No abstract

The organochlorine pesticide 1,1'-(2,2,2-trichloroethylidene) bis(4-chlorobenzene) (DDT) and four structural analogues (bromopropylate, chlorobenzilate, dicofol and fenarimol) were investigated for their ability to inhibit gap junctional intercellular communication both in the Chinese hamster V79 metabolic co-operation assay and in the scrape-loading/dye-transfer assay in WB-F344 rat liver epithelial cells. The pesticides were also studied for their ability to enhance the development of gamma-glutamyltranspeptidase-positive altered hepatic foci and induce cytochrome P450 monooxygenase isoenzymes in nitrosamine-initiated male Sprague-Dawley rats. The in vitro studies showed all organohalogens except fenarimol to be potent inhibitors of cell-cell communication in both test systems used. Concomitant results were recorded in the in vivo study. Thus, all potent inhibitors of intercellular communication were found to enhance significantly foci development and fenarimol was again without any significant effect. All pesticides studied were shown to be potent inducers of the phenobarbital-inducible cytochrome P450b isoenzyme and to cause hepatomegaly. Thus, no strict correlation between cytochrome P450b induction/liver growth and tumour promotion-related effects in vivo and in vitro was apparent for these organohalogen pesticides in the present study.

The cyclodiene pesticides endosulfan, chlordane and heptachlor have been reported to be non-genotoxic rodent hepatocarcinogens. These three compounds and several metabolites of endosulfan (endosulfan sulfate, endosulfan ether and endosulfan lactone) were examined for their effects on gap junctional intercellular communication (GJIC) in primary cultured male F344 rat hepatocytes and B6C3F1 mouse hepatocytes. GJIC was evaluated by Lucifer Yellow CH dye-coupling. Endosulfan and endosulfan sulfate inhibited rat and mouse hepatocyte GJIC in a dose-responsive manner (50-200 microM) after 4 h treatment. Endosulfan ether inhibited rat hepatocyte GJIC only at 200 microM and had no effect on mouse hepatocytes. Endosulfan lactone did not affect rat or mouse hepatocyte GJIC. Chlordane and heptachlor inhibited both mouse and rat hepatocyte GJIC at concentrations of 50-200 microM. The inhibition of GJIC by the cyclodienes showed similar dose-response relationships and kinetics of onset of inhibition and reversibility for both mouse and rat hepatocytes. Concomitant treatment of the cells with inhibitors of cytochrome P450 monooxygenases (SKF-525A, piperonyl butoxide or carbon monoxide) did not alter the inhibition of GJIC by the cyclodienes, suggesting that cytochrome P450 metabolism was not involved in the inhibitory mechanism. Dibutyryl cyclic AMP (0.5 mM), however, decreased the inhibition of GJIC by the cyclodienes and may indicate that these compounds inhibit intercellular communication through a cAMP-dependent process. The inhibition of mouse and rat hepatocyte GJIC by the cyclodienes correlated with previous reports indicating that these compounds are non-genotoxic rodent liver carcinogens.

Male Sprague-Dawley rats dosed with N-nitrosodiethylamine (NDEA) 24 h after two-thirds partial hepatectomy were treated with the pyrethroid insecticides fenvalerate, flucythrinate or cypermethrin in the diet for 20 weeks. Altered hepatic foci were analyzed by quantitative stereology from paraffin-embedded sections stained for gamma-glutamyltranspeptidase (GGT) or glutathione S-transferase P (GST-P). The present results demonstrate that the pyrethroids tested all enhance the development of NDEA-initiated, GGT-positive foci in rat liver at non-hepatotoxic doses. On the contrary, the volume fractions of GST-P-positive foci were not elevated as compared to the control group. The three pyrethroids tested all inhibited the transfer of Lucifer Yellow CH between WB-F344 rat liver epithelial cells in culture, supporting the increase of GGT-positive foci and suggesting that these substances can act as tumour promoters. The discrepancy between the results from analyses using GGT or GST-P as markers emphasizes the importance of understanding the mechanism underlying the expression of different markers for preneoplastic lesions and the importance of such effects in tumour promotion.

