Sustained expression of CYPs and DNA adduct accumulation with continuous exposure to PCB126 and PCB153 through a new delivery method: Polymeric implants.
ABSTRACT: A new delivery method via polymeric implants was used for continuous exposure to PCBs. Female Sprague-Dawley rats received subcutaneous polymeric implants containing PCB126 (0.15% load), PCB153 (5% load), or both, for up to 45 days and release kinetics and tissue distribution were measured. PCB153 tissue levels on day 15 were readily detected in lung, liver, mammary and serum, with highest levels in the mammary tissue. PCB126 was detected only in liver and mammary tissues. However, a completely different pharmacokinetics was observed on co-exposure of PCB153 and PCB126, with a 1.8-fold higher levels of PCB153 in the liver whereas a 1.7-fold lower levels in the mammary tissue. PCB126 and PCB153 caused an increase in expression of key PCB-inducible enzymes, CYP 1A1/2 and 2B1/2, respectively. Serum and liver activities of the antioxidant enzymes, PON1 and PON3, and AhR transcription were also significantly increased by PCB126. 32 P-Postlabeling for polar and lipophilic DNA-adducts showed significant quantitative differences: PCB126 increased 8-oxodG, an oxidative DNA lesion, in liver and lung tissues. Adduct levels in the liver remained upregulated up to 45 days, while some lung DNA adducts declined. This is the first demonstration that continuous low-dose exposure to PCBs via implants can produce sustained tissue levels leading to the accumulation of DNA-adducts in target tissue and induction of indicator enzymes. Collectively, these data demonstrate that this exposure model is a promising tool for long-term exposure studies.
Project description:Previous reports suggested that non-dioxin-like (NDL) PCB153 effects on cytochrome P450 3A (Cyp3a) expression in Atlantic killifish (Fundulus heteroclitus) gills differed between F0 generation fish from a PCB site (New Bedford Harbor; NBH) and a reference site (Scorton Creek; SC). Here, we examined effects of PCB153, dioxin-like (DL) PCB126, or a mixture of both, on Cyp3a56 mRNA in killifish generations removed from the wild, without environmental PCB exposures. PCB126 effects in liver and gills differed between populations, as expected. Gill Cyp3a56 was not affected by either congener in NBH F2 generation fish, but was induced by PCB153 in SC F1 fish, with females showing a greater response. PCB153 did not affect Cyp3a56 in liver of either population. Results suggest a heritable resistance to NDL-PCBs in killifish from NBH, in addition to that reported for DL PCBs. Induction of Cyp3a56 in gills may be a biomarker of exposure to NDL PCBs in fish populations that are not resistant to PCBs.
Project description:To assess the impact of a mixture containing dioxin-like and non-dioxin-like polychlorinated biphenyls (PCBs), male mice were initiated with N-nitroso-diethylamine and subsequently treated with PCB126, an Ah-Receptor agonist, and PCB153, acting via activation of the constitutive androstane receptor. The two congeners were given at two dose levels: the low dose was adjusted to induce ~150-fold increases in cytochrome P450 (Cyp)1a1 (PCB126) and Cyp2b10 mRNAs (PCB153), and the high dose was chosen as twice the low dose. To keep the liver PCB levels constant, mice were given initial loading doses followed by weekly maintenance doses calculated on the basis of the PCBs' half-lives. Mice were treated with the individual congeners (low and high dose) or with a mixture consisting of the low doses of the 2 PCBs. The following results were obtained: (1) the 2 PCBs produced dose-dependent increases in Cyp1a1 and Cyp2b10 mRNA, protein, and activity when given individually; (2) combined treatment caused more than additive effects on Cyp1a1 mRNA expression, protein level, and ethoxyresurofin activity; (3) changes in the levels of several proteins were detected by proteome analysis in livers of PCB-treated mice; (4) besides these biological responses, the individual PCBs caused no significant increase in the number of glucose-6-phospatase (G6Pase)-deficient neoplastic lesions in liver, whereas a moderate significant effect occurred in the combination group. These results suggest weak but significant response-additive effects of the 2 PCBs when given in combination. They also suggest that the Cyp biomarkers tend to overestimate the carcinogenic response produced by the PCBs in mouse liver.
