PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells.

Dioxins and polychlorinated biphenyls (PCBs) have been shown to accumulate in the adrenal glands when incorporated into the body. However, the impacts of exposure on adrenal steroidogenesis have not been thoroughly investigated. In this study, we demonstrated that dioxin-like PCB126 altered androgen, cortisol, and aldosterone biosynthesis differentially in human adrenocortical H295R cells. PCB126 diminished basal and cAMP-induced androstenedione production as well as CYP17 mRNA expression in a dose-dependent and time-dependent manner. The CYP17 repression was accompanied with decreases in the encoded 17 alpha-hydroxylase and 17,20-lyase activities, particularly the latter. In contrast, high concentrations of PCB126 stimulated basal cortisol and aldosterone biosynthesis, including induction of CYP21B, CYP11B1, and CYP11B2 mRNA expression and elevation of the conversion of cortisol from 17-OH-progesterone and aldosterone from progesterone. cAMP abolished the positive effect of PCB126 on cortisol synthesis, while it synergistically enhanced PCB126 stimulation on CYP11B2 expression and aldosterone production. It seemed likely that the downregulation of CYP21B caused by the combination of PCB126 and cAMP counteracted the CYP11B1 induction stimulated by the co-treatment. In addition, high concentrations of PCB126 might sensitize the regulation of adrenocorticotropin (ACTH) on the adrenocortical cells by increasing ACTH receptor levels. Because adrenal steroids have profound influences on glucose tolerance, insulin sensitivity, lipid metabolism, obesity, vascular function, and cardiac remodeling, this article also discusses the potential association of the detected adrenocortical alterations with increased diabetic and cardiovascular risk found among highly exposed people.     

Polychlorinated biphenyl exposure and CYP19 gene regulation in testicular and adrenocortical cell lines.

Exposure to polychlorinated biphenyls (PCBs) disturbs many estrogen-mediated biochemical processes. PCBs may cause these abnormalities by altering expression of the aromatase gene CYP19. This study demonstrated that high concentrations of PCB126 increased basal CYP19 mRNA abundance in mouse testicular Leydig I-10 cells and human adrenocortical H295R cells. Stimulating the cells with chorionic gonadotropin or 8-Br-cAMP concealed the estrogenic effect of PCB126. PCB126 is a powerful ligand for nuclear receptor AhR. Antagonizing the AhR activity of H295R by an inhibitor abolished PCB126-elicited CYP19 induction. However, PCB126 elevated basal CYP19 expression and aromatase activity in a slow progressive manner contrary to the sharp induction of the classic AhR target gene CYP1A1. Exposure of H295R to PCBs with different AhR activation abilities also varied CYP19 and CYP1A1 expression in dissimilar patterns, although the CYP19 mRNA levels were in line with the AhR activation abilities of the congeners. In contrast to PCB126, PCB39, which could not activate AhR and lacked effect on CYP1A1, significantly reduced CYP19 mRNA expression. AhR apparently played an important role in CYP19 gene regulation, but it might regulate CYP19 differently from CYP1A1 in the adrenocortical cells. Regardless of the action mechanism, PCB exposure increases risk for CYP19 dysregulation.     

Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14-21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3-100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4-2.7nM, Lowest observed effect concentration [LOEC]: 0.3-1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.     

Comparison of hepatic and extra hepatic induction of cytochrome P4501A by graded doses of aryl hydrocarbon receptor agonists in Atlantic tomcod from two populations