The polychlorinated dibenzo-p-dioxins/dibenzofurans 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) were studied for liver tumour promoting activity in a medium-term altered foci assay in nitrosamine-initiated female Sprague-Dawley rats. The congeners under study were administered by weekly subcutaneous injections at three dose levels for 20 weeks. Evaluation of gamma-glutamyltranspeptidase (GGT+), altered hepatic foci development, showed that all congeners studied acted as potent promoters of hepatocarcinogenesis. TCDD and PeCDD were virtually equipotent as enhancers of foci development while PeCDF displayed approximately ten per cent of the activity of the dioxins. Analysis of the dioxin- and furan-congeners by gas chromatography/mass spectroscopy (GC/MS) technique showed that the retention of PeCDD and PeCDF in liver tissue was approximately 7 and 20 times, respectively, as high as the retention of TCDD. Based on the concentration of the respective congener in liver tissue, PeCDD and PeCDF were 0.14 and 0.007 times as active as TCDD as promoters of foci development. The dose related enhancement of GGT+ foci development induced by the PCDD/PCDF congeners was accompanied by an increased incidence of histological changes in the liver.

The synthetic pyrethroids cypermethrin, delta-methrin, fenvalerate, permethrin, and the fenvalerate metabolite p-chlorophenylisovaleric acid were investigated for inhibition of gap-junctional intercellular communication in vitro in the Chinese hamster lung fibroblast (V79) metabolic cooperation assay. Fenvalerate was furthermore studied for enhancement of gamma-glutamyl transpeptidase-positive enzyme altered foci incidence in partially hepatectomized, nitrosodiethylamine-initiated male Sprague Dawley rats. The in vitro studies showed that fenvalerate and p-chlorophenylisovaleric acid were inhibitors of intercellular communication at non-cytotoxic concentrations while cypermethrin, deltamethrin, and permethrin were inactive. In the in vivo study in rat liver, fenvalerate administered p.o. (75 mg/kg/day) 5 days a week for 10 weeks induced significantly more foci per cm3 and a larger percentage of liver tissue occupied by foci tissue compared to a vehicle control group. Analysis of size distributions of foci in fenvalerate- and vehicle-treated rats showed elevated foci incidences in fenvalerate-treated rats at all foci sizes. Fenvalerate induced no hepatotoxic effects as judged by plasma transaminase activities and histopathology. The results of this study suggest fenvalerate to be a potential tumour promoter.

An in vitro assay measuring inhibition of metabolic cooperation between 6-thioguanine sensitive and 6-thioguanine resistant Chinese hamster (V79) cells in co-culture was used to detect chemically induced inhibition of gap-junctional intercellular communication. Inhibition of this cellular process by xenobiotics has been suggested to be an important event in tumour promotion. This study was undertaken to determine the effect on metabolic cooperation by the bioflavonoid quercetin alone and in co-exposure experiments with two recognized tumour promoters, the phorbol ester TPA and the organochlorine pesticide DDT. Furthermore, co-exposure experiments with TPA and DDT were performed. Quercetin alone did not affect metabolic cooperation at noncytotoxic doses. Treatment of the cells with either TPA, DDT or TPA together with DDT caused significant inhibition of metabolic cooperation. This effect was dose-dependently decreased by addition of quercetin. These findings suggest that quercetin inhibits or compensates a common effect induced by both TPA and DDT. Treatment of the cells with a fixed dose of TPA and increasing doses of DDT, or a fixed dose of DDT and increasing doses of TPA, caused significantly higher recovery of mutant cells than a calculated additive response. The data indicate that TPA and DDT induce a synergistic response with respect to affecting intercellular communication. The results suggest that there are different pathways of action for TPA and DDT.

The non-genotoxic, chlorinated cyclodiene insecticide endosulfan was studied for its ability to act as a tumour promoter in a two-stage, altered hepatic foci bioassay in male Sprague-Dawley rats. Two stereoisomers of endosulfan, alpha-endosulfan (ENDO alpha) and beta-endosulfan (ENDO beta) were used, as well as a commercially-occurring mixture of the alpha- and beta-isomers (ENDO alpha beta). The animals were initiated by intraperitoneal injection of nitrosodiethylamine 24 h after a two-thirds-partial hepatectomy. Five weeks later the animals were transferred to diets containing 30, 100 and 300 p.p.m. of either ENDO alpha beta, ENDO alpha or ENDO beta. The study was terminated 25 weeks after initiation and the development of foci of gamma-glutamyltranspeptidase-positive hepatocytes was evaluated by stereological methods. The results show that endosulfan and its two stereoisomers promote the development of altered hepatic foci, suggesting that endosulfan is a tumour-promoting agent acting by clonal expansion of initiated cells.