactive oxygen species (ROS) production, inhibited cell proliferation, and induced toxicity in HaCaT cells. Results from a Human Antioxidant Mechanism PCR array and quantitative RT-PCR assay showed that the mRNA levels of antioxidant gene selenoprotein P (sepp1) and catalase were significantly downregulated by the treatment, which correlated with evident decreases in their protein levels and catalase enzymatic activity. Pharmacological (sodium selenite supplementation) and molecular (sepp1overexpression) manipulation of SEPP1 expression significantly suppressed 4-ClBQ induced oxidative stress and toxicity. Additional results demonstrated that decreased catalase expression was associated with an inhibition in transcriptional coactivator peroxisome proliferator activated receptor Γ coactivator 1α (PGC-1α) expression. Overexpression of pgc-1α restored catalase expression and activity and consequently protected HaCaT cells from 4-ClBQ induced oxidative stress and toxicity. Furthermore, results from metabolic flux analysis using Seahorse XF96 Analyzer showed that 4-ClBQ treatment increased extracellular acidification rate, proton production rate, and oxygen consumption rate, which were associated with increases in glucose uptake and in the expression of glucose metabolism regulatory gene hexokinase 2, pyruvate kinase M2, and glucose-6-phosphate dehydrogenase (G6PD). G6PD is the rate-limiting enzyme of the pentose phosphate pathway. The enhanced expression of G6PD correlated with an increase in cellular glutathione content; and inhibition of G6PD activity sensitized HaCaT cells to 4-ClBQ induced toxicity, suggesting that the protective function of the pentose phosphate pathway is active in 4-ClBQ treated cells. Interestingly, we also found that 4-ClBQ selectively and significantly decreased mitochondrial complex II subunits C (sdhc) and D (sdhd) mRNA expression and subsequently reduced complex II activity leading to metabolic oxidative stress and toxicity, which were significantly suppressed by overexpressing sdhc and sdhd in HaCaT cells. Taken together, findings from this project demonstrate that 4-ClBQ treatment increases ROS production through perturbing cellular metabolism and mitochondrial function and decreases antioxidant capacity by inhibiting SEPP1 and catalase expression in HaCaT cells. This imbalance due to increased mitochondrial prooxidant production and decreased antioxidant capacity leads to oxidative stress and toxicity. Importantly, antioxidant supplementation could abrogate 4-ClBQ induced toxicity, suggesting that antioxidants, especially nutrient-based manipulation of selenoproteins could be promising countermeasures for PCB induced adverse health effects in humans.

Recently, persistent organic pollutants such as polychlorinated biphenyls (PCBs) were classified as “metabolic disruptors” for their suspected roles is altering metabolic and energy homeostasis through bioaccumulation in liver and adipose tissues. Among PCBs, a specific congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), is a potent arylhydrocarbon receptor (AhR) agonist and elicits toxicity similar to the classic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). PCB126 levels found in human blood are particularly associated with diabetes and nonalcoholic fatty liver disease (NAFLD) in humans, however the mechanisms are unclear. We hypothesized that the accumulation of PCB126 disrupts carbohydrate and lipid metabolism by altering the functions of liver and adipose tissues. Hence, our objective was to characterize PCB126 induced-metabolic disruption and the underlying molecular mechanisms that cause toxicity. Separate animal studies were performed using a rat model to understand the time- and dose-dependent effects after PCB126 administration. The chronology of PCB126 toxicity showed early decreases in serum glucose level at 9 h, worsened in a time-dependent way until the end of the study at 12 d. Lipid accumulation and the liver pathology also worsened over time between 3 d and 12 d post administration. These observed effects in the liver were also found to be dose-dependent. The decrease in serum glucose was a result of a decrease in the transcript levels of gluconeogenic and glycogenolytic enzymes, necessary for hepatic glucose production and hence the maintenance of steady glucose levels in the blood. Phosphoenolpyruvate carboxykinase (PEPCK-C), the rate limiting enzyme of gluconeogenesis, was found to be significantly decreased upon exposure to PCB126. The expression levels of peroxisome proliferator-activated receptor alpha (Pparα) and some of its targets involved in fatty acid oxidation were also found to be time and dose-dependently decreased upon exposure to PCB126. In an attempt to understand the molecular targets that may cause these dual effects on both gluconeogenic and fatty acid oxidation, we found that PCB126 significantly decreases phosphorylation of the cAMP response element-binding protein (CREB). CREB is a nuclear transcription factor that is activated in the liver through phosphorylation; to switch-on the transcription of enzymes that catalyze gluconeogenesis and fatty acid oxidation, in order to meet energy demands, especially during fasting. Further, to understand the toxicity of PCB126 on adipose tissue, a human pre-adipocyte model that can be differentiated into mature adipocytes was used. In these studies, we found that exposure of preadipocytes to PCB126 resulted in a significant reduction in their ability to differentiate into adipocytes. This results in decreased lipid accumulation in the adipocyte. Reduction in the differentiation by PCB126 was associated with down regulation in transcript levels of a key adipocyte transcription factor, PPARγ and its transcriptional targets necessary for adipogenesis and adipocyte function. These inhibitory effects of PCB126 on the regulation of PPARγ and the initiation of adipogenesis were mediated through activation of AhR. Overall, this work shows that PCB126 disrupts nutrient homeostasis through its effects on the function of target tissues; liver and adipose. PCB126 significantly alters the nutrient homeostasis through its effects on gluconeogenesis and fatty-acid oxidation necessary for glucose and energy regulation during fasting. In addition, PCB126 interrupts the storage functions of adipose tissue by inhibiting adipogenesis and thus disrupts lipid storage and distribution

