Lars Wärngård

Inhibition of metabolic cooperation in vitro and enhancement of enzyme altered foci incidence in rat liver by the pyrethroid insecticide fenvalerate

The synthetic pyrethroids cypermethrin, deltamethrin, 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.

Protein S-glutathionylation correlates to selective stress gene expression and cytoprotection.

During situations of oxidative stress phenotypic adaptation to altered redox state is achieved by changes in expression of selected genes. The mechanisms regulating this may involve reversible S-glutathionylation of cellular proteins. In this study we compared and contrasted changes in gene expression patterns in human type II lung epithelial A549 cells and human endothelial ECV304 cells in correlation to glutathione oxidation and the formation of glutathione-protein mixed disulphides, after exposure to subtoxic levels of hydrogen peroxide, formed in the medium by addition of glucose oxidase, or the thiol oxidant diamide. Both the number of specific mRNAs and their levels of induction were grossly correlated to the degree of S-glutathionylation of cellular protein. Thus, diamide induced the expression of a variety of protein and DNA chaperones and transcriptional regulators, particularly in ECV304 cells. On the other hand, the peroxide failed to induce many of these species, in association with only minimal disturbances to glutathione homeostasis. The induction of the chaperone responses at the level of mRNA was clearly shown to translate into a more resistant morphological phenotype in response to both heat shock and oxidative stress induced by the DNA-damaging pro-oxidant potassium bromate.

Inhibition of cell-cell communication by methylsulfonyl metabolites of polychlorinated biphenyl congeners in rat liver epithelial IAR 20 cells

The effects of three polychlorinated biphenyl (PCB) congeners and their six methylsulfonyl (MeSO2)-metabolites on cell communication have been investigated in the scrape-loading/dye-transfer assay in IAR 20 rat liver epithelial cells. The results demonstrated that at non-cytotoxic concentrations 2,2′,4′,5-tetrachlorobiphenyl, 2,2′,4′,5,5′-pentachlorobiphenyl (2,2′,4′,5,5′-pentaCB), 2,2′,4′,5,5′,6-hexachlorobiphenyl (2,2′,4′,5,5′, 6-hexaCB), and their 3- and 4-MeSO2 derivatives completely inhibited the cell communication within 1 h. 4-MeSO2-2,2′,4′,5,5′-pentaCB and 4-MeSO2-2,2′,4′,5, 5′,6-hexaCB appeared to inhibit the cell communication at slightly lower concentration than their parental PCB congeners and 3-MeSO2 derivatives. The results show that 3- and 4-MeSO2 derivatives of the PCB congeners tested inhibit gap junction intercellular communication at about the same potency as their parental compounds. Since inhibition of cell communication is often observed after treatment with many tumor promoters, our findings suggest that the metabolites may also act as tumor promoters.

Calmodulin involvement in TPA and DDT induced inhibition of intercellular communication.

The organochlorine pesticide DDT is a liver tumour promoter and a potent inhibitor of intercellular communication. Present knowledge of the mechanism by which DDT inhibits intercellular communication is limited but it has been suggested that increased intracellular free calcium induced by DDT could be of importance. As the effects of calcium are closely associated with the multifunctional protein calmodulin (CaM) in most cells the potential binding of DDT to CaM and subsequent effects on CaM-stimulated Ca2+/Mg2+-ATPase activity were studied. DDT inhibited CaM-stimulated Ca2+/Mg2+-ATPase activity and bound to CaM in a manner similar to established CaM-inhibitors. Subsequently an in vitro assay for measuring inhibition of metabolic cooperation between 6-thioguanine (TG)-sensitive and TG-resistant Chinese hamster (V79) cells was used to investigate the possible involvement of CaM in the regulation of intercellular communication. Calmidazolium (CzM), a potent CaM inhibitor, was tested alone or in combination with the tumour promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) or DDT known inhibitors of intercellular communication. The results showed that CzM alone was without effect with regard to inhibition of metabolic cooperation but potentiated the response induced by TPA, an effect not noticed with DDT. These results suggest different mechanisms of action of TPA and DDT on metabolic cooperation and support the hypothesis that with calcium CaM may be of importance for drug-induced inhibition of intercellular communication and tumour promotion.

Inhibition of metabolic cooperation in Chinese hamster lung fibroblast cells (V79) in culture by various DDT-analogs.

Anin vitro assay measuring inhibition of metabolic cooperation in Chinese hamster lung fibroblast cells (V79) was used to investigate the effects of DDT and five DDT analogs (dicofol, chlorobenzilate, bromopropylate, chloropropylate and fenarimol). These compounds were studied primarily because of the nongenotoxic but simultaneously carcinogenic properties of DDT and its analogs (chlorobenzilate and dicofol) and, secondly, to investigate how variations in the metabolic cooperation of hamster cells could be related to the chemical structure of the compounds. The results clearly demonstrate that all tested pesticides influenced the metabolic cooperation in a dose-dependent manner. The weak response of fenarimol suggests that sterical hindrance might influence the inhibition of metabolic cooperation.