Project description:Following absorption, polychlorinated biphenyls (PCBs) bind to albumin and are transported via blood into the target tissues. PCBs then accumulate in tissues and induce a variety of harmful chronic and developmental effects. The aim of the present study is to determine binding parameters, such as binding constant, quenching constant, and number of binding sites for three PCB congeners (PCB118, PCB126 and PCB153) in complex with human serum albumin (HSA). The binding parameters for the complexes of HSA-PCB118, HSA-PCB126, and HSA-PCB153 excited at 280 nm were compared with those excited at 295 nm. Quenching (static and dynamic) of HSA fluorescence was analyzed based on the Stern-Volmer method. Binding (Ka) constants were calculated according to the Scatchard method and analysis of non-linear regression was based on a two-component model with the Lavenberg-Marquardt algorithm. For all analyzed complexes, a single independent class of binding site for PCB congeners was found in HSA subdomain IIA. Tyrosine residues appear to play the most prominent role in binding of PCB126 to HSA, while tryptophan-214 played a dominant role in interactions of PCB153 with HSA. Among studied PCB congeners, PCB118 formed the most stable complexes with HSA. These results illustrate the importance of studies targeting the binding of PCBs to serum albumin as part of the strategy to understand and protect against toxicity of these environmental toxicants.
Project description:Sentinel species such as the Atlantic killifish (Fundulus heteroclitus) living in urban waterways can be used as toxicological models to understand impacts of environmental metabolism disrupting compound (MDC) exposure on both wildlife and humans. Exposure to MDCs is associated with increased risk of metabolic syndrome, including impaired lipid and glucose homeostasis, adipogenesis, appetite control, and basal metabolism. MDCs are ubiquitous in the environment, including in aquatic environments. New Bedford Harbor (NBH), Massachusetts is polluted with polychlorinated biphenyls (PCBs), and, as we show for the first time, tin (Sn). PCBs and organotins are ligands for two receptor systems known to regulate lipid homeostasis, the aryl hydrocarbon receptor (AHR) and the peroxisome proliferator-activated receptors (PPARs), respectively. In the current study, we compared lipid homeostasis in laboratory-reared killifish from NBH (F2) and a reference location (Scorton Creek, Massachusetts; F1 and F2) to evaluate how adaptation to local conditions may influence responses to MDCs. Adult killifish from each population were exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126, dioxin-like), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153, non-dioxin-like), or tributyltin (TBT, a PPAR? ligand) by a single intraperitoneal injection and analyzed after 3 days. AHR activation was assessed by measuring cyp1a mRNA expression. Lipid homeostasis was evaluated phenotypically by measuring liver triglycerides and organosomatic indices, and at the molecular level by measuring the mRNA expression of pparg and ppara and a target gene for each receptor. Acute MDC exposure did not affect phenotypic outcomes. However, overall NBH killifish had higher liver triglycerides and adiposomatic indices than SC killifish. Both season and population were significant predictors of the lipid phenotype. Acute MDC exposure altered hepatic gene expression only in male killifish from SC. PCB126 exposure induced cyp1a and pparg, whereas PCB153 exposure induced ppara. TBT exposure did not induce ppar-dependent pathways. Comparison of lipid homeostasis in two killifish populations extends our understanding of how MDCs act on fish and provides a basis to infer adaptive benefits of these differences in the wild.
