Inhibition of gap junctional intercellular communication (GJIC) and the activation of intracellular mitogenic pathways are common hallmarks of epithelial derived cancer cells. We previously determined that the 1-methyl and not the 2-methyl isomer of anthracene, which are prominent cigarette smoke components, activated extracellular receptor kinase, and inhibited GJIC in WB-F344 rat liver epithelial cells. Using these same cells, we show that an immediate upstream response to 1-methylanthracene was a rapid (<1 min) release of arachidonic acid. Inhibition of phosphatidylcholine-specific phospholipase C prevented the inhibition of GJIC by 1-methylanthracene. In contrast, inhibition of phosphatidylinositol specific phospholipase C, phospholipase A 2 , diacylglycerol lipase, phospholipase D, protein kinase C, and tyrosine protein kinases had no effect on 1-methylanthracene-induced inhibition of GJIC. Inhibition of protein kinase A also prevented inhibition of GJIC by 1-methylanthracene. Direct measurement of phosphatidylcholine-specific phospholipase C and sphingomyelinase indicated that only phosphatidylcholinespecific phospholipase C was activated in response to 1-methylanthracene, while 2-methylanthracene had no effect. 1-methylanthracene also activated p38-mitogen activated protein kinase; however, like extracellular kinase, its activation was not involved in 1-methylanthracene-induced regulation of GJIC, and this activation was independent of phosphatidylcholine-specific phospholipase C. Although mitogen activated protein kinases were activated, Western blot analyzes indicated no change in connexin43 phosphorylation status. Our results indicate that phosphatidylcholinespecific phospholipase C is an important enzyme in the induction of a tumorigenic phenotype, namely the inhibition of GJIC; whereas mitogen activated protein kinases triggered in response to 1-methylanthracene, were not involved in the deregulation of GJIC. (Cancer Sci 2008; 99: 696-705)

Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens, and a considerable amount of research has been devoted to predicting the tumor-initiating potential of PAHs based on chemical structure. However, there has been little research into the effects of PAHs on the epigenetic events of tumor promotion and no structural correlation has been made thereof. Gap junctional intercellular communication (GJIC) activity was used in this study as an epigenetic biomarker to determine the structure-activity relationships of twelve different PAHs. The PAHs used were naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9, 10-dimethylanthracene, phenanthrene, fluorene, 1-methylfluorene, and fluoranthene. Results showed that PAHs containing bay or baylike regions inhibited GJIC more than did the linear PAHs. The nonnaphthalene PAHs were not cytotoxic as determined by a vital dye uptake assay, but the naphthalene compounds were cytotoxic at the higher doses, indicating that the down regulation of GJIC by these naphthalenes could be a consequence of general membrane damage. Inhibition of GJIC by all the inhibitory PAHs was reversed when the cells were refreshed with PAH-free growth medium. Inhibition of GJIC occurred within 0.5-5 min and correlated with the aqueous solubility of the PAHs. The present study revealed that there are structural determinants of epigenetic toxicity as determined by GJIC activity.ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7Figure 8Figure 9Figure 10

Cancer is a multistage multimechanism process involving gene and/or chromosomal mutations (genotoxic events), altered gene expression at the transcriptional, translational, and posttranslational levels (epigenetic events), and altered cell survival (proliferation and apoptosis or necrosis), resulting in an imbalance of the organism's homeostasis. Maintenance of the organism's homeostasis depends on the intricate coordination of genetic and metabolic events between cells via extracellular and intercellular communication mechanisms. The release of a quiescent cell, whether normal or premalignant, from the suppressing effects of communicating neighbors requires the downregulation of intercellular communication via gap junctions, thereby allowing factors that control intracellular events to exceed a critical mass necessary for the cell to either proliferate or undergo apoptosis. Therefore, determining the role an environmental pollutant must play in the multistage carcinogenic process includes mechanisms of epigenetic toxicity such as the effects of a compound on gap junctional intercellular communication (GJIC). A classic example of a class of compounds in which determination of carcinogenicity focused on genotoxic events and ignored epigenetic events is polycyclic aromatic hydrocarbons (PAHs). The study of structure-activity relationships of PAHs has focused exclusively on the genotoxic and tumorinitiating properties of the compound. We report on the structure-activity relationships of two-to four-ringed PAHs on GJIC in a rat liver epithelial cell line. PAHs containing a bay or baylike region were more potent inhibitors of GJIC than the linear PAHs that do not contain these regions. These are some of the first studies to determine the epigenetic toxicity of PAHs at the epigenetic level.Environ Health Perspect 1 06(Suppl 4): 975-981 (1998). http.//ehpnetl.niehs.nih.gov/docs/ 1998/Suppl4/975-981upham/abstract.html

Cancer is a multistage multimechanism process involving gene and/or chromosomal mutations (genotoxic events), altered gene expression at the transcriptional, translational, and post-translational levels (epigenetic events), and altered cell survival (proliferation and apoptosis or necrosis), resulting in an imbalance of the organism's homeostasis. Maintenance of the organism's homeostasis depends on the intricate coordination of genetic and metabolic events between cells via extracellular and intercellular communication mechanisms. The release of a quiescent cell, whether normal or premalignant, from the suppressing effects of communicating neighbors requires the downregulation of intercellular communication via gap junctions, thereby allowing factors that control intracellular events to exceed a critical mass necessary for the cell to either proliferate or undergo apoptosis. Therefore, determining the role an environmental pollutant must play in the multistage carcinogenic process includes mechanisms of epigenetic toxicity such as the effects of a compound on gap junctional intercellular communication (GJIC). A classic example of a class of compounds in which determination of carcinogenicity focused on genotoxic events and ignored epigenetic events is polycyclic aromatic hydrocarbons (PAHs). The study of structure-activity relationships of PAHs has focused exclusively on the genotoxic and tumor-initiating properties of the compound. We report on the structure-activity relationships of two- to four-ringed PAHs on GJIC in a rat liver epithelial cell line. PAHs containing a bay or baylike region were more potent inhibitors of GJIC than the linear PAHs that do not contain these regions. These are some of the first studies of determine the epigenetic toxicity of PAHs at the epigenetic level.ImagesFigure 1

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