Atlantic tomcod Microgadus tomcod from the Hudson River, New York, are exposed to high levels of polycyclic aromatic hydrocarbons (PAHs) and bioaccumulate mixtures of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and polychlorinatedfurans (PCDD/Fs). Previous studies demonstrated that hepatic cytochrome P4501A (CYP1A) mRNA was not inducible in tomcod from the Hudson River treated with single doses of PCB77 or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but was inducible with PAHs. In this study, we sought to determine if CYP1A mRNA was inducible with higher doses of these and other halogenated aromatic hydrocarbons (HAHs) in Hudson River tomcod and if decreased sensitivity to gene inducibility occurs across all tissues. Tomcod from the Hudson River and the cleaner Miramichi River, New Brunswick, were treated individually with graded doses of TCDD and coplanar PCBs (PCB77, PCB81, PCB126, PCB169) and profiles of hepatic CYP1A mRNA expression were compared between the two populations. CYP1A mRNA inducibility was also compared in multiple tissues of tomcod from the two rivers that were treated with PCB77. Additionally, hepatic CYP1A mRNA was characterized in Miramichi River tomcod treated with pairs of PCB congeners that included aryl hydrocarbon receptor (AHR) agonists and antagonists. Hepatic CYP1A mRNA was significantly inducible by all agonists in tomcod from the Miramichi River and TCDD and two of four PCBs in tomcod from the Hudson River. CYP1A mRNA was also significantly inducible in four of five tissues of tomcod from the Miramichi River but only in liver of Hudson River tomcod. In summary, CYP1A mRNA inducibility was approximately two orders of magnitude less sensitive in tomcod from the Hudson River than in those from the Miramichi River. But when achieved, maximum levels of CYP1A expression were similar in tomcod from the two populations. Co-administration of PCB126 and PCB77 did not produce significantly greater CYP1A mRNA induction than administration of PCB126 alone and co-administration of mono-ortho-substituted PCB105 significantly decreased CYP1A mRNA inducibility by PCB77. These results indicate that CYP1A mRNA expression is significantly inducible by HAHs in tomcod from the Hudson River and suggest that all components of the AHR pathway are present and functional, but that the pathway is less sensitive to activation than in tomcod from the Miramichi River. Our results also indicate that CYP1A mRNA levels in environmentally exposed fish may not reflect additive tissue burdens of PCB congeners.     

New CYP1 genes in the frog Xenopus (Silurana) tropicalis: induction patterns and effects of AHR agonists during development

The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), β-naphthoflavone (βNF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6- and 1.4-fold induction versus the control, respectively). βNF induced CYP1A and CYP1C1 (26- and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and βNF was positively correlated to the number of putative dioxin response elements 0-20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 μM PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 μM PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene- and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions.     

Antagonism of aryl hydrocarbon receptor-dependent induction of CYP1A1 and inhibition of IgM expression by di-ortho-substituted polychlorinated biphenyls

Halogenated aromatic hydrocarbons (HAHs) are ubiquitous environment contaminants that produce many of their toxic effects by binding to the aryl hydrocarbon receptor (AhR). However, several investigations have demonstrated that certain polychlorinated biphenyl (PCB) congeners, principally di-ortho-chlorinated PCB congeners, or mixtures containing multiple di-ortho-chlorinated PCBs, inhibit AhR-mediated responses induced by other toxic HAHs. Most relevant to the present study are past reports demonstrating antagonism by these uniquely acting PCB congeners on AhR agonist-mediated inhibition of humoral immune responses. The mechanism responsible for antagonism of AhR agonists by certain PCBs is presently unknown. The present study evaluated the antagonist activity of several di-ortho-substituted PCB congeners [PCB47 (2,2',4,4'), PCB52 (2,2',5,5'), PCB128 (2,2',3,3',4,4'), and PCB153 (2,2',4,4',5,5')] when present in combination with AhR agonists [TCDD (2,3,7,8,-tetrachlorodibenzo-p-dioxin), PCB126 (3,3',4,4',5), and PCB77 (3,3',4,4')] on CYP1A1 induction and inhibition of lipopolysaccharide (LPS)-induced immunoglobulin production in the CH12.LX B cell line. In contrast to non-ortho-substituted PCB (PCB77), which showed additive effects on CYP1A1 induction in combination with TCDD, all of the di-ortho-substituted PCBs examined produced antagonism. Di-ortho-substituted PCB (PCB52) also antagonized TCDD- or PCB126- mediated inhibition of IgM secretion and immunoglobulin heavy chain mRNA expression in the LPS-activated B cells. In addition, PCB52 inhibited TCDD-induced AhR DNA binding to a dioxin-responsive element. Collectively, these results suggest that the mechanism responsible for antagonism by di-ortho-substituted PCB congeners of AhR agonist-mediated CYP1A1 induction and inhibition of antibody responses in B cells occurs through interference with agonist activation of the cytosolic AhR complex.     