Project description:PCBs are persistent organic pollutants that are carcinogenic and immunotoxic and have developmental toxicity. This suggests that they may interfere with normal cell maturation. Cancer and stem/progenitor cells have telomerase activity to maintain and protect the chromosome ends, but lose this activity during differentiation. We hypothesized that PCBs interfere with telomerase activity and the telomere complex, thereby disturbing cell differentiation and stem/progenitor cell function. HL-60 cells are cancer cells that can differentiated into granulocytes and monocytes. We exposed HL-60 cells to PCB126 (dioxin-like) and PCB153 (nondioxin-like) 6 days before and during 3 days of differentiation. The differentiated cells showed G0/G1 phase arrest and very low telomerase activity. hTERT and hTR, two telomerase-related genes, were downregulated. The telomere shelterins TRF1, TRF2, and POT1 were upregulated in granulocytes, and TRF2 was upregulated and POT1 downregulated in monocytes. Both PCBs further reduced telomerase activity in differentiated cells, but had only small effects on the differentiation and telomere-related genes. Treatment of undifferentiated HL-60 cells for 30 days with PCB126 produced a downregulation of telomerase activity and a decrease of hTERT, hTR, TRF1, and POT1 gene expression. With PCB153, the effects were less pronounced and some shelterin genes were increased after 30 days of exposure. With each PCB, no differentiation of cells was observed and cells continued to proliferate despite reduced telomerase activity, resulting in shortened telomeres after 30 days of exposure. These results indicate cell-type and PCB congener-specific effects on telomere/telomerase-related genes. Although PCBs do not seem to strongly affect differentiation, they may influence stem or progenitor cells through telomere attrition with potential long-term consequences for health.
Project description:Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with metabolic disruption and non-alcoholic fatty liver disease (NAFLD). Based on their ability to activate the aryl hydrocarbon receptor (AhR), PCBs are subdivided into two classes: dioxin-like (DL) and non-dioxin-like (NDL) PCBs. Previously, we demonstrated that NDL PCBs compromised the liver to promote more severe diet-induced NAFLD. Here, the hepatic effects and potential mechanisms (by untargeted liver proteomics) of DL PCBs, NDL PCBs or co-exposure to both in diet-induced NAFLD are investigated. Male C57Bl/6 mice were fed a 42% fat diet and exposed to vehicle control; Aroclor1260 (20 mg/kg, NDL PCB mixture); PCB126 (20 μg/kg, DL PCB congener); or a mixture of Aroclor1260 (20 mg/kg)+PCB126 (20 μg/kg) for 12 weeks. Each exposure was associated with a distinct hepatic proteome. Phenotypic and proteomic analyses revealed increased hepatic inflammation and phosphoprotein signaling disruption by Aroclor1260. PCB126 decreased hepatic inflammation and fibrosis at the molecular level; while altering cytoskeletal remodeling, metal homeostasis, and intermediary/xenobiotic metabolism. PCB126 attenuated Aroclor1260-induced hepatic inflammation but increased hepatic free fatty acids in the co-exposure group. Aroclor1260+PCB126 exposure was strongly associated with multiple epigenetic processes, and these could potentially explain the observed non-additive effects of the exposures on the hepatic proteome. Taken together, the results demonstrated that PCB exposures differentially regulated the hepatic proteome and the histologic severity of diet-induced NAFLD. Future research is warranted to determine the AhR-dependence of the observed effects including metal homeostasis and the epigenetic regulation of gene expression.
Project description:Exposure to polychlorinated biphenyls (PCBs) is associated with numerous adverse health effects. Although the main route of exposure to PCBs is through the gastrointestinal tract, little is known about the contribution of the gut to the health effects of PCBs. We hypothesize that PCBs can disrupt intestinal integrity, causing lipopolysaccharide (LPS) translocation into the bloodstream and potentiation of the systemic toxicity of PCBs. C57BL/6 mice were exposed to individual PCB congeners by oral gavage, followed by the assessment of small intestine morphology and plasma levels of proinflammatory mediators. In addition, mice and human brain endothelial cells were exposed to PCB118 in the presence or absence of LPS to evaluate the contribution of LPS to PCB-induced toxicity at the blood-brain barrier (BBB) level. Oral administration of PCB153, PCB118, or PCB126 disrupted intestinal morphology and increased plasma levels of LPS and proinflammatory cytokines. Direct injection of LPS and PCB118 into the cerebral microvasculature resulted in synergistic disruption of BBB integrity and decreased expression of tight junction proteins in brain microvessels. In vitro experiments confirmed these effects and indicated that stimulation of the toll-like receptor 4 (TLR4) pathway can be responsible for these effects via activation of interferon regulatory factor-3 (IRF-3). These results indicate that LPS may be a contributing factor in PCB-induced dysfunction of the brain endothelium via stimulation of the TLR4/IRF-3 pathway.
