Inhibition Of Metabolic Cooperation Research Articles

Inhibition of metabolic cooperation in Chinese hamster cells by various chemical including tumor promoters.

The effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and other chemicals on the contact-feeding phenomenon were investigated. The death of 6-TGr cells was prevented by TPA even upon culture with 3 times as many wild-type cells as in TPA-free medium. Seven out of 18 chemicals tested were shown to inhibit the phenomenon of metabolic cooperation. They were concanavalin A, lithocholic acid, chaetoglobosin A, 3-hydroxyanthranilic acid, urea, anthralin and saccharin. Among known tumor promoters, sodium deoxycholate, estradiol and phenobarbital did not inhibit the phenomenon of metabolic cooperation.

Absence of tumor promoting activity of Euphorbia milii latex on the mouse back skin

Euphorbia milii (Euphorbiaceae) is a decorative plant used in gardens and living fences. In China, it has also been employed in herbal remedies for hepatitis and abdominal edema. Since E. milii latex—lyophilized or in natura—proved to be a potent plant molluscicide, its toxicity to non-target organisms has been comprehensively studied. Concerns on a possible tumor promoting activity have discouraged its use as a locally-available alternative molluscicide in schistosomiasis control programs. Two in vitro assays (inhibition of metabolic cooperation in V79 cells and Epstein-Barr virus induction in Raji cells) had suggested that E. milii latex contained tumor-promoting substances. This study was undertaken to verify whether the latex acts as a tumor promoter in vivo as well. A single dose of the initiating agent DMBA (400 nmol) was applied on the back skin of male and female DBA/2 mice. Testing for tumor promoting activity began 10 days after initiation. Tetradecanoyl phorbol acetate (TPA) (5 nmol, positive control), lyophilized latex (20, 60 and 200 μg per mouse) or acetone (vehicle control) were applied on mouse back skin twice a week for 20 weeks. In TPA-treated mice, papillomas were firstly noted during the 11th week, and by the 17th week all animals exhibited skin tumors. No tumors developed in mice treated with the solvent alone and in those exposed to latex. Findings from the present study therefore indicated that E. milii crude latex does not act as a tumor promoting agent on the mouse back skin assay.

Review of the genotoxicity of 2-butoxyethanol

The available data on the genotoxicity of 2-butoxyethanol have been reviewed. 2-Butoxyethanol has been examined for genotoxic activity in a range of in vitro and in vivo assays, including the mouse bone marrow micronucleus assay. The in vitro assays used range from well validated and generally accepted assays such as the Salmonella/microsome and in vitro cytogenetic assay, through to less well validated assays such as assessment of the inhibition of metabolic cooperation in V79 cells. The levels of experimental details and data reporting vary across the studies with some papers presenting only limited information. Taking the above factors into consideration, the available data indicate that 2-butoxyethanol has no significant genotoxic activity. This conclusion is also consistent with the chemical structure of 2-butoxyethanol, which is not alerting for likely genotoxic activity. These collected considerations indicate that 2-butoxyethanol is unlikely to be a genotoxic carcinogen to rodents, a prediction that supplements seventeen published predictions of the outcome of the ongoing NTP rodent carcinogenicity bioassays.

Lack of tumor-promoting effects of flavonoids: studies on rat liver preneoplastic foci and on in vivo and in vitro gap junctional intercellular communication.

Possible tumor-promoting activity of four flavonoids, quercetin (QC), tangeretin (TG), flavone (FO), and flavanone (FN), was examined in a rat liver short-term carcinogenesis assay as well as with in vivo and in vitro assays of inhibition of gap junctional intercellular communication (GJIC). Rat hepatocarcinogenesis was induced by aflatoxin B1 treatment followed by a selection phase (2-acetylaminofluorene treatment and partial hepatectomy), then treatment with or without test chemicals (in vivo studies of antipromotion were not performed). Using glutathione S-transferase placental form (GST-P)-positive foci, we compared the effects of flavonoids (at 1,000 ppm in the diet) with the effects of phenobarbital (PB) on the occurrence of liver preneoplastic lesions. In addition, we studied the effects of flavonoids on GJIC in the livers derived from these experiments and in two types of cultured cells. No significant difference in the number and area of GST-P-positive foci was found after one or three months of treatment between any flavonoid group and control group. In the positive control group, PB markedly increased the numbers and areas of preneoplastic lesions at three months. Whereas PB also decreased by 60% the average size of lucifer yellow dye spread in slices of liver parenchyma free of preneoplastic lesions among the different flavonoids, only TG decreased the dye transfer in vivo: by 30% at one month and 50% at three months. With the dye transfer assay applied to a rat liver epithelial cell line (REL) and the Chinese hamster V79 metabolic cooperation assay, none of the tested flavonoids (< or = 25 microM) inhibited GJIC. Conversely, protective properties were seen for some of the compounds in antipromotion in vitro studies, because TG and FN enhanced the dye transfer in REL cells and FO, TG, and QC partly prevented the inhibition of metabolic cooperation by 12-O-tetradecanoylphorbol-13-acetate. Thus, taken together, our results suggest that QC, FO, and FN do not show tumor-promoting activity. Concerning TG, some discrepancies in the in vivo data are observed. Some of them (GJIC inhibition in liver slices) are probably more relevant to promotion of hepatocarcinogenesis.

