Mechanistical studies on gap junctions and their role in tumour promotion : Structure-activity relationships of polychlorinated biphelyls

Chemically induced carcinogenesis is a multi-stage process that can be divided into at least three major stages, initiation, promotion and progression. The promotion stage involves clonal expansion of genetically altered cells (i.e. initiated cells). The final stage, progression, includes a karyotypic instability of cells that may result in malignancy. Several lines of evidence support the theory that inhibition of gap junctional intercellular communication plays a significant role in carcinogenesis by favouring the clonal expansion of initiated cells by isolating them from growth control mechanisms usually mediated by surrounding normal cells. The aim of the present study has been to study tumour promotion related effects induced by polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to elucidate possible mechanisms by which these compounds cause these effects and evaluate test assays for identifying tumour promoters. The study focused on an important mechanism, gap junctional intercellular communication, and its role in tumour promotion, in attempt to clarify structure-activity relationships for different PCB- congeners. The present study shows that the dioxin-like 3,4,5,3',4'-pentachlorobiphenyl (PCB 126), the non dioxin-like 2,4,5,2',4',5'-hexachlorobiphenyl (PCB 153) and TCDD can act as tumour promoters in the livers of female Sprague-Dawley rats. PCB 126 treatment is about 10% as efficient as TCDD in tumour promotion activity, which is in agreement with a toxic equivalency factor (TEF) of 0 1 for this PCB- congener. The use of the TEF concept assumes that different congeners in a mixture act via the same mechanistic pathways as TCDD and also cause additive effects. The results from the present study demonstrate that co-exposure to PCB 126 and TCDD can result in an additive response in altered hepatic foci formation. However, exposure of rats to a mixture of PCB 153 and PCB 126 can cause a more than additive effect, suggesting a different mode of action for PCB 153 compared to PCB 126. Thus, the TEF concept may be preferentially used for the dioxin-like PCBs. Exposure of rats to PCB 126 and TCDD demonstrates that these potent tumour promoters reduce the two major liver gap junction proteins outside altered hepatic foci. Furthermore, the decrease in gap junctions precedes the development of foci. The down-regulation of the gap junctions induced by PCB 126 and TCDD is suggested to be at the post-transcriptional level, since no changes in mRNA amounts were observed. Treatment with the weaker tumour promoters PCB 153 and PCB 118 (2,4,5,3',4'- pentachlorobiphenyl, which has properies of both dioxin-like and non dioxin-like PCBs) caused no alteration of gap junctions outside foci. In the non-tumourigenic rat liver epithelial cell line, IAR 20, aD tested PCB-congeners (PCB 126, PCB 153 and PCB 118) and TCDD down-regulate gap juncional intercellular communication. However, down-regulaion occurs after different lengths of exposure and by different mechanisms. PCB 153 treatment can decrease cell-cell communicaion after 1 hour exposure by reducing one of the phosphorylated isofomms of the gap junction protein (which is believed to be the functional form). PCB 126 and TCDD can reduce the amount of this phosphorylated isoform after a longer period of exposure (48 hours) by decreasing the mRNA level. Exposure of cells to PCB 118 demonstrates that this congener can act both as PCB 153 and as PCB 126 in gap junction regulation, since both the phosphorylation pattern and the mRNA level are altered. In summary, all tested PCB-congeners and TCDD inhibit gap junctional intercellular communication in IAR 20 cells and enhance the development of altered hepatic foci in rats, suggesting that inhibition of gap junctional intercellular communication in vitro can be used to identify potential tumour promoters. The tumour promotion potency of the PCB-congeners tested and of TCDD can be associated with their ability to decrease gap junctions in rat liver. This further supports the significant role of gap junctions in carcinogenesis. Key words: gap junctional intercellular communication, phosphorylation, tumour promotion, altered hepatic foci, rat liver, potency, PCBs, TCDD. Doctoral Thesis Yvonne Bager ISBN 91-628-2326-