Project description:We employed DNA microarray to identify unique hepatic gene expression patterns associated with subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other halogenated aromatic hydrocarbons (HAHs). Female Harlan Sprague-Dawley rats were exposed for 13 weeks to toxicologically equivalent doses of four different HAHs based on the toxic equivalency factor of each chemical: TCDD (100 ng/kg/day), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF; 200 ng/kg/day), 3,3',4,4',5-pentachlorobiphenyl (PCB126; 1,000 ng/kg/day), or 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153; 1,000 microg/kg/day). Global gene expression profiles for each exposure, which account for 8,799 gene probe sets contained on Affymetrix RGU34A GeneChips, were compared by principal components analysis. The aryl hydrocarbon receptor (AhR) ligands TCDD, PeCDF, and PCB126 produced very similar global gene expression profiles that were unique from the nonAhR ligand PCB153, underscoring the extensive impact of AhR activation and/or the resulting hepatic injury on global gene expression in female rat liver. Many genes were co-expressed during the 13-week TCDD, PeCDF, or PCB126 exposures, including classical AhR-regulated genes and some genes not previously characterized as being AhR regulated, such as carcinoembryonic-cell adhesion molecule 4 (C-CAM4) and adenylate cyclase-associated protein 2 (CAP2). Real-time reverse-transcriptase polymerase chain reaction confirmed the increased expression of these genes in TCDD-, PeCDF-, and PCB126-exposed rats as well as the up- or down-regulation of several other novel dioxin-responsive genes. In summary, DNA microarray successfully identified dioxin-responsive genes expressed after exposure to AhR ligands (TCDD, PeCDF, PCB126) but not after exposure to the non-AhR ligand PCB153. Together, these findings may help to elucidate some of the fundamental features of dioxin toxicity and may further clarify the biologic role of the AhR signaling pathway.
Project description:Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ?400 times higher, and the levels of non-dioxin-like PCBs ?3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.
Project description:Environmental exposure to polychlorinated biphenyls (PCBs) is associated with an increased risk of incidence of metabolic disease, however the molecular mechanisms underlying this phenomenon are not fully understood. Our study provides new insights into molecular interactions between PCBs and retinoids (vitamin A and its metabolites) by defining a role for constitutive androstane receptor (CAR) in the disruption of retinoid homeostasis by non-coplanar 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153). Administration of four weekly 50?mg/kg doses of PCB153 to C57BL/6 male mice resulted in a significant decline in the tissue concentrations of retinyl esters, retinol and all-trans-retinoic acid (atRA), while no decline in hepatic and adipose tissue retinoid levels were detected in Car-null littermates. Our data imply that disrupted retinoid homeostasis occurs as a consequence of PCB153-induced activation of CAR, and raise the possibility that CAR signaling can affect atRA homeostasis in vivo. A strong correlation between the changes in retinoid metabolism and extensive upregulation of hepatic CAR-driven Cyp2b10 expression implicates this CYP isoform as contributing to retinoid homeostasis disruption via atRA oxidation during PCB153 exposure. In response to PCB153-induced CAR activation and disruption of retinoid homeostasis, expression of hepatic Pepck, Cd36 and adipose tissue Ppar?, Cd36, Adipoq, and Rbp4 were altered; however, this was reversed by administration of exogenous dietary retinoids (300?IU daily for 4?weeks). Our study establishes that PCB153 exposure enables a significant disruption of retinoid homeostasis in a CAR-dependent manner. We propose that this contributes to the obesogenic properties of PCB153 and may contribute to the predisposition to the metabolic disease.