Role of cytoplasmic membrane in the screening of potential tumour promoters.

The functional changes of cytoplasmic membrane of mammalian cells as an alternative approach for evaluating the tumor promoter activity were investigated. The V79 metabolic cooperation assay, as the standard method for detection of tumor promoters in vitro was used. From three known tumor promoters only phenobarbital increased the efflux of [3H]2-deoxy-D-glucose-6-phosphate from the cells in the same concentration range where the inhibition of metabolic cooperation was estimated. Neither TPA, saccharin nor theophylline caused any functional changes of cytoplasmic membrane of V79 cells. On the basis of the results with known tumor promoters we suggest that following the functional changes of cytoplasmic membrane is not a convenient approach for screening potential tumor promoters.

Studies on the tumor-promoting activity of biomaterials: inhibition of metabolic cooperation by polyetherurethane and silicone.

When V79 metabolic cooperation (MC) assay was performed to detect tumor-promoting activities, inhibitory activities on the gap-junctional intercellular communication were first detected in both extracts prepared from polyetherurethane (PEU) and silicone. The former inhibited more strongly than the latter. Furthermore, the lowest effective concentrations in MC assay correlated well with the values of the total active incidences obtained by histologic evaluation of the 2-year implantation test in rats. Poly(tetramethylene oxide) (PTMO) showed the highest inhibitory activities among the constituents of PU. Thus, the shorter the chain length of PTMO, the stronger the inhibitory activities of PTMO in MC assay. PTMO moiety may play an important role in the tumor-promotion stage during tumorigenesis induced by PU.

Studies on the tumor‐promoting activity of additives in biomaterials: Inhibition of metabolic cooperation by phenolic antioxidants involved in rubber materials

For the detection of tumor-promoting activities of phenolic antioxidants, the inhibitory activities on the intercellular gap-junctional communication were investigated using the V79 metabolic cooperation (MC) assay. Among eight antioxidants, 4,4'-butylidene-bis(3-methyl-6-tert-butyl-phenol), 2,2'-methylene-bis(4-methyl-6-tert-butylphenol) (MBMBP), and styrenated phenol (SP) showed stronger inhibitory activities than lithocholic acid, which is known to be a tumor promotor. However, 4,4'-thio-bis(3-methyl-6-tert-butylphenol), Irganox 1010, and 1330 did not inhibit at any concentrations. When the single-electron oxidation potentials were compared among antioxidants, the electrochemical ease estimated with the first oxidation potential was correlated with the cytotoxic potentials (r = 0.88), but not with the inhibitory activities in an MC assay. The tumor-promoting activity of MBMBP was also investigated using an in vitro, two-stage Balb/c 3T3 transformation assay. MBMBP did not show initiating activity, but significant promoting activity at concentrations of both 1 and 2.5 micrograms/ml were noted. These concentrations were close to the lowest effective inhibitory concentration (1.3 micrograms/ml) of MBMBP in an MC assay. In conclusion, there is a possibility that the phenolic antioxidants that show inhibitory activities in an MC assay contribute to the enhancement of tumor incidence induced by biomaterials.

Effect of lipids and aldehydes on gap-junctional intercellular communication between human smooth muscle cells.