The ability to alter the gap junction protein expression outside GST-P positive foci in liver of rats was associated to the tumour promotion potency of different polychlorinated biphenyls

The results demonstrate different modes of action by a dioxin-like polychlorinated biphenyl (PCB 126) and a non dioxin-like PCB (PCB 153) in the alteration of connexin (cx) 26 and cx 32 expression outside GST-P positive foci in liver of female Sprague-Dawley rats, treated according to an initiation-promotion protocol. A decreased relative amount of immunopositive cx 26 and cx 32 spots in the parenchymal cell plasma membranes was observed after treatment with the potent tumour promoters PCB 126 or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). No reduction of cx 26 or cx 32 was noted after administration with the weaker tumour promoters PCB 153 or PCB 118 (PCB 118; both dioxin- and non dioxin-like). Additionally, we found that the down-regulation of connexins also occurred in rats treated with PCB 126 or TCDD without partial hepatectomy and initiation with nitrosodiethylamine. In summary, the results indicate that the ability to reduce the gap junction protein level in liver of rats can be associated to the tumour promotive potency of the different PCB-congeners and TCDD.

Inhibition of intercellular communication in cells in culture by polychlorinated compounds

Abstract In the present study, the scrape loading and microinjection dye transfer techniques and the V79 metabolic cooperation assay were used in order to investigate inhibition of cell-cell communication induced by different PCDD-, PCDF- and PCB-congeners. The results further support the hypothesis that the molecular structure is one important characteristic for substances inhibiting intercellular communication. Substitution in the ortho-position in the biphenyl molecule seem to be necessary for the ability to inhibit intercellular communication. The PCDDs and the PCDF studied and the co-planar, non-orthosubstituted PCBs were inactive.

Initiation and promotion of altered hepatic foci in female rats and inhibition of cell-cell communication by the imidazole fungicide prochloraz.

The imidazole fungicide prochloraz (1-[N-propyl-N-2-(2,4,6-trichlorophenoxy) ethyl carbamoyl] imidazole) was investigated for its ability to inhibit gap junctional intercellular communication in the scrape-loading/dye-transfer assay in IAR 20 rat liver epithelial cells and for effects on the initiation and promotion stages of hepatocarcinogenesis. Female Sprague-Dawley rats initiated with N-nitrosodiethylamine 24-hr after partial hepatectomy were administered prochloraz five days a week by oral gavage (30 or 150 mg/kg) for 10 weeks. Altered hepatic foci (AHF) were analyzed by quantitative stereology from liver sections stained for gamma-glutamyltranspeptidase (GGT) and glutathione S-transferase P (GST-P). The fungicide was also studied for its ability to initiate the development of GGT-positive AHF in rat liver. The in vitro studies showed prochloraz to be an inhibitor of cell-cell communication in the test system used. In the in vivo studies, prochloraz showed no effect on the initiation of GGT-positive foci. However, significant increases in the percentage of liver tissue occupied by GGT-positive AHF and the number of GST-P-positive AHF per cm3 in initiated animals were recorded in the low dose group. The present data suggest that prochloraz acts as a weak tumour promoter of hepatocarcinogenesis but does not initiate this process.

Effects of tetradecanoyl phorbol acetate, pyrethroids and DDT in the V79

The effects of the pyrethroids fucythrinate, cyfluthrin, bioallethrin and resmethrin on metabolic cooperation between V79 cells were investigated. Addition offucythrinate to cocultures of 6-thioguanine-resistant and 6-thioguanine-sensitive V79 cells significantly increased the mutant cell recovery, indicating inhibition of intercellular communication. No such effect was observed by the other pyrethroids tested. To compare the modes of action of TPA-, DDT-, and pyrethroid-induced inhibition of intercellular communication, co-exposure experiments were undertaken. Addition of TPA, together with increasing doses of fenvalerate or fucythrinate, produced a synergistic response. Various combinations of fenvalerate-, fucythrinate- and DDT-exposure gave results in accordance with an additive response. The result suggest different pathways of action for TPA and the insecticides investigated in this study.

Chlorobenzilate-induced effects on enzyme-altered foci in rat liver and intercellular communication in rat liver WB-F344 epithelial cells

The mouse liver carcinogen chlorobenzilate (CB), a DDT-related pesticide, was investigated for enhancement of enzyme altered foci incidence in partially hepatectomized, diethyl-nitrosamine-initiated rats. In this in vivo experiment, CB administered per os (25 or 100 mg/kg per day for 10 weeks) enhanced foci incidence at the high dose level. In order to study potential mechanisms involved, CB was investigated for inhibition of gap-junctional intercellular communication in rat liver epithelial WB-F344 cells and Chinese hamster V79 cells in vitro. CB abolished dye transfer in WB-F344 cells and inhibited metabolic cooperation in V79 cells. Two CB metabolites were unable to induce such tumor promotion related effects. The results of this investigation provide support for the involvement of an epigenetic, tumor promoting mechanism in CB-induced liver tumors in laboratory animals.