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1
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Leroy K, Pieters A, Tabernilla A, Cooreman A, Van Campenhout R, Cogliati B, Vinken M. Targeting gap junctional intercellular communication by hepatocarcinogenic compounds. JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH. PART B, CRITICAL REVIEWS 2020; 23:255-275. [PMID: 32568623 DOI: 10.1080/10937404.2020.1781010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
Gap junctions in liver, as in other organs, play a critical role in tissue homeostasis. Inherently, these cellular constituents are major targets for systemic toxicity and diseases, including cancer. This review provides an overview of chemicals that compromise liver gap junctions, in particular biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. The focus in this review is placed upon the mechanistic scenarios that underlie these adverse effects. Further, the potential use of gap junctional activity as an in vitro biomarker to identify non-genotoxic hepatocarcinogenic chemicals is discussed.
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Affiliation(s)
- Kaat Leroy
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel , Brussels, Belgium
| | - Alanah Pieters
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel , Brussels, Belgium
| | - Andrés Tabernilla
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel , Brussels, Belgium
| | - Axelle Cooreman
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel , Brussels, Belgium
| | - Raf Van Campenhout
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel , Brussels, Belgium
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Cidade Universitária , São Paulo, Brazil
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel , Brussels, Belgium
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2
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Doktorova TY, Oki NO, Mohorič T, Exner TE, Hardy B. A semi-automated workflow for adverse outcome pathway hypothesis generation: The use case of non-genotoxic induced hepatocellular carcinoma. Regul Toxicol Pharmacol 2020; 114:104652. [PMID: 32251711 DOI: 10.1016/j.yrtph.2020.104652] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Revised: 01/10/2020] [Accepted: 03/29/2020] [Indexed: 02/07/2023]
Abstract
The utility of the Adverse Outcome Pathway (AOP) concept has been largely recognized by scientists, however, the AOP generation is still mainly done manually by screening through evidence and extracting probable associations. To accelerate this process and increase the reliability, we have developed an semi-automated workflow for AOP hypothesis generation. In brief, association mining methods were applied to high-throughput screening, gene expression, in vivo and disease data present in ToxCast and Comparative Toxicogenomics Database. This was supplemented by pathway mapping using Reactome to fill in gaps and identify events occurring at the cellular/tissue levels. Furthermore, in vivo data from TG-Gates was integrated to finally derive a gene, pathway, biochemical, histopathological and disease network from which specific disease sub-networks can be queried. To test the workflow, non-genotoxic-induced hepatocellular carcinoma (HCC) was selected as a case study. The implementation resulted in the identification of several non-genotoxic-specific HCC-connected genes belonging to cell proliferation, endoplasmic reticulum stress and early apoptosis. Biochemical findings revealed non-genotoxic-specific alkaline phosphatase increase. The explored non-genotoxic-specific histopathology was associated with early stages of hepatic steatosis, transforming into cirrhosis. This work illustrates the utility of computationally predicted constructs in supporting development by using pre-existing knowledge in a fast and unbiased manner.
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Affiliation(s)
- Tatyana Y Doktorova
- Edelweiss Connect GmbH, Hochbergerstrasse 60C, Technology Park Basel, Basel, Switzerland.
| | - Noffisat O Oki
- American Association for the Advancement of Science, Science & Technology Policy Fellow, USA; National Institutes of Health, Rockville, MD, USA
| | - Tomaž Mohorič
- Edelweiss Connect GmbH, Hochbergerstrasse 60C, Technology Park Basel, Basel, Switzerland
| | - Thomas E Exner
- Edelweiss Connect GmbH, Hochbergerstrasse 60C, Technology Park Basel, Basel, Switzerland
| | - Barry Hardy
- Edelweiss Connect GmbH, Hochbergerstrasse 60C, Technology Park Basel, Basel, Switzerland
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3
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Gouesse RJ, Lavoie M, Dianati E, Wade MG, Hales BF, Robaire B, Plante I. Gestational and Lactational Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants Downregulates Junctional Proteins, Thyroid Hormone Receptor α1 Expression, and the Proliferation-Apoptosis Balance in Mammary Glands Post Puberty. Toxicol Sci 2019; 171:13-31. [PMID: 31241157 PMCID: PMC6735962 DOI: 10.1093/toxsci/kfz147] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2019] [Revised: 06/11/2019] [Accepted: 06/11/2019] [Indexed: 12/21/2022] Open
Abstract
Mammary gland development requires hormonal regulation during puberty, pregnancy, and lactation. Brominated flame retardants (BFRs) are endocrine disruptors; they are added to consumer products to satisfy flammability standards. Previously, we showed that gestational and lactational exposure to an environmentally relevant mixture of BFRs disrupts proteins of the adherens junctions in rat dam mammary glands at weaning. Here, we hypothesize that perinatal exposure to the same BFR mixture also disrupts junctional proteins and signaling pathways controlling mammary gland development in pups. Dams were exposed through diet to a BFR mixture based on the substances in house dust; doses of the mixture used were 0, 0.06, 20, or 60 mg/kg/day. Dams were exposed continuously beginning prior to mating until pups' weaning; female offspring were euthanized on postnatal day (PND) 21, 46, and 208. The lowest dose of BFRs significantly downregulated adherens junction proteins, E-cadherin, and β-catenin, and the gap junction protein p-Cx43, as well as thyroid hormone receptor alpha 1 protein at PND 46. No effects were observed on estrogen or progesterone receptors. The low dose also resulted in a decrease in cleaved caspase-3, a downward trend in PARP levels, proteins involved in apoptosis, and an upward trend in proliferating cell nuclear antigen, a marker of proliferation. No effects were observed on ductal elongation or on the numbers of terminal end buds. Together, our results indicate that gestational and lactational exposure to an environmentally relevant mixture of BFRs disrupts cell-cell interactions, thyroid hormone homeostasis and the proliferation-apoptosis balance at PND 46, a critical stage for mammary gland development.
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Affiliation(s)
| | - Mélanie Lavoie
- INRS, Centre Armand-Frappier Santé Bioscience, Laval, Quebec, Canada
| | - Elham Dianati
- INRS, Centre Armand-Frappier Santé Bioscience, Laval, Quebec, Canada
| | - Mike G Wade
- Health Canada, Environmental Health Science and Research Bureau, Ottawa, Ontario, Canada
| | | | - Bernard Robaire
- Department of Pharmacology & Therapeutics
- Department of Obstetrics & Gynecology, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
| | - Isabelle Plante
- INRS, Centre Armand-Frappier Santé Bioscience, Laval, Quebec, Canada
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4
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Crobeddu B, Ferraris E, Kolasa E, Plante I. Di(2-ethylhexyl) phthalate (DEHP) increases proliferation of epithelial breast cancer cells through progesterone receptor dysregulation. ENVIRONMENTAL RESEARCH 2019; 173:165-173. [PMID: 30909102 DOI: 10.1016/j.envres.2019.03.037] [Citation(s) in RCA: 53] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/13/2018] [Revised: 02/20/2019] [Accepted: 03/15/2019] [Indexed: 05/05/2023]
Abstract
The di(2-ethylhexyl) phthalate (DEHP) is a plasticizer incorporated to plastic matrices of widely used consumer products. However, it is gradually released from these products, resulting in a chronic exposure for humans. Although DEHP, similar to other members of the phthalates family, is generally considered as an endocrine disruptor, the mechanisms implicated in its toxicity are yet poorly understood. Our objective was to determine the effects of an exposure to DEHP and to one of its major metabolite, the mono(2-ethylhexyl) phthalate (MEHP) on markers involved in breast carcinogenesis. T-47D cells were exposed to environmentally relevant and higher doses of DEHP and MEHP (0.1-10 000 nM) for 4 days. Our results showed that an exposure to 10 000 nM of DEHP and 0.1 nM of MEHP significantly increased the proliferation of T-47D cells, without inducing apoptosis. In addition, a significant increase in the protein levels of the isoform A of the progesterone receptor (PR) and of nuclear levels of PR were observed in T-47D cells exposed to 10 000 nM of DEHP. Importantly, the increased proliferation and nuclear levels of PR were totally and partially inhibited, respectively, by Mifepristone, a PR antagonist. These results suggest that an exposure to DEHP or MEHP increase cell proliferation by activating PR signaling, which could potentially increase the risks to develop breast cancer. The mechanism of activation of the progesterone pathway by DEHP and the long-term consequences of this activation remained to be elucidated.
