Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing

Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework

Tumor promoting properties of a cigarette smoke prevalent polycyclic aromatic hydrocarbon as indicated by the inhibition of gap junctional intercellular communication via phosphatidylcholine-specific phospholipase C

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 ( LT 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 phosphatidylcholine-specific 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 phosphatidylcholine-specific 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

A Paradigm Shift in the Understanding of Oxidative Stress and its Implications to Exposure of Low-level Ionizing Radiation

For many years, research on oxidative stress focused primarily on determining how reactive oxygen species (ROS) damage cells by indiscriminate reactions with its macromolecular machinery, particularly lipids, proteins and DNA. However, many chronic diseases affiliated with oxidative stress are not always a consequence of tissue necrosis, DNA, or protein damage but rather to altered gene expression. Gene expression is highly regulated by the coordination of extra-, intra- and inter-cellular communication systems that typically maintain tissue homeostasis by sustaining a balance between proliferation, differentiation and apoptosis. Therefore, much research has shifted to the understanding of how ROS reversibly controls gene expression at noncytotoxic doses through cell signaling mechanisms. Cell proliferation typically involves a transient inhibition of gap junctional intercellular communication (GJIC) and the activation of mitogen activated protein kinase pathways (MAPK). We demonstrate that epidermal growth factor (EGF) inhibited GJIC in normal rat liver epithelial cells in addition to activating extracellular signal regulatory kinase, a MAPK. Inhibition of NADPH oxidase, which reduces oxygen to H2O2 with the very selective inhibitor diphenyleneiodonium, prevented EGF from inhibiting GJIC, suggesting that the generation of H2O2 is an essential component of the intracellular pathway controlling GJIC. We previously demonstrated that reduced-glutathione (GSH) was also a necessary cofactor of H2O2-induced inhibition of GJIC. These results demonstrate that ROS and GSH play essential roles in controlling EGF-dependent control of GJIC. Therefore, the overly simplistic approach of either preventing the generation of ROS or accelerate the removal by antioxidants could deleteriously alter normal signaling functions

A Paradigm Shift in the Understanding of Oxidative Stress and its Implications to Exposure of Low-level Ionizing Radiation

For many years, research on oxidative stress focused primarily on determining how reactive oxygen species (ROS) damage cells by indiscriminate reactions with its macromolecular machinery, particularly lipids, proteins and DNA. However, many chronic diseases affiliated with oxidative stress are not always a consequence of tissue necrosis, DNA, or protein damage but rather to altered gene expression. Gene expression is highly regulated by the coordination of extra-, intra- and inter-cellular communication systems that typically maintain tissue homeostasis by sustaining a balance between proliferation, differentiation and apoptosis. Therefore, much research has shifted to the understanding of how ROS reversibly controls gene expression at noncytotoxic doses through cell signaling mechanisms. Cell proliferation typically involves a transient inhibition of gap junctional intercellular communication (GJIC) and the activation of mitogen activated protein kinase pathways (MAPK). We demonstrate that epidermal growth factor (EGF) inhibited GJIC in normal rat liver epithelial cells in addition to activating extracellular signal regulatory kinase, a MAPK. Inhibition of NADPH oxidase, which reduces oxygen to H2O2 with the very selective inhibitor diphenyleneiodonium, prevented EGF from inhibiting GJIC, suggesting that the generation of H2O2 is an essential component of the intracellular pathway controlling GJIC. We previously demonstrated that reduced-glutathione (GSH) was also a necessary cofactor of H2O2-induced inhibition of GJIC. These results demonstrate that ROS and GSH play essential roles in controlling EGF-dependent control of GJIC. Therefore, the overly simplistic approach of either preventing the generation of ROS or accelerate the removal by antioxidants could deleteriously alter normal signaling functions