Document b3e7Y8XRb0J0BkMGe6BEv0zg

G Model CBI-6056; No. of Pages 4 ARTICLE IN PRESS Chemico-Biological Interactions xxx (2009) xxxxxx Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Gap junction intercellular communication and benzene toxicity Edgar Rivedal a,, Gisela Witz b, Edward Leithe a a Department of Cancer Prevention, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital and Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway b Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School/UMDNJ, USA article info Article history: Available online xxx Keywords: Benzene Muconaldehyde Cancer Hematopoiesis Gap junctions Connexin43 abstract Aberrant regulation of gap junction intercellular communication (GJIC) has been linked to several human diseases, including cancer and abnormal hematopoietic development. Benzene exposure has been shown to cause hematotoxicity and leukemia, but the underlying mechanisms involved remain unclear. We have observed that several metabolites of benzene have the ability to block gap junction intercellular communication. The ring-opened trans,trans-muconaldehyde (MUC) was found to be the most potent inhibitor of gap junction channels. MUC was found to induce cross-linking of the gap junction protein connexin43, which seemed to be responsible for the induced inhibition of GJIC. Glutaraldehyde, which has a similar molecular structure as MUC, was found to possess similar effects on gap junctions as MUC, while the mono-aldehyde formaldehyde shows lower potency, both as a connexin cross-linker, and as an inhibitor of GJIC. Both glutaraldehyde and formaldehyde have previously been associated with induction of leukemia and disturbance of hematopoiesis. Taken together, the data support a possible link between the effect of MUC on gap junctions, and the toxic effects of benzene. 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Gap junctions are specialized plasma membrane domains enriched in intercellular channels that provide for transport of ions, metabolites and cell signalling molecules between adjacent cells [1]. The gap junction channels consist of transmembrane proteins called connexins and have been shown to play an important role in maintaining normal cell growth and tissue homeostasis [24]. Aberrant regulation of GJIC has been associated with several human pathological conditions [5,6], and compounds with the ability to block gap junction intercellular communication (GJIC) have been linked to cancer induction, as well as interference with normal hematopoietic development [712]. The best-studied connexin isoform, connexin43 (Cx43), is ubiquitously expressed in various tissues and is involved in bone marrow hematopoiesis [13]. Reduced Cx43 expression during embryogenesis has been shown to compromise normal progression of T and B lymphopoiesis [14]. Gap junction dependent interaction with stromal cells in the bone marrow and thymus seems necessary for normal hematopoietic development [15], and inhibitors of GJIC have been found to interfere with such normal regulation [1618]. The toxicity and cancer-causing abilities of benzene were recognized many years ago [19,20]. The underlying mechanisms are, however, poorly understood, although metabolism has been shown Corresponding author. Tel.: +47 22 78 17 18. E-mail address: edgarr@rr-research.no (E. Rivedal). 0009-2797/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.11.013 to be required for the induced toxicological effects. Chromosomal aberrations [2124], sister chromatid exchange [25], micronuclei formation [26,27], chromosomal loss and DNA strand breaks [28,29] have been observed, but the role of direct DNA reactivity, DNA adducts and mutations in benzene toxicity is still questioned [30]. The ring-opened six-carbon benzene metabolite trans,transmuconaldehyde (MUC) has been shown to cause bone marrow depression [31] and reduced erythropoiesis in mice [32], and is one of the most potent bone marrow depressive benzene metabolites. We have observed that MUC is a potent inhibitor of gap junction channels and studied its underlying mechanisms of action [33,34]. MUC-induced inhibition of GJIC is associated with loss of Cx43 visualized by Western blotting, mediated through its ability as a protein cross-linker. The ability of benzene metabolites to interfere with gap junction functionality could be involved in benzene-induced hematotoxicity and leukemia. 