A comparative study on the effects of 2,3,7,8,-tetrachlorodibenzo-p-dioxin polychlorinated biphenyl126 and estrogen in human bronchial epithelial cells

Epidemiological studies on 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) exposure indicated high incidences of pulmonary dysfunctions and lung cancer. Animal studies also demonstrated lung cancer development in female, but not in male, rats exposed to TCDD. Such effects, however, have not been reported in polychlorinated biphenyls (PCB) exposure. In our present study, we have investigated the effects of TCDD and PCB126, with or without cotreatment with 17 beta-estradiol (E2), on a human bronchial epithelial cell line BEAS-2B. We found that treatment with either TCDD or PCB126 alone reduced cell numbers as well as thymidine incorporation. Cell death, however, was only detected in PCB126-, but not TCDD-, treated cultures. The TCDD-induced cell reduction, therefore, could not be contributed to cell death. Meanwhile, because TCDD- and PCB126-enhanced CYP1A1 and CYP1B1 expressions were significantly reduced by the AhR antagonist and CYP1 inhibitor alpha-naphthoflavone (ANF), this indicated that the effects of TCDD and PCB126 were AhR and cytochrome p450 1 dependent. We also found that while E2 itself did not alter CYP1A1 and CYP1B1 expressions, cotreatment of E2 with TCDD or PCB126 would significantly enhance TCDD-, but not PCB126-, induced toxicity. We further demonstrated that in the presence of E2, 1 nM TCDD increased the production of E2 metabolites, 2-methoxyestradiol (2-MeOE2) and 4-methoxyestradiol (4-MeOE2). PCB126, however, only increased 2-MeOE2 formation without significant induction of 4-MeOE2. We believe that these metabolites, especially 4-MeOE2, interacted with TCDD to further suppress cell growth. Our data provided the first demonstration on the enhancement of TCDD-induced toxicity in human lung cells via interaction with estrogen.     

The chlorinated AHR ligand 3,3',4,4',5-pentachlorobiphenyl (PCB126) promotes reactive oxygen species (ROS) production during embryonic development in the killifish (Fundulus heteroclitus)

Exposure to dioxin-like chemicals that activate the aryl hydrocarbon receptor (AHR) can result in increased cellular and tissue production of reactive oxygen species (ROS). Little is known of these effects during early fish development. We used the fish model, Fundulus heteroclitus, to determine if the AHR ligand and pro-oxidant 3,3',4,4',5-pentachlorobiphenyl (PCB126) can increase ROS production during killifish development, and to test a novel method for measuring ROS non-invasively in a living organism. The superoxide-sensitive fluorescent dye, dihydroethidium (DHE), was used to detect in ovo ROS production microscopically in developing killifish exposed to PCB126 or vehicle. Both in ovo CYP1A activity (ethoxyresorufin-o-deethylase, EROD) and in ovo ROS were induced by PCB126. In ovo CYP1A activity was inducible by PCB126 concentrations as low as 0.003 nM, with maximal induction occurring at 0.3 nM PCB126. These PCB126 concentrations also significantly increased in ovo ROS production in embryonic liver, ROS being detectable as early as 5 days post-fertilization. These data demonstrate that the pro-oxidant and CYP1A inducer, PCB126, increases both CYP1A activity and ROS production in developing killifish embryos. The superoxide detection assay (SoDA) described in this paper provides a semi-quantitative, easily measured, early indicator of altered ROS production that can be used in conjunction with simultaneous in ovo measurements of CYP1A activity and embryo development to explore functional relationships among biochemical, physiological and developmental responses to AHR ligands.     

First Selective CYP11B1 Inhibitors for the Treatment of Cortisol-Dependent Diseases

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Aldosterone synthesis and cytokine production in human peripheral blood mononuclear cells