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Affiliation(s)
| | | | - Elise Kolasa
- INRS-Institut Armand-Frappier, Laval, Québec, Canada
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5
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Zhang Q, Wu S, Liu L, Hou X, Jiang J, Wei X, Hao W. Effects of bisphenol A on gap junctions in HaCaT cells as mediated by the estrogen receptor pathway. J Appl Toxicol 2018; 39:271-281. [DOI: 10.1002/jat.3717] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2017] [Revised: 07/11/2018] [Accepted: 07/25/2018] [Indexed: 12/23/2022]
Affiliation(s)
- Qi Zhang
- Department of Toxicology, School of Public Health; Peking University, Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety; Beijing 100191 China
| | - Shuang Wu
- Department of Toxicology, School of Public Health; Peking University, Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety; Beijing 100191 China
| | - Lu Liu
- Department of Genetics, School of Basic Medical Science; Peking University; Beijing 100191 China
| | - Xiaohong Hou
- Department of Toxicology, School of Public Health; Peking University, Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety; Beijing 100191 China
| | - Jianjun Jiang
- Department of Toxicology, School of Public Health; Peking University, Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety; Beijing 100191 China
| | - Xuetao Wei
- Department of Toxicology, School of Public Health; Peking University, Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety; Beijing 100191 China
| | - Weidong Hao
- Department of Toxicology, School of Public Health; Peking University, Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety; Beijing 100191 China
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6
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Dianati E, Wade MG, Hales BF, Robaire B, Plante I. From the Cover: Exposure to an Environmentally Relevant Mixture of Brominated Flame Retardants Decreased p-β-Cateninser675 Expression and Its Interaction With E-Cadherin in the Mammary Glands of Lactating Rats. Toxicol Sci 2018; 159:114-123. [PMID: 28903489 DOI: 10.1093/toxsci/kfx123] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Proper mammary gland development and function require precise hormonal regulation and bidirectional cross talk between cells provided by means of paracrine factors as well as intercellular junctions; exposure to environmental endocrine disruptors can disturb these processes. Exposure to one such family of chemicals, the brominated flame retardants (BFRs), is ubiquitous. Here, we tested the hypothesis that BFR exposures disrupt signaling pathways and intercellular junctions that control mammary gland development. Before mating, during pregnancy and throughout lactation, female Sprague-Dawley rats were fed diets containing that BFR mixture based on house dust, delivering nominal exposures of BFR of 0 (control), 0.06, 20, or 60 mg/kg/d. Dams were euthanized and mammary glands collected on postnatal day 21. BFR exposure had no significant effects on mammary gland/body weight ratios or the levels of proteins involved in milk synthesis, epithelial-mesenchymal transition, cell-cell interactions, or hormone signalling. However, BFR exposure (0.06 mg/kg/d) down-regulated phospho-ser675 β-catenin (p-β-catSer675) levels in the absence of any effect on total β-catenin levels. Levels of p-CREB were also down-regulated, suggesting that PKA inhibition plays a role. p-β-catSer675 co-localized with β-catenin at the mammary epithelial cell membrane, and its expression was decreased in animals from the 0.06 and 20 mg/kg/d BFR treatment groups. Although β-Catenin signaling was not affected by BFR exposure, the interaction between p-β-catSer675 and E-cadherin was significantly reduced. Together, our results demonstrate that exposure to an environmentally relevant mixture of BFR during pregnancy and lactation decreases p-β-catser675 at cell adhesion sites, likely in a PKA-dependant manner, altering mammary gland signaling.
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Affiliation(s)
- Elham Dianati
- INRS, Institut Armand-Frappier, Laval, Québec, Canada.,Centre de Recherche Biomed, Université du Québec à Montréal, Montréal, Québec, Canada
| | - Michael G Wade
- Health Canada, Environmental Health Science and Research Bureau, Ottawa, Ontario, Canada
| | | | - Bernard Robaire
- Department of Pharmacology and Therapeutics.,Department of Obstetrics and Gynecology, Faculty of Medicine, McGill University, Montreal, Québec, Canada
| | - Isabelle Plante
- INRS, Institut Armand-Frappier, Laval, Québec, Canada.,Centre de Recherche Biomed, Université du Québec à Montréal, Montréal, Québec, Canada
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7
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Yang Y, Zhu J, Zhang N, Zhao Y, Li WY, Zhao FY, Ou YR, Qin SK, Wu Q. Impaired gap junctions in human hepatocellular carcinoma limit intrinsic oxaliplatin chemosensitivity: A key role of connexin 26. Int J Oncol 2015; 48:703-13. [PMID: 26648344 DOI: 10.3892/ijo.2015.3266] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Accepted: 11/12/2015] [Indexed: 12/21/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is generally believed to have low sensitivity to chemotherapeutic agents including oxaliplatin (OXA). Studies have demonstrated that gap junctions (GJs) composed of connexin (Cx) proteins have the potential to modulate drug chemosensitivity in multiple tumor cells. In the present study, we investigated the characteristics of Cx and GJs in HCC at both histologic and cytologic levels, and the effects of GJ and its effective components on OXA cytotoxicity in HCC cells in vitro. Immunohistochemistry was performed in 76 HCCs and 20 normal liver tissues to detect and locate the expression of Cx26, Cx32 and Cx43. At cytologic levels, the expression and localization of Cxs were evaluated by RT-PCR, western blot and immunofluorescence assay, respectively. The GJ function between adjacent cells was detected using dye transfer assay. The role of GJs in the modulation of OXA toxicity in HCC cells was explored using pharmacologic and molecular biologic methods. We found that Cx expression in HCC tissues was significantly lower than in normal liver tissues, and the 'internalization' from cell membrane to cytoplasm was remarkable. In vitro experiments revealed the presence of functional GJs in the SMMC-7721 HCC cells due to a small amount of Cx protein along the plasma membrane at cell-cell contacts. Regulation of this part of GJs positively influenced OXA cytotoxicity. Using RNA interference, only specific inhibition of Cx26 but not Cx32 or Cx43 reduced OXA cytotoxicity. Conversely, Cx26 overexpression by transfection of Cx26 plasmid DNA enhanced OXA cytotoxicity. This study demonstrated that during hepatocarcinogenesis, the reduced expression and internalization of Cx proteins impaired the GJ function, which further attenuated OXA cytotoxicity. Impaired GJ function may contribute to low intrinsic chemosensitivity of HCC cells to OXA, mediated by Cx26.
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Affiliation(s)
- Yan Yang
- Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
| | - Jian Zhu
- Department of Cardiovasology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
| | - Na Zhang
- Department of Pathology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
| | - Yu Zhao
- Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
| | - Wan-Yun Li
- Department of Pathology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
| | - Fu-You Zhao
- Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
| | - Yu-Rong Ou
- Department of Pathology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
| | - Shu-Kui Qin
- Department of Medical Oncology, PLA Cancer Center, Nanjing Bayi Hospital, Nanjing, Jiangsu 210002, P.R. China
| | - Qiong Wu
- Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
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8
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Delisle A, Ferraris E, Plante I. Chronic exposure to hexachlorobenzene results in down-regulation of connexin43 in the breast. ENVIRONMENTAL RESEARCH 2015; 143:229-240. [PMID: 26519829 DOI: 10.1016/j.envres.2015.10.020] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/07/2015] [Revised: 09/29/2015] [Accepted: 10/19/2015] [Indexed: 06/05/2023]
Abstract
Decreased expression of connexins has been associated with cancer, but the underlying mechanisms are poorly understood. We have previously shown that a 5 day exposure to hexachlorobenzene (HCB) resulted in decreased connexins expression in hepatocytes 45 days later, and that this down-regulation was linked to activation of Akt through the ILK pathway. Because HCB promotes cancer in both the liver and breast, the present study aimed to determine if the mechanisms are similar in both tissues. MCF-12A breast cells were thus transfected with vectors coding for either Akt or a constitutively active form of Akt. In those cells, activation of Akt was correlated with decreased Cx43 levels. Female rats were then exposed to HCB by gavage either following the same protocol used previously for the liver or through a chronic exposure. While no changes were observed after the 5 days exposure protocol, chronic exposure to HCB resulted in increased Akt levels and decreased Cx43 levels in breast cells. In vitro, Akt was activated in MCF-12A cells exposed to HCB either for 7 days or chronically, but no changes were observed in junctional proteins. Together, these results suggested that, while activation of Akt can decrease Cx43 expression in breast cells in vitro, other mechanisms are involved during HCB exposure, leading to a decrease in Cx43 levels in a model- and duration-dependent manner. Finally, we showed that HCB effects are tissue specific, as we did not observe the same results in breast and liver tissues.
