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. A considerable amount of research has been devoted to predicting the genotoxic, tumor-initiating potential of PAHs based on chemical structure. However, information on the correlation of structure with the non-genetoxic, epigenetic events of tumor promotion is sparse. PAHs containing a bay or bay-like region were shown to be potent inhibitors of gap-junctional intercellular communication (GJIC), an epigenetic event involved in the removal of an initiated cell from growth suppression. We tested the epigenetic toxicity of PAHs containing bay-like regions by comparing the effects of methylated vs. chlorinated isomers of anthracene on the temporal activation of mitogen-activated protein kinase (MAPK) and the regulation of GJIC. Specifically, we used anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9,10-dimethylanthracene, 1-chloroanthracene, 2-chloroanthracene, and 9-chloroanthracene. We determined the effect of these compounds on GJIC and on the activation of extracellular receptor kinase (ERK 1 and 2), a MAPK, in F344 rat liver epithelial cells. Results showed that bay or bay-like regions, formed by either chlorine or a methyl group, reversibly inhibited GJIC at the same doses, time, and time of recovery, whereas the linear-planar isomers had no effect on GJIC. Similarly, the GJIC-inhibitory isomers also induced the phosphorylation of ERK 1 and ERK 2, while the non-inhibitory isomers had no effect on the activation of these MAPKs. MAPK activation occurred 10-20 min after the inhibition of GJIC, which indicates that MAPK is not involved in the initial regulation of GJIC; instead altered GJIC may be affecting MAPK activation. The present study revealed that there are structural determinants of PAHs, which clearly affect epigenetic events known to be involved in the non-genetoxic steps of tumor promotion. These events are the release of a cell from growth suppression involving the reduction of GJIC, followed by the activation of intracellular mitogenic events.

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

BackgroundPerfluoroalkanoates, [e.g., perfluorooctanoate (PFOA)], are known peroxisome proliferators that induce hepatomegaly and hepatocarcinogenesis in rodents, and are classic non-genotoxic carcinogens that inhibit in vitro gap-junctional intercellular communication (GJIC). This inhibition of GJIC is known to be a function of perfluorinated carbon lengths ranging from 7 to 10.ObjectivesThe aim of this study was to determine if the inhibition of GJIC by PFOA but not perfluoropentanoate (PFPeA) observed in F344 rat liver cells in vitro also occurs in F344 rats in vivo and to determine mechanisms of PFOA dysregulation of GJIC using in vitro assay systems.MethodsWe used an incision load/dye transfer technique to assess GJIC in livers of rats exposed to PFOA and PFPeA. We used in vitro assays with inhibitors of cell signaling enzymes and antioxidants known to regulate GJIC to identify which enzymes regulated PFOA-induced inhibition of GJIC.ResultsPFOA inhibited GJIC and induced hepatomegaly in rat livers, whereas PFPeA had no effect on either end point. Serum biochemistry of liver enzymes indicated no cytotoxic response to these compounds. In vitro analysis of mitogen-activated protein kinase (MAPK) indicated that PFOA, but not PFPeA, can activate the extracellular receptor kinase (ERK). Inhibition of GJIC, in vitro, by PFOA depended on the activation of both ERK and phosphatidylcholine-specific phospholipase C (PC-PLC) in the dysregulation of GJIC in an oxidative-dependent mechanism.ConclusionsThe in vitro analysis of GJIC, an epigenetic marker of tumor promoters, can also predict the in vivo activity of PFOA, which dysregulated GJIC via ERK and PC-PLC.

Dicumyl peroxide (di-CuOOH) and benzoyl peroxide (BzOOH) act as tumor promoters in SENCAR mice, whereas di-tert-butylhydroperoxide does not. Tumor promotion requires the removal of growth suppression by inhibition of gap junctional intercellular communication (GJIC) and the induction of mitogenic intracellular pathways. We showed that di-CuOOH and BzOOH both reversibly inhibited GJIC and transiently activated mitogen-activated protein kinase, specifically, the extracellular receptor kinase at noncytotoxic conditions in WB-F344 rat liver epithelial cells, whereas the non-tumor-promoting di-tert-butylhydroperoxide did not inhibit GJIC or activate extracellular receptor kinase. di-CuOOH but not BzOOH inhibited GJIC through a phosphatidylcholine-specific phospholipase C-dependent mechanism. N-acetylcysteine (NAC) was needed to prevent a cytotoxic, glutathione-depleting effect of BzOOH, whereas di-CuOOH was noncytotoxic and did not alter glutathione levels at all doses and times tested. Pretreatment of WB-F344 cells with resveratrol, a polyphenolic antioxidant present in red wine, prevented at physiological doses the inhibition of GJIC by di-CuOOH but not from BzOOH and was effective in significantly preventing extracellular receptor kinase activation by both peroxides. NAC did not prevent any of the peroxide effects on either GJIC or extracellular receptor kinase, suggesting a specific antioxidant effect of resveratrol.