Previously, we reported that spironolactone reduced cytokine production in cultured human peripheral blood mononuclear cells (PBMCs) with angiotensin (Ang) II stimulation. To address the mechanisms underlying this effect, we examined the contribution of aldosterone to cytokine production in cultured human PBMCs with Ang II stimulation. PBMCs expressed the messenger RNA (mRNA) of Ang II type 1 receptor (AT1R) and mineralocorticoid receptor (MR) both spontaneously and after Ang II stimulation, but expressed Ang II type 2 receptor (AT2R) under neither condition. After 24 h of incubation, exogenous Ang II induced the expression of CYP11B2 (a key enzyme of aldosterone synthesis) mRNA and caused aldosterone synthesis. CV-11974 (an AT1R antagonist) reduced Ang II-induced aldosterone synthesis, whereas PD-123319 (an AT2R antagonist) had no effect. The concentration of aldosterone peaked earlier than those of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha). After 48 h of incubation (under the influence of synthesized aldosterone), CV-11974 and spironolactone significantly reduced the Ang II-enhanced production of MCP-1 and TNF-alpha, whereas PD-123319 also had no effect. In conclusion, Ang II induces aldosterone synthesis through AT1R and enhances cytokine production through an AT1R-dependent mechanism and, at least partly, through a MR-dependent mechanism in human PBMCs.     

Probing the control elements of the CYP1A1 switching module in H4IIE hepatoma cells.

Previous research from our laboratory has shown a switch-like response to PCB 126 mediated CYP1A1 induction in primary rat hepatocytes and in H4IIE rat hepatoma cells. On a single cell level, cells appear to be either "on" or "off" for CYP1A1 induction at a given dose; some cells never respond to PCB 126. These cells represent a non-responding population. Cells that are switched "on" by PCB 126 display varying levels of induction, much like the dimmer on a light switch. The goal of the present research is to begin to uncover the mechanism for this switch-like response to CYP1A1 induction in H4IIE rat hepatoma cells. The AhR pathway is modulated by multiple co-activators and by phosphorylation. This research focuses on the phosphorylation cascades initiated by PCB 126 and the role they play in CYP1A1 induction. Our research reveals a likely role for protein kinase C (PKC) in this switch response. Inhibition of PKC by H-7 dramatically reduced the percent of cells that express CYP1A1 in response to PCB 126 treatment, as determined by flow cytometry. The effect of H-7 was concentration dependent, decreasing the number of cells expressing CYP1A1 rather than decreasing the level of CYP1A1 in all cells. This finding provides further evidence for the switch-like behavior of CYP1A1 induction and implicates PKC in this response to PCB126. The protein kinase inhibitor, HA-1004, had only a minor effect on CYP1A1 induction. A high-throughput immunoblot screen for 40 proteins revealed the regulation of several proteins/phosphoproteins by PCB 126. Most importantly, two proteins containing phosphoserine/phoshothreonine residues were increased by PCB126 treatment. However, PKC translocation studies and activity studies failed to verify that PCB126 activates PKC. It is possible that constitutive PKC activity is sufficient to maintain phosphorylation of critical components of the AhR pathway. Immunoblotting studies showed that MAP kinases ERK and JNK are not activated by PCB 126 in H4IIE cells and the ERK inhibitor U0126 did not impair CYP1A1 induction. Additional studies are planned to further investigate the role of PKC in the switch-like response to PCB 126.     

Induction of cytochrome P450 1B1 in MDA-MB-231 human breast cancer cells by non-ortho-substituted polychlorinated biphenyls

The effects of 12 non-ortho-substituted polychlorinated biphenyl (PCB) congeners on the induction of human cytochrome P450 1B1 (CYP1B1), an estradiol 4-hydroxylase, were investigated in MDA-MB-231 breast cancer cells. Three independent quantitative assays were used, in which the rates of estrogen metabolism, the levels of the CYP1B1 and CYP1A1 mRNAs, and luciferase activities under the control of the CYP1B1 promoter were measured. Of the congeners investigated, 3,4,4',5-tetrachlorobiphenyl (PCB 81), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 3,4',5-trichlorobiphenyl (PCB 39) and 3,3',4,5-tetrachlorobiphenyl (PCB 78) were the most potent in each assay, causing four to 10-fold increases in response. Exposure to 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) resulted in elevated CYP1B1 mRNA and increased CYP1B1-promoter driven luciferase activity, but caused depressed rather than elevated rates of E(2) metabolism due to inhibition of CYP1B1. The relative magnitudes of CYP1B1 induction by the PCB congeners, as determined by the three assays, were in close agreement, with the exception noted for PCB 169. These results indicate that PCB structure-activity relationships for the induction of human CYP1B1 are similar to those observed for human CYP1A1, but differ somewhat from what has been reported for induction of rat CYP1A1.