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Affiliation(s)
- Ariane Delisle
- INRS-Institut Armand-Frappier, Laval, Québec, Canada H7V 1B7
| | | | - Isabelle Plante
- INRS-Institut Armand-Frappier, Laval, Québec, Canada H7V 1B7. http://www.inrs.ca
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9
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Nahta R, Al-Mulla F, Al-Temaimi R, Amedei A, Andrade-Vieira R, Bay SN, Brown DG, Calaf GM, Castellino RC, Cohen-Solal KA, Colacci A, Cruickshanks N, Dent P, Di Fiore R, Forte S, Goldberg GS, Hamid RA, Krishnan H, Laird DW, Lasfar A, Marignani PA, Memeo L, Mondello C, Naus CC, Ponce-Cusi R, Raju J, Roy D, Roy R, Ryan EP, Salem HK, Scovassi AI, Singh N, Vaccari M, Vento R, Vondráček J, Wade M, Woodrick J, Bisson WH. Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression. Carcinogenesis 2015; 36 Suppl 1:S2-18. [PMID: 26106139 DOI: 10.1093/carcin/bgv028] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.
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Affiliation(s)
- Rita Nahta
- Departments of Pharmacology and Hematology & Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA 30322, USA, Department of Pathology, Kuwait University, Safat 13110, Kuwait, Department of Experimental and Clinical Medicine, University of Firenze, 50134 Florence, Italy, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada, Program in Genetics and Molecular Biology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA, Department of Environmental and Radiological Health Sciences/Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA, Center for Radiological Research, Columbia University Medical Center, New York, NY 10032, USA, Instituto de Alta Investigacion, Universidad de Tarapaca, Arica 8097877, Chile, Division of Hematology and Oncology, Department of Pediatrics, Children's Healthcare of Atlanta and Emory University, Atlanta, GA 30322, USA, Department of Medicine/Medical Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08901-1914, USA, Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy, Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 980033, USA, Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, 90127 Palermo, Italy, Mediterranean Institute of Oncology, 95029 Viagrande, Italy, Graduate School of Biomedical Sciences and Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084-1501, USA, Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra, Serdang, Selangor 43400, Malaysia, Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontari
| | - Fahd Al-Mulla
- Department of Pathology, Kuwait University, Safat 13110, Kuwait
| | | | - Amedeo Amedei
- Department of Experimental and Clinical Medicine, University of Firenze, 50134 Florence, Italy
| | - Rafaela Andrade-Vieira
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
| | - Sarah N Bay
- Program in Genetics and Molecular Biology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA
| | - Dustin G Brown
- Department of Environmental and Radiological Health Sciences/Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA
| | - Gloria M Calaf
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032, USA, Instituto de Alta Investigacion, Universidad de Tarapaca, Arica 8097877, Chile
| | - Robert C Castellino
- Division of Hematology and Oncology, Department of Pediatrics, Children's Healthcare of Atlanta and Emory University, Atlanta, GA 30322, USA
| | - Karine A Cohen-Solal
- Department of Medicine/Medical Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08901-1914, USA
| | - Annamaria Colacci
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy
| | - Nichola Cruickshanks
- Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 980033, USA
| | - Paul Dent
- Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 980033, USA
| | - Riccardo Di Fiore
- Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, 90127 Palermo, Italy
| | - Stefano Forte
- Mediterranean Institute of Oncology, 95029 Viagrande, Italy
| | - Gary S Goldberg
- Graduate School of Biomedical Sciences and Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084-1501, USA
| | - Roslida A Hamid
- Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra, Serdang, Selangor 43400, Malaysia
| | - Harini Krishnan
- Graduate School of Biomedical Sciences and Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084-1501, USA
| | - Dale W Laird
- Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada
| | - Ahmed Lasfar
- Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, NJ 60503, USA
| | - Paola A Marignani
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
| | - Lorenzo Memeo
- Mediterranean Institute of Oncology, 95029 Viagrande, Italy
| | - Chiara Mondello
- Institute of Molecular Genetics, National Research Council, 27100 Pavia, Italy
| | - Christian C Naus
- Department of Cellular & Physiological Sciences, Life Sciences Institute, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
| | - Richard Ponce-Cusi
- Instituto de Alta Investigacion, Universidad de Tarapaca, Arica 8097877, Chile
| | - Jayadev Raju
- Toxicology Research Division, Bureau of Chemical Safety Food Directorate, Health Products and Food Branch Health Canada, Ottawa, Ontario K1A0K9, Canada
| | - Debasish Roy
- Department of Natural Science, The City University of New York at Hostos Campus, Bronx, NY 10451, USA
| | - Rabindra Roy
- Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA
| | - Elizabeth P Ryan
- Department of Environmental and Radiological Health Sciences/Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA
| | - Hosni K Salem
- Urology Dept., kasr Al-Ainy School of Medicine, Cairo University, El Manial, Cairo 12515, Egypt
| | - A Ivana Scovassi
- Institute of Molecular Genetics, National Research Council, 27100 Pavia, Italy
| | - Neetu Singh
- Advanced Molecular Science Research Centre, King George's Medical University, Lucknow, UP 226003, India
| | - Monica Vaccari
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy
| | - Renza Vento
- Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, 90127 Palermo, Italy, Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, Philadelphia, PA 19122, USA
| | - Jan Vondráček
- Department of Cytokinetics, Institute of Biophysics AS CR, Brno 612 65, Czech Republic
| | - Mark Wade
- Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT), Milan 16163, Italy and
| | - Jordan Woodrick
- Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA
| | - William H Bisson
- Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, USA
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10
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Arstikaitis J, Gagné F, Cyr DG. Exposure of fathead minnows to municipal wastewater effluent affects intracellular signaling pathways in the liver. Comp Biochem Physiol C Toxicol Pharmacol 2014; 164:1-10. [PMID: 24747326 DOI: 10.1016/j.cbpc.2014.04.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2014] [Revised: 04/01/2014] [Accepted: 04/04/2014] [Indexed: 12/12/2022]
Abstract
Municipal wastewater effluent can impact its receiving environment. In the St. Lawrence River, male fish living downstream from Montreal exhibit increased hepatic vitellogenin, intersex, delayed spermatogenesis and altered immune function. Few studies have examined genome-wide effects associated with municipal effluent exposure in fish to decipher the mechanisms of toxicity. The present objective was to identify hepatic cellular signaling pathways in fathead minnows following exposure to municipal wastewater effluent. Immature minnows were exposed for 21 days to either 0% (Control) or 20% municipal effluent, the highest concentration in the St. Lawrence River. Hepatic RNA was extracted and used to hybridize a fathead minnow oligonucleotide microarray containing approximately 15k gene sequences. A total of 1300 genes were differentially expressed, of which 309 genes had more than 2-fold change in expression level between control and MWWE-exposed fish. Of those, 118 were up-regulated and 191 were down-regulated. Altered genes grouped according to function, indicated effects on various signaling pathways, apoptosis, immune responses, and cellular metabolism. Pathway analysis software predicted at least 5 signaling pathways that were altered by treatment: cell adhesion, inflammation, various kinases, estrogen receptor signaling and WNT signaling. Various components of the canonical Wnt pathway were dramatically down-regulated, while several other genes involved in the non-canonical Wnt pathway, such as Wnt4, LRP6, and PPP2R5E, which are known to inhibit the canonical Wnt pathway, were increased. These results indicate that municipal wastewater effluent from Montreal can target and inhibit various signaling including those implicated in hepatic Wnt signaling pathway in fathead minnows.
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Affiliation(s)
- Jennifer Arstikaitis
- INRS-Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, Québec H7V 1B7,Canada
| | - François Gagné
- INRS-Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, Québec H7V 1B7,Canada; Centre Saint-Laurent, Environment Canada, Montreal, Québec H2Y 2E7, Canada
| | - Daniel G Cyr
- INRS-Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, Québec H7V 1B7,Canada.