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix PER/ARNT/SIM (bHLH-PAS) transcription factor that binds ligands typified by 2,3,7,8-tetracholordibenzo-p-dioxin, translocates to the nucleus, dimerizes with the aryl hydrocarbon nuclear translocator (ARNT) and associates with specific cis xenobiotic response elements to activate transcription of genes involved with xenobiotic metabolism. AHR-mediated signal transduction has been evaluated thoroughly in the C57BL/6J mouse model system. This model system, however, may not be the most accurate model for human comparisons as the AHRb-1 allele carried by C57BL/6J contains a point mutation that prematurely truncates the receptor at 805 amino acids, while the AHRb-2, rat, and human AHR all contain an additional 42-45 amino acids at their carboxy-terminus that have 70% identity. This carboxy-terminal region could be functionally significant and the analysis of AHR-mediated signal transduction in the rat, human, or other mouse strains may better represent the physiology of the AHR pathway. ARNT is another member of the bHLH-PAS family of proteins that is essential in several distinct signal transduction pathways mediated by its dimerization with a variety of bHLH-PAS proteins. Several isoforms of ARNT have been identified in mammalian and aquatic species. While ARNT and ARNT2 exhibit >90% amino acid identity in the bHLH and PAS domains, gene knock-out of either ARNT or ARNT2 results in embryonic/perinatal lethality characterized by distinct phenotypes. This suggests that neither protein can compensate fully for the loss of the other. Since overlapping tissue specific expression of ARNT and ARNT2 does exist, but neither ARNT can compensate fully for loss of the other, this suggests that the two proteins have distinct functions in the presence of various dimerization partners. Thus, the focus of these studies is to examine the discrepancies between the rat, human, or AHRb-2 possessing the extended carboxy-terminal region and that of the AHRb-1 and also to examine the role of both ARNT and ARNT2 during AHR-mediated signal transduction.

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.

Most members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.

Most members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.

It is hypothesized that exposing endothelial cells to steady or non-reversing pulsatile shear stress produces a healthy, anti-atherogenic endothelium, whereas a reversing pulsatile shear stress promotes an unhealthy, pro-atherogenic endothelium. To further investigate this hypothesis, a novel parallel plate flow chamber system was used to expose human endothelial cells to a pro-atherogenic reversing shear stress waveform designed to simulate the wall shear stress at the carotid sinus, a region prone to atherosclerosis. Cells exposed to this reversing shear stress were compared to cells exposed to high levels of steady shear stress (15 dynes/cm²), low steady shear stress (1 dyne/cm², the time-average of the carotid shear stress), and static culture conditions. Functional analysis confirmed previous findings that reversing shear stress increases cell proliferation and monocyte adhesion. Microarray results indicate that although there are unique sets of genes controlled by both low average shear stress and by reversing flow, more genes were controlled by low average shear stress. We propose that low-time average shear stress, and not fluid reversal/oscillation, may be the more significant mechanical force. The reversing shear stress system was also used to investigate two shear stress-responsive genes, CYP1A1 and CYP1B1. Both were maximally up-regulated at arterial steady shear stresses of at least 15 dynes/cm² and reversing pulsatile shear stress attenuated expression of both genes. Furthermore, AhR nuclear localization and CYP1A1 protein expression correlate with the flow patterns in the mouse aortic arch. The data strongly suggest that the AhR/CYP1 pathway promotes an anti-atherogenic phenotype in the endothelium. Changes in free zinc were measured under different shear stresses. High steady shear stress dramatically increases the levels of free zinc in endothelial cells as compared to cells grown in static culture. This increase in free zinc is attenuated under reversing shear stress and low steady shear stress, which correlates with an increase in zinc-binding metallothinein proteins and zinc exporter Znt-1. Overall, the findings provide further insight into endothelial responses to mechanical forces and may be important in understanding mechanisms of atherosclerotic development and localization to regions of disturbed flow.

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.

Diss.--Philosophische Fakultät--Albert-Ludwig-Universität zu Freiburg i. Br., 1988.

The aryl hydrocarbon receptor (AhR) has been reported to interact with multiple signaling pathways during prostate development including the androgen receptor. AhR was overexpressed in LNCaP using PLNCX2 retrovirus vector containing AhR cDNA to determine if ectopic overexpression induces castrate resistant phenotype. The highly overexpressed AhR clone illustrated further increase in transcriptional and promotor activity for AhR and AR compared to the moderately overexpressed AhR clone and control. Western blot analysis showed more AhR, AR, cSrc, and pSrc protein expression in clones. AhR overexpression was found to induce several biological properties such as migration, invasion, proliferation, and promotion of G1 to S phase during the cell cycle. Bicalutamide treatment had no effect on AR transcriptional activity in either clone, proving resistance to anti-androgen therapy. Our results confirm that overexpression of AhR induces constitutive activity and stimulates androgen receptor signaling. This suggests a role for AhR in the development of CRPC.