2. Materials and methods 2.1. Cells The rat liver epithelial cell line IAR20 obtained from International Agency for Research on Cancer, Lyon, France were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells are well coupled by connexin43-containing gap junctions. Please cite this article in press as: E. Rivedal, et al., Gap junction intercellular communication and benzene toxicity, Chem. Biol. Interact. (2009), doi:10.1016/j.cbi.2009.11.013 G Model CBI-6056; No. of Pages 4 2 ARTICLE IN PRESS E. Rivedal et al. / Chemico-Biological Interactions xxx (2009) xxxxxx 2.2. Chemicals MUC was obtained from Calbiochem (San Diego, CA, USA), benzene and formaldehyde from Merck (Darmstadt, Germany), chlordane from Sulpelco (Bellefonte, PA, USA), and glutaraldehyde and TPA from Sigma. The chemicals were dissolved in DMSO. 2.3. Measurement of gap junction intercellular communication GJIC was determined by scrape loading of Lucifer yellow (LY) and quantitative determination of dye spreading by image analysis [35]. 106 IAR20 cells were plated onto 60 mm Petri-dishes (Costar, Cambridge, MA, USA). Two days later the dishes were washed with PBS and 0.05% (w/v) LY dissolved in PBS was added prior to cutting the monolayer with a surgical scalpel and incubating 3.5 min at room temperature. The dye was removed, cells rinsed and fixed in 4% formalin. Digital mages were acquired and analysed for Lucifer yellow diffusion. 2.4. Western blotting Cells were seeded and treated as described in the cell communication experiments. Following exposure as indicated the cells were scraped into 500 l SDS electrophoresis sample buffer (10 mM Tris pH 6.8, 15% (w/v) glycerol, 3% (w/v) SDS, 0.01% (w/v) bromophenol blue and 5% (v/v) 2-mercaptoethanol), sonicated, heated at 95 C and loaded on SDS-polyacryamide gels. Following electrophoresis protein was electro blotted onto nitrocellulose membranes and incubated with the relevant antibodies. 3. Results We have shown that several metabolites of benzene block GJIC [33]. Benzene itself had no effect on GJIC even at very high concentrations. The most potent benzene metabolite, MUC, blocks GJIC with increasing effect with time up to 5 h. This is different from what is observed for many other GJIC inhibiting compounds such as the phorbol ester TPA and chlordane, which induce complete block in cell communication via gap junctions after a few minutes [36]. Exposure to communication inhibiting concentrations of MUC for more than 1 h results in the disappearance of Cx43 on Western blots [33]. As shown in Western blots in Fig. 1A, Cx43 disappears after exposure to 10 M or higher concentrations of MUC after 5 h exposure. Slot blot analysis revealed however no loss (data not shown). We have previously shown that TPA as well as epidermal growth factor (EGF) induce internalization and degradation of Cx43, in a process involving Cx43 ubiquitination [37,38]. Our data suggest, however, that the loss of Cx43 in MUC-exposed cells is mediated through a different mechanism. One explanation is that MUC exposure causes cross-linking of Cx43. MUC has previously been shown to function as a cross-linking agent [39], and this could explain why Cx43 from MUC-exposed cells did not appear in the normal position in homogeneous SDS poly-acrylamide gels with stacking gel. When samples were run on gradient poly-acrylamide gels without stacking gel, Cx43 bands with molecular weights of approximately 80 and 120 kD appeared (Fig. 1D), suggesting cross-linking of Cx43. MUC and glutaraldehyde are dialdehydes with similar molecular structure, MUC being one carbon atom longer (Fig. 1B). Due to more double bonds MUC is also conformationally restrained compared to glutaraldehyde, which could have an impact on the efficiency of connexin cross-linking. We therefore tested the effect of glutaraldehyde on gap junctions and observed that it induced the same disappearance of Cx43 in Western blots as shown for MUC Fig. 1. Concentration-dependent effect of MUC (A), glutaraldehyde (B) and formaldehyde (C) on Cx43 in Western blot after 5 h exposure of IAR20 cells. (D) Western blotting of Cx43 in gradient SDS poly-acrylamide gel. Exposure to 50 M MUC resulted in bands with 2 and 3 increased molecular weight, suggesting cross-linking of Cx43. (Fig. 1B) with approximately three times lower potency; i.e. three times higher concentration was required to induce the same effect as for MUC. Formaldehyde is a short one-carbon mono-aldehyde, known to cross-link via CH2 bridges. Short cross-linking distance may prohibit cross-linking between individual Cx43 molecules and explain why Cx43 does not disappear from the Western blots in formaldehyde-exposed cells (Fig. 1C). The effect of the three aldehydes on GJIC is shown in Fig. 2. Glutaraldehyde completely blocks GJIC at concentrations where Cx43 disappears on Western blots and 50% at about 50 M, while MUC was about three times more potent. This difference in potency is in concordance with the difference in ability to cross-link Cx43 as judged from the Western blots in Fig. 1. Formaldehyde was also far less effective than MUC and glutaraldehyde in inhibiting GJIC, and only 2030% inhibition was observed at the most effective concentrations (Fig. 2). Fig. 2. Effect of exposure to different concentrations of MUC (5 h), glutaraldehyde (2 h), formaldehyde (2 h) and chlordane (1 h) on GJIC in IAR20 cells. Mean SD, n = 15. Please cite this article in press as: E. Rivedal, et al., Gap junction intercellular communication and benzene toxicity, Chem. Biol. Interact. (2009), doi:10.1016/j.cbi.2009.11.013 G Model CBI-6056; No. of Pages 4 ARTICLE IN PRESS E. Rivedal et al. / Chemico-Biological Interactions xxx (2009) xxxxxx 3 4. Discussion Conflict of interest Benzene causes myelodysplastic syndrome, aplastic anaemia, and acute myelogenous leukaemia [17,18]. The mechanism underlying these effects is however poorly understood although previous studies have focused on chromosomal effects [20,40], DNA-protein cross-links and DNA strand breaks [39]. We have shown that some benzene metabolites are potent inhibitors of gap junction intercellular communication [33]. The strongest GJIC inhibitor of the benzene metabolites is the dialdehyde trans,trans-muconaldehyde. The mono-aldehyde benzene metabolites, 6-hydroxy-trans,trans2,4-hexadienal and 6-oxo-trans,trans-2,4-hexadienoic acid, are weaker inhibitors of GJIC, while trans,trans-muconic acid has no inhibitory effect [41]. A similar order of potency has been shown for hematotoxic effects in mice [42]. Loss of GJIC has under different circumstances been related to downregulated transcription or post-translational modification of connexins. A number of kinases, including MAP kinase and Protein kinase C have been shown to inhibit GJIC through direct phosphorylation of Cx43. Our data suggest that the apparent loss of Cx43 observed in Western blots is caused by cross-linking of Cx43. Immunohistochemistry has shown that remaining Cx43 after MUC exposure is localized in the plasma membrane [33], differently from what is observed after exposure to TPA, where the Cx43 plaques are endocytosed [43]. We have previously shown that TPAinduced internalization and degradation of Cx43 involves a phosphorylation- and ubiquitination-dependent mechanism [37]. MUC seems not to have this capability due to cross-linking in the plasma membrane. The slow removal of plasma membraneassociated Cx43 plaques in MUC-exposed cells may suggest that such plaques are more prone to being cross-linked than intracellular Cx43. Glutaraldehyde, as well as MUC are potent activators of ERK1/2, but it seems that Cx43 phosphorylation is of lesser importance than Cx43 cross-linking for their induced inhibition of GJIC [34]. Formaldehyde, being a mono-aldehyde, is inefficient as a cross-linker of Cx43 and far less efficient as inhibitor of GJIC. The major inhibitory effect of formaldehyde on GJIC seems to be related to activation of ERK1/2 [34]. Glutaraldehyde has been extensively tested for toxicological effects and conflicting data is reported regarding its genetic activity [44]. Bone marrow hyperplasia and enhanced levels of leukemia were observed in a chronic drinking water study in rats [45]. This is of interest since this is similar to the effects observed after benzene exposure, and highlights the possible role of MUC in benzene toxicity. Formaldehyde is classified as Group 1 carcinogen by IARC [46,47] based on the finding of enhanced levels of different types of cancer in exposed humans, including nasopharyngeal cancer, leukemia and sinonasal cancer. Inhalation studies in rats have shown induction of squamous carcinomas of the nasal cavities. When exposed to formaldehyde in drinking water, forestomach and gastrointestinal tumors were observed, in addition to lymphomas and leukemias. Co-exposure with different model carcinogens resulted in enhanced levels of different tumors, indicating cocarcinogenic or tumor promoting effects of formaldehyde [46,47]. It is difficult to compare doses of reactive compounds used in in vitro cell assays with the relevant in vivo situation. Aldehydes and other reactive substances will be able to reach other cellular targets when being formed through metabolism, such as acetaldehyde through the metabolism of ethanol, or muconaldehyde from benzene. The presented data may suggest that effects of MUC on gap junction function, caused by cross-linking of connexin, could be involved in the hematotoxic effects of benzene. The authors declare that there are no conflicts of interest. Acknowledgements We would like to thank Astri Nordahl for excellent technical assistance. The work is supported by the Norwegian Cancer Society. References [1] C.J. Wei, X. Xu, C.W. Lo, Connexins and cell signaling in development and disease, Annu. Rev. Cell Dev. Biol. 20 (2004) 811838. [2] H. Yamasaki, C.C. Naus, Role of connexin genes in growth control, Carcinogenesis 17 (1996) 11991213. [3] E. McLachlan, Q. Shao, H.L. Wang, S. 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