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Hsiao PJ, Jao JC, Tsai JL, Chang WT, Jeng KS, Kuo KK. Inorganic arsenic trioxide induces gap junction loss in association with the downregulation of connexin43 and E-cadherin in rat hepatic "stem-like" cells. Kaohsiung J Med Sci 2013; 30:57-67. [PMID: 24444534 DOI: 10.1016/j.kjms.2013.10.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2013] [Accepted: 07/01/2013] [Indexed: 11/26/2022] Open
Abstract
Chronic exposure to inorganic arsenic trioxide causes tumors of the skin, urinary bladder, lung, and liver. Several cancer initiators and promoters have been shown to alter cell-cell signaling by interference with gap junction intercellular communication (GJIC) and/or modulation of cell adhesion molecules, such as connexin43 (Cx43), E-cadherin, and β-catenin. The aim of this study was to determine whether the disruption of cell-cell interactions occurs in liver epithelial cells after exposure to arsenic trioxide. WB-F344 cells were treated with arsenic trioxide (6.25-50 μM) for up to 8 hours, and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, the changes in mRNA and protein levels of Cx43, E-cadherin, and β-catenin were determined. A significant dose- and time-dependent decrease in GJIC was observed when WB-F344 cells were exposed to arsenic trioxide (p < 0.05). Consistent with the inhibition of GJIC, cells' exposure to arsenic trioxide resulted in dose- and time-dependent decreases in Cx43 and E-cadherin mRNA expression and protein levels. However, arsenic trioxide did not alter the mRNA or protein levels of β-catenin. In an immunofluorescence study, nuclei were heavily stained with anti-β-catenin antibody, indicating significant nuclear translocation. In this study, we also demonstrated that arsenic trioxide-induced GJIC loss was a reversible process. Taken together, these data support the hypothesis that disruption of cell-cell communication may contribute to the tumor-promoting effect of inorganic arsenic trioxide.
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Affiliation(s)
- Pi-Jung Hsiao
- Division of Endocrinology and Metabolism, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Jo-Chi Jao
- College of Health Science, Kaohsiung Medical University, Department of Medical Imaging and Radiological Sciences, Kaohsiung, Taiwan
| | - Jin-Lian Tsai
- Graduate Institute of Occupational Safety, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Wen-Tsan Chang
- Division of Hepatobiliopancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Kuo-Shyang Jeng
- Department of Surgery, Far Eastern Memorial Hospital, Taipei, Taiwan
| | - Kung-Kai Kuo
- Division of Hepatobiliopancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
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Addae C, Cheng H, Martinez-Ceballos E. Effect of the environmental pollutant hexachlorobenzene (HCB) on the neuronal differentiation of mouse embryonic stem cells. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 2013; 10:5244-56. [PMID: 24157519 PMCID: PMC3823326 DOI: 10.3390/ijerph10105244] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/31/2012] [Revised: 02/06/2013] [Accepted: 02/16/2013] [Indexed: 11/21/2022]
Abstract
Exposure to persistent environmental pollutants may constitute an important factor on the onset of a number of neurological disorders such as autism, Parkinson’s disease, and Attention Deficit Disorder (ADD), which have also been linked to reduced GABAergic neuronal function. GABAergic neurons produce γ-aminobutyric acid (GABA), which is the main inhibitory neurotransmitter in the brain. However, the lack of appropriate models has hindered the study of suspected environmental pollutants on GABAergic function. In this work, we have examined the effect of hexachlorobenzene (HCB), a persistent and bioaccumulative environmental pollutant, on the function and morphology of GABAergic neurons generated in vitro from mouse embryonic stem (ES) cells. We observed that: (1) treatment with 0.5 nM HCB did not affect cell viability, but affected the neuronal differentiation of ES cells; (2) HCB induced the production of reactive oxygen species (ROS); and (3) HCB repressed neurite outgrowth in GABAergic neurons, but this effect was reversed by the ROS scavenger N-acetylcysteine (NAC). Our study also revealed that HCB did not significantly interfere with the function of K+ ion channels in the neuronal soma, which indicates that this pollutant does not affect the maturation of the GABAergic neuronal soma. Our results suggest a mechanism by which environmental pollutants interfere with normal GABAergic neuronal function and may promote the onset of a number of neurological disorders such as autism and ADD.
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Affiliation(s)
- Cynthia Addae
- Department of Biological Sciences and Environmental Toxicology Program, Southern University and A&M College, Baton Rouge, LA 70813, USA; E-Mail:
| | - Henrique Cheng
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA; E-Mail:
| | - Eduardo Martinez-Ceballos
- Department of Biological Sciences and Environmental Toxicology Program, Southern University and A&M College, Baton Rouge, LA 70813, USA; E-Mail:
- Author to whom correspondence should be addressed; E-Mail: ; Tel.: +1-225-771-3606; Fax: +1-225-771-3606
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Dubé E, Dufresne J, Chan PTK, Cyr DG. Epidermal growth factor regulates connexin 43 in the human epididymis: role of gap junctions in azoospermia. Hum Reprod 2012; 27:2285-96. [PMID: 22611165 DOI: 10.1093/humrep/des164] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
BACKGROUND Gap junctions (GJs) allow for direct communication between adjacent cells. They are composed of connexons consisting of transmembrane proteins, connexins (Cxs). The objectives of this study were to determine if GJ proteins GJA1 (Cx43), GJB1 (Cx32) and GJB2 (Cx26) are present in the epididymis of men with a normal epididymis, to assess whether or not Cx expression and localization are altered in azoospermic patients, and to determine if epidermal growth factor (EGF) regulates GJA1 expression. METHODS Epididymides were obtained from men with localized testis cancer with active spermatogenesis and histologically normal epididymal tubule (group 1), men with non-obstructive azoospermia secondary to Sertoli-cell only syndrome (group 2) and from azoospermic men with normal spermatogenesis and epididymal obstruction (group 3). Epididymides were subdivided into three segments: caput, corpus and cauda. Quantitative real-time RT-PCR was performed to assess GJA1, GJB1, GJB2 and EGF receptor (EGFR) mRNA levels in epididymides from patients from each group (all n=3, except n=1 for caput blockage). A human caput epididymal cell line was then used to determine the role of EGFR signaling on the regulation of human epididymal GJA1. RESULTS Real-time RT-PCR analysis revealed that GJA1, GJB1, GJB2 and EGFR were expressed along the human epididymis. In the cauda epididymidis of group 2 and 3 men, we observed a significant decrease in GJA1 (P=0.0456 and P=0.0465, respectively) and GJB1 (P=0.0450 and P=0.0497, respectively) mRNA levels when compared with group 1 men. We also observed a decrease in EGFR mRNA levels (P=0.0358) in the cauda epididymidis of group 3 men when compared with group 1. Immunocytochemistry revealed that in the epididymis, GJA1 and EGFR were localized between basal and principal cells and between adjacent principal cells. In group 2 and 3 patients, however, we noted a dramatic increase in cytosolic immunostaining for both GJA1 and EGFR in both principal and basal cells. Using a human caput epididymal cell line derived from fertile men, we demonstrated that changes in GJA1 phosphorylation could be regulated by EGF (P=0.015) and the extracellular regulated kinase 1/2 signaling pathway (P=0.03). Furthermore, while the phosphoinositide-3-kinase (PI3K)/AKT signaling pathway did not alter GJA1 phosphorylation, treatment with PI3K/AKT inhibitor LY294002 significantly (P=0.024) inhibited the EGF-stimulated increase in GJA1 total protein levels at 24 h. Immunolocalization indicated that loss of PI3K/AKT signaling was associated with increased cytosolic localization of Cx43 in this cell line. CONCLUSIONS Together, these data suggest that in azoospermic men decreased expression of EGFR may be responsible for decreasing GJA1 levels and increasing its cytosolic localization via the PI3K/AKT signaling pathway.
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Affiliation(s)
- Evemie Dubé
- INRS-Institut Armand Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, QC, Canada H7V 1B7
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Naiki-Ito A, Kato H, Asamoto M, Naiki T, Shirai T. Age-dependent carcinogenic susceptibility in rat liver is related to potential of gap junctional intercellular communication. Toxicol Pathol 2012; 40:715-21. [PMID: 22569583 DOI: 10.1177/0192623312441402] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Connexin 32 (Cx32) is a major gap junction protein in the liver. The authors previously demonstrated that transgenic rats carrying a dominant negative mutant of Cx32 (Cx32ΔTg) have much decreased capacity for gap junctional intercellular communication (GJIC) and increased susceptibility to diethylnitrosamine (DEN)-induced hepatocarcinogenesis as compared to littermate wild-type (wt) rats. To evaluate the age-dependent susceptibility to DEN-induced hepatocarcinogenesis and alteration of GJIC function, male Cx32ΔTg and wt rats at 10, 30, or 85 weeks old were given a single intraperitoneal administration of DEN (40 mg/rat) and sacrificed 12 weeks later. The number and area of glutathione S-transferase placental form (GST-P)-positive preneoplastic foci were significantly increased in the liver of 10- and 30-wk-old Cx32ΔTg rats compared with age-matched wt. However, in the 85-wk-old rats, both Cx32ΔTg and wt rats had similarly large number and area of GST-P-positive foci, and the difference was not significant. Interestingly, function of hepatic GJIC was reduced and protein and mRNA expression of Cx32 were decreased with aging in wt rats. These results suggest that a decline of hepatic intercellular communication through gap junction results in increased susceptibility to DEN-induced hepatocarcinogenesis in aged rats.
