Sten Flodström

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.

Modulation of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Phenobarbital Induced Promotion of Hepatocarcinogenesis in Rats by the Type of Diet and Vitamin A Deficiency

This study was undertaken to investigate the influence of dietary vitamin A deficiency and type of diet on tetrachlorodibenzo-p-dioxin (TCDD)- and phenobarbital-induced liver tumor promotion in rats. Female Sprague-Dawley rats were partially hepatectomized and subsequently initiated with nitrosodiethylamine. One week later the rats were allocated to five different dietary regimens for the duration of the study: three purified (casein-based) diets containing 200, 1200, and 10000 IU vitamin A per kilogram, respectively, and two conventional (cereal-based) rat diets containing 2000 and 14000 IU vitamin A per kilogram, respectively. After an additional 4 weeks, groups of rats on each dietary regimen were started on one of four different promoter treatments: 0.07 micrograms TCDD/kg/week (sc); 0.7 micrograms TCDD/kg/week (sc); 500 ppm phenobarbital in the drinking water or vehicle only (arachis oil, sc). The study was terminated after 16 weeks of promoter treatment. Sections of liver were stained for gamma-glutamyltranspeptidase (GGT) activity and GGT-positive altered hepatic foci (AHF) were evaluated by stereological methods. All factors studied (TCDD, phenobarbital, dietary vitamin A content, and the type of diet) were shown to influence AHF development significantly. As expected, TCDD and phenobarbital enhanced foci development. Vitamin A deficiency enhanced foci development in its own right and increased the TCDD-induced response markedly. Dietary vitamin A content did not modulate phenobarbital promotion of AHF in the same manner. The enhancement of TCDD-induced effects on foci development by vitamin A deprivation was accompanied by an increased incidence of histological changes marking degeneration in the liver (e.g., oval cell hyperplasia) and accentuation of other TCDD-related toxic responses. In addition, the groups of rats maintained on the cereal-based diets and subjected to the various promoter/vitamin A regimens exhibited significantly higher AHF incidence as compared to correspondingly treated rats fed the purified, casein-based diets. In conclusion, vitamin A deficiency alone may promote hepatocarcinogenesis and enhance the promoting effect of TCDD treatment. However, TCDD-induced depletion of hepatic vitamin A stores was not implicated as a major cause of promotion by TCDD. Nevertheless, vitamin A deficiency brought about by TCDD alone may well act as a promotive stimulus concertedly with an as yet unidentified cellular mechanism in TCDD-induced liver tumor promotion. The differential effects of the two types of diets recorded in the study remain undocumented.

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.

Relative liver tumor promoting activity of some polychlorinated dibenzo-p-dioxin-, dibenzofuran- and biphenyl-congeners in female rats

Abstract Some pentachlorinated dibenzo- p -dioxin-, dibenzofuran- and biphenyl-congeners and TCDD were studied for liver tumor promoting activity in a medium-term altered foci assay with nitrosamine initiated female SD rats. The congeners under study were administered by weekly subcutaneous injections for 20 weeks. Evaluation of GGT + or GST-P + altered foci development showed that all congeners studied acted as promoters of hepatocarcinogenesis. TCDD and 1,2,3,7,8-pentachloro-dibenzo- p -dioxin (PeCDD) were virtually equipotent as enhancers of foci development while 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) displayed approximately 10% of the activity of the dioxins. The PCB congener 2,3,3′,4,4′-pentachlorobiphenyl (PCB105) also enhanced foci development but was more than 1000 times less potent than TCDD. Analysis of the dioxin- and furan-congeners by 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.

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.

Enhancement of altered hepatic foci development by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) — Effects of vitamin A supplementation and the type of diet

Abstract In this study we investigated the modification of TCDD-induced enhancement of nitrosamine-initiated altered hepatic foci (AHF) development by dietary vitamin A supplementation and the type of diet. Vitamin A deficiency was shown to potentiate the effect of TCDD. Furthermore, severe vitamin A deficiency alone enhanced the development of AHF. A differential effect on AHF-development by the two types of diet used (purified vs cereal diet) was apparent. However, it was concluded that TCDD-induced depletion of hepatic stores of vitamin A is not a primary mechanism in liver tumour promotion induced by this compound. However, it may well be that TCDD-induced vitamin A deficiency acts as a tumour promoting stimulus in concert with an, as yet, unidentified mechanism of tumour promotion by TCDD.