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Affiliation(s)
- Aya Naiki-Ito
- Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences, Mizuho-ku, Nagoya, Japan.
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de Montgolfier B, Audet C, Cyr DG. Regulation of the connexin 43 promoter in the brook trout testis: role of the thyroid hormones and cAMP. Gen Comp Endocrinol 2011; 170:110-8. [PMID: 20932836 DOI: 10.1016/j.ygcen.2010.09.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/22/2010] [Revised: 09/09/2010] [Accepted: 09/21/2010] [Indexed: 11/18/2022]
Abstract
Gap junctions are critical for spermatogenesis. They are composed of integral proteins, the connexins. In mammals, a loss of Cx43 expression results in the inhibition of spermatogenesis. We have shown that Cx43 is expressed in the Sertoli cells of rainbow trout and that cAMP and triiodothyronine (T(3)) regulate testicular Cx43 expression in brook trout testis. The objective of this study was to determine if cAMP and T(3) act at the level of the cx43 promoter to regulate its expression. A 607 bp 5' flanking sequence of the cx43 promoter was obtained by Genome Walking. A TATA box was predicted to be located between positions -36 and -30 relative to the transcriptional initiation site. 5'-Rapid amplification of cDNA ends indicated a single transcriptional start site. Single C/EBP (-164 to -156) and tr-beta (-112 to -107) response elements were identified and electrophoretic mobility shift assays indicated the presence of competitive protein binding sites at each region. Immortalized rainbow trout gonadal cell line (RTG-2) which express cx43 and tr-beta transcripts were transfected with a vector containing the Cx43 promoter inserted into a luciferase expression vector. Transactivation of the reporter genes was stimulated by either cAMP or T(3). Sequential deletion and point mutations in either the C/EBP or tr-beta response element indicated that T(3) but not cAMP directly induced luciferase transactivation of the luciferase gene by acting on different sites of the Cx43 promoter. Together, these data indicate that T(3) stimulates cx43 expression via direct regulation of gene transcription.
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Pontillo CA, García MA, Peña D, Cocca C, Chiappini F, Alvarez L, Kleiman de Pisarev D, Randi AS. Activation of c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration by hexachlorobenzene in MDA-MB-231 human breast cancer cell line. Toxicol Sci 2010; 120:284-96. [PMID: 21205633 DOI: 10.1093/toxsci/kfq390] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Hexachlorobenzene (HCB) is a widespread environmental pollutant. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR) protein. HCB is a tumor cocarcinogen in rat mammary gland and an inducer of cell proliferation and c-Src kinase activity in MCF-7 breast cancer cells. This study was carried out to investigate HCB action on c-Src and the human epidermal growth factor receptor (HER1) activities and their downstream signaling pathways, Akt, extracellular-signal-regulated kinase (ERK1/2), and signal transducers and activators of transcription (STAT) 5b, as well as on cell migration in a human breast cancer cell line, MDA-MB-231. We also investigated whether the AhR is involved in HCB-induced effects. We have demonstrated that HCB (0.05μM) produces an early increase of Y416-c-Src, Y845-HER1, Y699-STAT5b, and ERK1/2 phosphorylation. Moreover, our results have shown that the pesticide (15 min) activates these pathways in a dose-dependent manner (0.005, 0.05, 0.5, and 5μM). In contrast, HCB does not alter T308-Akt activation. Pretreatment with a specific inhibitor for c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine [PP2]) prevents Y845-HER1 and Y699-STAT5b phosphorylation. AG1478, a specific HER1 inhibitor, abrogates HCB-induced STAT5b and ERK1/2 activation, whereas 4,7-orthophenanthroline and α-naphthoflavone, two AhR antagonists, prevent HCB-induced STAT5b and ERK1/2 phosphorylation. HCB enhances cell migration evaluated by scratch motility and transwell assays. Pretreatment with PP2, AG1478, and 4,7-orthophenanthroline suppresses HCB-induced cell migration. These results demonstrate that HCB stimulates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways in MDA-MB-231. c-Src, HER1, and AhR are involved in HCB-induced increase in cell migration. The present study makes a significant contribution to the molecular mechanism of action of HCB in mammary carcinogenesis.
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Affiliation(s)
- Carolina A Pontillo
- Laboratorio de Efectos Biológicos de Contaminantes Ambientales, Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
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Vinken M, Doktorova T, Decrock E, Leybaert L, Vanhaecke T, Rogiers V. Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity. Crit Rev Biochem Mol Biol 2009; 44:201-22. [PMID: 19635038 DOI: 10.1080/10409230903061215] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Direct communication between hepatocytes, mediated by gap junctions, constitutes a major regulatory platform in the control of liver homeostasis, ranging from hepatocellular proliferation to hepatocyte cell death. Inherent to this pivotal task, gap junction functionality is frequently disrupted upon impairment of the homeostatic balance, as occurs during liver toxicity and carcinogenicity. In the present paper, the deleterious effects of a number of chemical and biological toxic compounds on hepatic gap junctions are discussed, including environmental pollutants, biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. Particular attention is paid to the molecular mechanisms that underlie the abrogation of gap junction functionality. Since hepatic gap junctions are specifically targeted by tumor promoters and epigenetic carcinogens, both in vivo and in vitro, inhibition of gap junction functionality is considered as a suitable indicator for the detection of nongenotoxic hepatocarcinogenicity.
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Affiliation(s)
- Mathieu Vinken
- Department of Toxicology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium.
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Uehara T, Hirode M, Ono A, Kiyosawa N, Omura K, Shimizu T, Mizukawa Y, Miyagishima T, Nagao T, Urushidani T. A toxicogenomics approach for early assessment of potential non-genotoxic hepatocarcinogenicity of chemicals in rats. Toxicology 2008; 250:15-26. [PMID: 18619722 DOI: 10.1016/j.tox.2008.05.013] [Citation(s) in RCA: 95] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2008] [Revised: 05/15/2008] [Accepted: 05/20/2008] [Indexed: 11/16/2022]
Abstract
For assessing carcinogenicity in animals, it is difficult and costly, an alternative strategy has been desired. We explored the possibility of applying a toxicogenomics approach by using comprehensive gene expression data in rat liver treated with various compounds. As prototypic non-genotoxic hepatocarcinogens, thioacetamide (TAA) and methapyrilene (MP) were selected and 349 commonly changed genes were extracted by statistical analysis. Taking both compounds as positive with six compounds, acetaminophen, aspirin, phenylbutazone, rifampicin, alpha-naphthylisothiocyanate, and amiodarone as negative, prediction analysis of microarray (PAM) was performed. By training and 10-fold cross validation, a classifier containing 112 probe sets that gave an overall success rate of 95% was obtained. The validity of the present discriminator was checked for 30 chemicals. The PAM score showed characteristic time-dependent increases by treatment with several non-genotoxic hepatocarcinogens, including TAA, MP, coumarin, ethionine and WY-14643, while almost all of the non-carcinogenic samples were correctly predicted. Measurement of hepatic glutathione content suggested that MP and TAA cause glutathione depletion followed by a protective increase, but the protective response is exhausted during repeated administration. Therefore, the presently obtained PAM classifier could predict potential non-genotoxic hepatocarcinogenesis within 24 h after single dose and the inevitable pseudo-positives could be eliminated by checking data of repeated administrations up to 28 days. Tests for carcinogenicity using rats takes at least 2 years, while the present work suggests the possibility of lowering the time to 28 days with high precision, at least for a category of non-genotoxic hepatocarcinogens causing oxidative stress.
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Affiliation(s)
- Takeki Uehara
- Toxicogenomics Project, National Institute of Biomedical Innovation, 7-6-8 Asagi, Ibaraki, Osaka 567-0085, Japan
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de Montgolfier B, Fournier M, Audet C, Marcogliese DJ, Cyr DG. Influence of municipal effluents on the expression of connexins in the brook trout (Salvelinus fontinalis) testis. AQUATIC TOXICOLOGY (AMSTERDAM, NETHERLANDS) 2008; 86:38-48. [PMID: 18006156 DOI: 10.1016/j.aquatox.2007.09.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/11/2007] [Revised: 09/12/2007] [Accepted: 09/21/2007] [Indexed: 05/06/2023]
Abstract
Gap junctions are essential for spermatogenesis. Exposure to municipal wastewater effluent can modify spermatogenesis in fish. The present aim was to determine if municipal wastewater effluent could alter the expression of testicular connexins (Cxs) in brook trout. Trout were exposed for 4 and 12 weeks to various concentrations of municipal effluent (0, 1, 10 and 20%, v/v). Hepatic vitellogenin (vtg) mRNA levels were not different between exposure groups after 4 weeks. At this time, testicular cx43 and cx31 mRNA levels increased in the 1% group, but cx30 and cx43.4 levels were not different at any concentration. Immunolocalization of each Cx did not differ between groups after 4 weeks. After 12 weeks, spermatogenesis in the 1% group was more advanced than in other groups, and hepatic vtg mRNA levels were significantly increased at the higher exposure concentrations. Testicular cx43 mRNA levels were higher than controls at all doses, while cx43.4 levels increased in a dose-dependent manner but remained lower than controls. cx31 mRNA levels were significantly lower in the 1 and 10% groups than in control and 20% group, while cx30 levels did not vary. Immunolocalization of Cxs did not differ between groups except for Cx43.4, which was expressed between spermatocytes in the 1% group. Furthermore, the Cx31 immunoreaction appeared to decrease in testicular blood vessels of fish exposed to the highest dose. Furthermore, vegf mRNA levels were unaltered by treatment at both time points. Thus, long-term exposure to environmental concentrations of wastewater effluent can alter the expression of testicular Cxs.
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Llambías EBC, Mazzetti MB, Lelli SM, Aldonatti C, San Martín de Viale LC. Melatonin formation in pineal gland from rats with hexachlorobenzene experimental porphyria. Int J Toxicol 2007; 26:545-51. [PMID: 18066970 DOI: 10.1080/10915810701707643] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
Hexachlorobenzene produces an experimental hepatic porphyria in rats, which is similar to human porphyria cutanea tarda, with hyperpigmentation as one of its characteristic features. Alterations in tryptophan metabolism have been previously observed in this chronic porphyria. Melatonin formation from tryptophan via serotonin shows diurnal rhythmicity in the pineal gland, and higher values are observed during the dark phase of an imposed light-dark cycle. The purpose of this study was to determine the contents of tryptophan and its metabolites in pineal gland of normal and hexachlorobenzene-treated rats in order to find alterations potentially related to porphyria cutanea tarda. Results show that in animals with this experimental porphyria some tryptophan metabolite levels (serotonin and 5-hydroxyindoleacetic acid) increase only during the light period, whereas tryptophan content remained equal to the controls. Hydroxyindole-O-methyltransferase activity also increases by light in pineal gland from hexachlorobenzene-treated rats. On the other hand, tryptophan is converted to melatonin in the dark period, but this route is not exacerbated in hexachlorobenzene porphyria. The relevance of these alterations is discussed in relation to hyperpigmentation, neoplastic and oxidative stress processes associated with this porphyria.
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Affiliation(s)
- Elena B C Llambías
- Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales,, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina.
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Lu Y, Lohitnavy M, Reddy M, Lohitnavy O, Eickman E, Ashley A, Gerjevic L, Xu Y, Conolly RB, Yang RSH. Quantitative analysis of liver GST-P foci promoted by a chemical mixture of hexachlorobenzene and PCB 126: implication of size-dependent cellular growth kinetics. Arch Toxicol 2007; 82:103-16. [PMID: 17874069 DOI: 10.1007/s00204-007-0238-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2007] [Revised: 08/04/2007] [Accepted: 08/15/2007] [Indexed: 11/30/2022]
Abstract
The objectives of this study were twofold: (1) evaluating the carcinogenic potential of the mixture of two persistent environmental pollutants, hexachlorobenzene (HCB) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126), in an initiation-promotion bioassay involving the development of pi glutathione S-transferase (GST-P) liver foci, and (2) analyzing the GST-P foci data using a biologically-based computer model (i.e., clonal growth model) with an emphasis on the effect of focal size on the growth kinetics of initiated cells. The 8-week bioassay involved a series of treatments of initiator, two-thirds partial hepatectomy, and daily oral gavage of the mixture of two doses in male F344 rats. The mixture treatment significantly increased liver GST-P foci development, indicating carcinogenic potential of this mixture. Our clonal growth model was developed to simulate the appearance and development of initiated GST-P cells in the liver over time. In the model, the initiated cells were partitioned into two subpopulations with the same division rate but different death rates. Each subpopulation was further categorized into single cells, mini- (2-11 cells), medium- (12-399 cells), and large-foci (>399 cells) with different growth kinetics. Our modeling suggested that the growth of GST-P foci is size-dependent; in general, the larger the foci, the higher the rate constants of division and death. In addition, the modeling implied that the two doses promoted foci development in different manners even though the experimental foci data appeared to be similar between the two doses. This study further illustrated how clonal growth modeling may facilitate our understanding in chemical carcinogenic process.
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Affiliation(s)
- Yasong Lu
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523-1681, USA
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Kinoshita A, Wanibuchi H, Wei M, Fukushima S. Hormesis in Carcinogenicity of Non-genotoxic Carcinogens. J Toxicol Pathol 2006. [DOI: 10.1293/tox.19.111] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Affiliation(s)
- Anna Kinoshita
- Department of Pathology, Osaka City University Medical School
| | | | - Min Wei
- Department of Pathology, Osaka City University Medical School
| | - Shoji Fukushima
- Department of Pathology, Osaka City University Medical School
- Japan Bioassay Research Center
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Plante I, Cyr DG, Charbonneau M. Involvement of the integrin-linked kinase pathway in hexachlorobenzene-induced gender-specific rat hepatocarcinogenesis. Toxicol Sci 2005; 88:346-57. [PMID: 16162845 DOI: 10.1093/toxsci/kfi323] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Overexpression of the integrin-linked kinase (ILK) pathway disrupts cell-cell interactions, an epigenetic event leading to epithelial cell transformation. Female rats exposed to hexachlorobenzene (HCB) for 5 consecutive days and sampled 45 days later show a decrease in liver gap junctional intercellular communication. We hypothesized that HCB also alters E-cadherin expression and that this alteration is mediated by the ILK pathway. Hepatic ILK levels were markedly increased in HCB-treated female rats. Cytoplasmic/membrane levels of protein kinase B (Akt), a target of ILK, and its phosphorylated active form were decreased in treated female rats. Flow cytometric analysis showed a concomitant increase in nuclear Akt levels. Both ILK and Akt can phosphorylate glycogen synthetase kinase-3beta (GSK3beta), rendering it inactive. Phosphorylated-GSK3beta levels were higher in treated females and resulted in a twofold decrease in the activity of GSK3beta. The inactivation of GSK3beta in HCB-treated female rats resulted in the nuclear translocation of beta-catenin, as demonstrated by both immunocytochemistry and flow cytometric analyses. Western blot analysis showed an 84% decrease in E-cadherin levels in HCB-treated rats as compared to controls, and this decrease was not mediated by Snail activation. Mimicking the activation of ILK with specific GSK3beta inhibitors resulted in downregulation of E-cadherin levels but had no effect on Cx32 expression in the MH(1)C(1) cells. Overall, these results indicate that hepatic E-cadherin is downregulated as a result of an overexpression of the ILK pathway. The concomitant HCB-induced downregulation of intercellular communication does not occur as a result of either E-cadherin downregulation or GSK3beta inactivation.
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Affiliation(s)
- Isabelle Plante
- INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, Canada
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24
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Mesnil M, Crespin S, Avanzo JL, Zaidan-Dagli ML. Defective gap junctional intercellular communication in the carcinogenic process. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2005; 1719:125-45. [PMID: 16359943 DOI: 10.1016/j.bbamem.2005.11.004] [Citation(s) in RCA: 253] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/15/2005] [Revised: 11/07/2005] [Accepted: 11/10/2005] [Indexed: 01/07/2023]
Abstract
Gap junctions are membrane structures made of intercellular channels which permit the diffusion from cytoplasm to cytoplasm of small hydrophilic molecules. Nearly 40 years ago, the loss of functional gap junctions has been described in cancer cells and led to the hypothesis that such type of intercellular communication is involved in the carcinogenesis process. From this time, a lot of data has been accumulated confirming that gap junctions are frequently decreased or absent in cancer cells whatever their tissue and species origins. Here, we review such data by insisting on the possible links existing between altered gap-junctional intercellular communication capacity (or the altered expression of their constitutive proteins, the connexins) and the stages of cancer progression in various cancer models. Then, we analyse particular aspects of the disturbance of connexin-mediated communication in cancer such as the cytoplasmic localization of connexins, the lack of heterologous communication between cancer cells and normal cells, the role of connexin gene mutations in cancer. In a separate part of the review, we also analyse the disturbance of gap-junctional intercellular communication during the late stages of cancer (invasion and metastasis processes).
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Affiliation(s)
- Marc Mesnil
- Equipe Interactions et Communications Cellulaires, Institut de Physiologie et Biologie Cellulaires, CNRS-UMR 6187, Université de Poitiers, 40 avenue du Recteur Pineau, 86022 Poitiers cedex, France.
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25
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Kushida M, Sukata T, Uwagawa S, Ozaki K, Kinoshita A, Wanibuchi H, Morimura K, Okuno Y, Fukushima S. Low dose DDT inhibition of hepatocarcinogenesis initiated by diethylnitrosamine in male rats: possible mechanisms. Toxicol Appl Pharmacol 2005; 208:285-94. [PMID: 15885732 DOI: 10.1016/j.taap.2005.03.018] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2004] [Revised: 02/15/2005] [Accepted: 03/04/2005] [Indexed: 11/15/2022]
Abstract
Previously we reported a tendency for reduction of the development of glutathione-S-transferase placental form (GST-P) positive foci, recognized as preneoplastic changes in rat liver, by a low dose of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), which belongs to the same group of hepatic cytochrome P-450 inducers as phenobarbital and is itself a non-genotoxic hepatocarcinogen. In order to clarify the biological significance of this phenomenon, we investigated the reproducibility and changes in other parameters using an initiation-promotion model in which male F344 rats were treated with DDT at doses of 0, 0.005, 0.5, 500 ppm in the diet for 11 or 43 weeks after initiation of hepatocarcinogenesis with N-diethylnitrosamine (DEN). When 500 ppm DDT was applied, the formation of GST-P positive foci and tumor were markedly elevated. In contrast, induction of GST-P positive foci and liver tumors tended to be inhibited at a dose of 0.005 ppm, correlating with protein levels of cytochrome P450 2B1 and 3A2 (CYP2B1 and 3A2) and generation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage. mRNA levels for 8-oxoguanine glycosylase 1 (OGG1), an 8-OHdG repair enzyme, connexin 32 (Cx32), a major component of Gap junctions, and hepatic nuclear factor 1alpha (HNF-1alpha), a Cx32 regulator, were inversely correlated with GST-P positive foci and tumor formation. These results indicate that low dose DDT may indeed exhibit inhibitory effects on chemically initiated-rat hepatocarcinogenicity, in contrast to the promotion observed with high doses, and that this is related to changes in metabolizing enzymes, cell communication, and DNA damage and its repair.
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MESH Headings
- Administration, Oral
- Animals
- Apoptosis/drug effects
- Cell Proliferation/drug effects
- Connexins/drug effects
- Connexins/genetics
- Connexins/metabolism
- Cytochrome P-450 Enzyme System/drug effects
- Cytochrome P-450 Enzyme System/metabolism
- DDT/administration & dosage
- DDT/pharmacology
- DNA Damage/drug effects
- DNA Glycosylases/drug effects
- DNA Glycosylases/genetics
- DNA Glycosylases/metabolism
- DNA, Single-Stranded/drug effects
- Deoxyguanosine/antagonists & inhibitors
- Diethylnitrosamine/administration & dosage
- Diethylnitrosamine/antagonists & inhibitors
- Diethylnitrosamine/toxicity
- Disease Models, Animal
- Dose-Response Relationship, Drug
- Drug Administration Schedule
- Gene Expression
- Glutathione S-Transferase pi/antagonists & inhibitors
- Glutathione S-Transferase pi/drug effects
- Glutathione S-Transferase pi/metabolism
- Glutathione Transferase/antagonists & inhibitors
- Glutathione Transferase/drug effects
- Glutathione Transferase/metabolism
- Hepatocyte Nuclear Factor 1-alpha/drug effects
- Hepatocyte Nuclear Factor 1-alpha/genetics
- Hepatocyte Nuclear Factor 1-alpha/metabolism
- Immunochemistry/methods
- Injections, Intraperitoneal
- Liver Neoplasms, Experimental/chemically induced
- Liver Neoplasms, Experimental/metabolism
- Liver Neoplasms, Experimental/prevention & control
- Male
- Proliferating Cell Nuclear Antigen/drug effects
- RNA, Messenger/genetics
- Rats
- Rats, Inbred F344
- Time Factors
- Up-Regulation/drug effects
- Gap Junction beta-1 Protein
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Affiliation(s)
- Masahiko Kushida
- Department of Pathology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan; Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 1-98, 3-chome, Kasugade-Naka, Konohana-ku, Osaka 554-8558, Japan
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26
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Salameh A, Dhein S. Pharmacology of gap junctions. New pharmacological targets for treatment of arrhythmia, seizure and cancer? BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2005; 1719:36-58. [PMID: 16216217 DOI: 10.1016/j.bbamem.2005.09.007] [Citation(s) in RCA: 94] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/14/2005] [Revised: 08/25/2005] [Accepted: 09/06/2005] [Indexed: 11/26/2022]
Abstract
Intercellular communication in many organs is maintained via intercellular gap junction channels composed of connexins, a large protein family with a number of isoforms. This gap junction intercellular communication (GJIC) allows the propagation of action potentials (e.g., in brain, heart), and the transfer of small molecules which may regulate cell growth, differentiation and function. The latter has been shown to be involved in cancer growth: reduced GJIC often is associated with increased tumor growth or with de-differentiation processes. Disturbances of GJIC in the heart can cause arrhythmia, while in brain electrical activity during seizures seems to be propagated via gap junction channels. Many diseases or pathophysiological conditions seem to be associated with alterations of gap junction protein expression. Thus, depending on the target disease opening or closure of gap junctions may be of interest, or alteration of connexin expression. GJIC can be affected acutely by changing gap junction conductance or--more chronic--by altering connexin expression and membrane localisation. This review gives an overview on drugs affecting GJIC.
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Affiliation(s)
- Aida Salameh
- Clinic I for Internal Medicine, Department of Cardiology, University of Leipzig, Johannisallee 32, 04103 Leipzig, Germany.
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27
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Fukushima S, Kinoshita A, Puatanachokchai R, Kushida M, Wanibuchi H, Morimura K. Hormesis and dose-response-mediated mechanisms in carcinogenesis: evidence for a threshold in carcinogenicity of non-genotoxic carcinogens. Carcinogenesis 2005; 26:1835-45. [PMID: 15975961 DOI: 10.1093/carcin/bgi160] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Recently the idea of hormesis, a biphasic dose-response relationship in which a chemical exerts opposite effects dependent on the dose, has attracted interest in the field of carcinogenesis. With non-genotoxic agents there is considerable experimental evidence in support of hormesis and the present review highlights current knowledge of dose-response effects. In particular, several in vivo studies have provided support for the idea that non-genotoxic carcinogens may inhibit hepatocarcinogenesis at low doses. Here, we survey the examples and discuss possible mechanisms of hormesis using phenobarbital, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), alpha-benzene hexachloride (alpha-BHC) and other non-genotoxins. Furthermore, the effects of low and high doses of non-genotoxic and genotoxic compounds on carcinogenesis are compared, with especial attention to differences in mechanisms of action in animals and possible application of the dose-response concept to cancer risk assessment in humans. Epigenetic processes differentially can be affected by agents that impinge on oxidative stress, DNA repair, cell proliferation, apoptosis, intracellular communication and cell signaling. Non-genotoxic carcinogens may target nuclear receptors, cause aberrant DNA methylation at the genomic level and induce post-translational modifications at the protein level, thereby impacting on the stability or activity of key regulatory proteins, including oncoproteins and tumor suppressor proteins. Genotoxic agents, in contrast, cause genetic change by directly attacking DNA and inducing mutations, in addition to temporarily modulating the gene activity. Carcinogens can elicit a variety of changes via multiple genetic and epigenetic lesions, contributing to cellular carcinogenesis.
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Affiliation(s)
- Shoji Fukushima
- Department of Pathology, Osaka City University Medical School, Osaka, Japan.
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28
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Sheen IS, Jeng KS, Shih SC, Kao CR, Wang PC, Chen CZ, Chang WH, Wang HY, Shyung LR. Do the expressions of gap junction gene connexin messenger RNA in noncancerous liver remnants of patients with hepatocellular carcinoma correlate with postoperative recurrences? World J Gastroenterol 2005; 11:171-5. [PMID: 15633210 PMCID: PMC4205396 DOI: 10.3748/wjg.v11.i2.171] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate whether the changes of gap junction gene connexin messenger RNA in the noncancerous liver tissue of patients with hepatocellular carcinoma (HCC) could play a significant role in its postresection recurrence.
METHODS: Seventy-nine consecutive patients having undergone curative resection for HCC entered this study. Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, connexin (Cx) 26, connexin (Cx) 32 and connexin (Cx) 43 mRNAs were determined prospectively in noncancerous liver tissues from these 79 patients and in the liver tissues from 15 controls. The correlations between connexin mRNA expression and the clinicopathological variables and outcomes (tumor recurrence and recurrence related mortality) were studied.
RESULTS: Compared with liver tissues of control patients, the expression of Cx 32 mRNA in noncancerous liver tissues was significantly lower (mean: 0.715 vs control 1.225, P<0.01), whereas the decreased Cx 26 mRNA (mean: 0.700 vs of control 1.205, P>0.05) and increased Cx 43 mRNA (mean: 0.241 vs control 0.100, P>0.05) had no statistical significance. We defined the value of Cx 32 mRNA or Cx 26 mRNA below 0.800 as a lower value. By multivariate analysis for noncancerous livers, a lower value of Cx 32 mRNA correlated significantly with a risk of HCC recurrence and recurrence-related mortality. The lower value of Cx 26 mRNA did not correlate with recurrence and mortality. The increased value of Cx43 mRNA also did not correlate with postoperative recurrence and recurrence-related mortality. By multivariate analysis, other significant predictors of HCC recurrence included vascular permeation, cellular dedifferentiation, and less encaps-ulation. The other significant parameter of recurrence related mortality was vascular permeation.
CONCLUSION: The decreased expression of Cx 32 mRNA in noncancerous liver tissues plays a significant role in the prediction of postoperative recurrence of HCC.
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Affiliation(s)
- I-Shyan Sheen
- Division of Hepatogastroenterology, Chang Gung Memorial Hospital, Taipei, Taiwan, China
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29
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Aravindakshan J, Cyr DG. Nonylphenol alters connexin 43 levels and connexin 43 phosphorylation via an inhibition of the p38-mitogen-activated protein kinase pathway. Biol Reprod 2005; 72:1232-40. [PMID: 15647452 DOI: 10.1095/biolreprod.104.038596] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Endocrine-disrupting chemicals are exogenous compounds that mimic or inhibit the action of estrogens or other hormones. Nonylphenol, an environmental contaminant distributed along the St. Lawrence River, has been reported to act as a weak estrogen. Previous studies from our laboratory have shown that rats that were fed fish taken from nonylphenol contaminated sites have altered spermatogenesis and decreased sperm count. The mechanism responsible for this effect is unknown. Gap junctional intercellular communication (GJIC) in the testis is critical for coordinating spermatogenesis. The objectives of the study were to determine the effects of nonylphenol on GJIC and connexin 43 (Cx43) in a murine Sertoli cell line, TM4. Cells were exposed for 24 h to different concentrations (1 to 50 microM) of either nonylphenol or 17beta-estradiol. GJIC was determined using a microinjection approach in which Lucifer yellow was injected directly into a single cell, and GJIC was assessed 3 min postinjection. Nonylphenol exposure decreased GJIC between adjacent cells by almost 80% relative to controls. A significant concentration-dependent reduction in GJIC was observed at nonylphenol concentrations between 1 and 50 microM. Cx43 immunofluorescent staining was reduced at both 10 and 50 microM doses of nonylphenol. Cx43 phosphorylation, as determined by Western blot analysis, was reduced at both 10 and 50 microM concentrations, which may explain, at least in part, the inhibition of GJIC. In contrast, no effect on GJIC or Cx43 protein was observed in cells exposed to 17beta-estradiol at these concentrations. Cx43 has been reported to be phosphorylated via the p38-mitogen-activated protein kinase (MAPK) pathway. P38-MAPK activity was assessed in both control and nonylphenol-exposed cells. A dose-dependent decrease in p38-MAPK activity was observed in nonylphenol-exposed Sertoli cells. Protein kinase C activity was also measured and was not influenced by nonylphenol. These results suggest that nonylphenol inhibits GJIC between Sertoli cells and that this is modulated via nonestrogenic pathways.
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30
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Campagna C, Bailey JL, Sirard MA, Ayotte P, Maddox-Hyttel P. An environmentally-relevant mixture of organochlorines and its vehicle control, dimethylsulfoxide, induce ultrastructural alterations in porcine oocytes. Mol Reprod Dev 2005; 73:83-91. [PMID: 16206133 DOI: 10.1002/mrd.20365] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Organochlorine chemicals accumulate in the environment, particularly in the Arctic, and constitute potential developmental hazards to wildlife and human health. Although some of their harmful effects are recognized, their mechanisms of action within the target cells need to be better understood. This study was designed to test the hypothesis that an environmentally-relevant organochlorine mixture alters oocyte ultrastructure in the porcine model. Immature cumulus-oocyte complexes (COCs), partially cultured (18 hr) COCs without treatment or exposed to the organochlorine mixture or its vehicle (0.1% dimethysulfoxide; DMSO) during culture were processed for light and transmission electronic microscopy (TEM). The organochlorines induced major ultrastructural changes in the COCs: decreased density of the lipid droplets, increased smooth endoplasmic reticulum (SER) volume and increased interactions among SER, mitochondria, lipid droplets and vesicles. We suggest that these ultrastructural changes facilitate energy formation necessary to produce metabolizing enzymes. Other ultrastructural changes may reflect some degree of organochlorine toxicity: fewer gap junctions and decreased electron density of the cortical granules. Unexpectedly, the DMSO control treatment also induced similar ultrastructural changes, but to a lesser degree than the organochlorine mixture. This study is the first to demonstrate the effect of environmental contaminants on mammalian oocyte ultrastructure.
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Affiliation(s)
- Céline Campagna
- Centre de Recherche en Biologie de la Reproduction, Département des Sciences animales, Université Laval, Québec, Canada
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31
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Hadjab S, Maurel D, Cazals Y, Siaud P. Hexachlorobenzene, a dioxin-like compound, disrupts auditory function in rat. Hear Res 2004; 191:125-34. [PMID: 15109712 DOI: 10.1016/j.heares.2003.12.017] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/24/2003] [Accepted: 12/29/2003] [Indexed: 10/26/2022]
Abstract
Hexachlorobenzene (HCB) is a dioxin-like compound widely distributed in the environment. In this study, we investigated the effects of HCB on the cochlea. Conscious free-moving rats were given HCB per os daily for 4 weeks at doses of 0.16, 4 or 16 mg/kg in olive oil, whereas the control group received olive oil only. The effects of HCB were evaluated at various time intervals, by measuring auditory nerve acoustic thresholds and plasma thyroid hormone concentration by radioimmunoassay. Histological evaluation involved surface preparation and scanning electron microscopy observations of cochlear hair cells. At a dose of 0.16 mg/kg, HCB induced no loss of acoustic sensitivity, whereas at 4 mg/kg, it induced cochlear sensitivity deficits at the mid-frequencies (2-16 kHz) with complete recovery once treatment was stopped. At a dose of 16 mg/kg, permanent threshold shifts were observed at all frequencies tested (from 1 to 32 kHz). Morphological studies showed no cochlear hair cell loss or alteration of stereocilia. HCB treatment reduced circulating thyroxine concentrations. Thyroidectomy had no effect on cochlear sensitivity in control animals. Thus, HCB is a potent oto-toxicant, and its ototoxicity may be independent of its thyroidal effects.
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Affiliation(s)
- Saida Hadjab
- Laboratoire d'Otologie Neuro-Otologie, EMI 9902 INSERM, Faculté de Médecine Secteur Nord, Université de la Méditerranée, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France
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32
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Dufresne J, Finnson KW, Gregory M, Cyr DG. Expression of multiple connexins in the rat epididymis indicates a complex regulation of gap junctional communication. Am J Physiol Cell Physiol 2003; 284:C33-43. [PMID: 12388089 DOI: 10.1152/ajpcell.00111.2002] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
In the epididymis, Cx43 forms gap junctions between principal and basal cells but not between adjacent principal cells. Cx30.3, 31.1, and 32 were identified in adult rat epididymis by RT-PCR, whereas Cx26 was present in young rats. Postnatal development studies indicate that Cx26 mRNA was detectable only in the caput-corpus region of the epididymis and that levels increased by fivefold during the first 4 wk postnatally, when epithelial cells differentiate, and decrease to nondetectable levels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout the epididymis in young rats and began to increase in the second and third weeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32 were localized to the lateral plasma membranes between adjacent epithelial cells of the epididymis. Colocalization studies indicate that Cx26 and Cx32 exist either independently of one another or can colocalize along the lateral plasma membrane of epithelial cells in young rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.
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Affiliation(s)
- Julie Dufresne
- Institut National de la Recherche Scientifique-Institut Armand Frappier, Université du Québec, 245 Hymus Boulevard, Pointe-Claire, Québec, Canada H9R 1G6
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