Inhibition of intercellular communication is an important feature in the tumour promotion phase of a multistage carcinogenesis model. In atherosclerosis inhibition of cell-cell communication by atherogenic compounds, e.g., low density lipoproteins (LDL), also seems to be important. For testing atherogenic compounds we used an atherosclerosis relevant cell type, namely human smooth muscle cells. In order to investigate which part of the LDL particle would be involved in inhibition of metabolic co-operation between human smooth muscle cells in culture we tested several fatty acids and their breakdown products, namely aldehydes. Unsaturated C-18 fatty acids markedly influenced gap-junctional intercellular communication (GJIC), whereas saturated (C18:0, C16:0) and unsaturated fatty acids with > 20 carbon atoms did not inhibit GJIC. In the case of oleic and elaidic acid, orientation seemed important; however, after exposure to palmitoleic and palmitelaidic acid no differences were found. The most potent inhibitor of GJIC was linoleic acid, which inhibited GJIC by 75%. No correlation was found between degrees of unsaturation and ability to inhibit GJIC. Of the tested aldehydes, hexanal, propanal, butanal and 4-hydroxynonenal did significantly inhibit GJIC, while pentanal had no effect. Since modification of LDL was shown to be important in order for LDL to inhibit GJIC, these results show that fatty acids and their oxidative breakdown products may be of importance for the inhibition of GJIC by LDL.

Cytochrome P450-mediated metabolism of tumour promoters modifies the inhibition of intercellular communication: a modified assay for tumour promotion.

The role of metabolism of tumour promoters on the inhibition of intercellular communication was investigated in a modified V79 metabolic cooperation system. V79 cells, which stably express different rat cytochrome P450 enzymes (CYP1A1, CYP1A2 or CYP2B1), were used in the metabolic cooperation assay. The inhibitory effect on intercellular communication of four compounds was changed in cells expressing cytochrome P450 enzymes, compared to cells without. The phorbol ester TPA and di(2-ethylhexyl)phthalate blocked intercellular communication in all the cell lines tested, but expression of CYP1A1 enzyme reduced the inhibitory activity in these cells. Diethylstilbestrol caused inhibition only with cells containing cytochrome P450 enzymes. In contrast, the benzene metabolite hydroquinone inhibited metabolic cooperation preferentially in cells without cytochrome P450 enzymes. The inhibition of metabolic cooperation by another benzene metabolite, phenol, was not affected by the cytochrome P450 enzymes. The inhibitory activity of several chemicals that have not been tested previously was analysed in the new metabolic cooperation assay. The inhibitory activity of none of these chemicals was affected by cytochrome P450-associated metabolism. 7-Octylindolactam V was as potent as TPA, whereas the related indolactam V was 100-fold less active. The carcinogenic aromatic amine 4-aminobiphenyl, but not its primary metabolite 4-hydroxyaminobiphenyl, inhibited metabolic cooperation. Other known carcinogens, ochratoxin A, aflatoxin B1 and 4-nitrobiphenyl, did not inhibit metabolic cooperation in either V79 cells expressing or cells not expressing cytochrome P450. We conclude that cytochrome P450-associated metabolism plays an important role in the inhibition of gap junctional intercellular communication of some tumour promoters. The modified metabolic cooperation assay presented here is valuable for detecting some inhibitory chemicals which have been 'false negative' in previous assays for gap junctional intercellular communication. The assay also discloses that cytochrome P450 metabolism alters intercellular communication by a mechanism other than metabolism of the exogenous inhibitor.

Study of the biological effects of theophylline on V79 cells: Viability, membrane permeability, and metabolic cooperation

The effect of theophylline, a specific inhibitor of phosphodiesterase, on gap junction-mediated intercellular communication between Chinese hamster V79 cells was examined. It was found that addition of theophylline to coculture of 6-thioguanine-resistant (TGr) and 6-thioguanine-sensitive (TGs) V79 cells significantly increased the recovery of TGr cells. This finding indicates an inhibition of metabolic cooperation of V79 cells by theophylline. Theophylline was tested at concentrations less than 0.3 mg/ml, which were neither cytotoxic (after short or continuous exposure) nor inhibited the synthesis of DNA, RNA, and proteins. At the tested concentrations, no change was found in the membrane permeability of cells. Theophylline did not increase the incorporation of glucose into the cells.

Mutagenic effects of ferric nitrilotriacetate (Fe-NTA) on V79 Chinese hamster cells and its inhibitory effects on cell-cell communication

Ferric nitrilotriacetate (Fe-NTA) induced dose- and time-dependent mutation of V79 Chinese hamster cells to 6-thioguanine resistance. It also caused dose-related inhibition of metabolic cooperation. However, no significant induction of chromosome aberrations was detected in cells treated with Fe-NTA up to 100 micrograms Fe/ml of the drug even after treatment for 3 days. Our results indicate that Fe-NTA has mutagenic effects on V79 cells and inhibitory effects on cell--cell communication, and these effects may contribute to NTA-Fe-induced neoplastic transformation of mammalian cells.

Synergistic inhibition of metabolic cooperation by oleic acid or 12-0-tetradecanoylphorbol-13-acetate and dichlorodiphenyltrichlorethane (DDT) in Chinese hamster V79 cells: implication of a role for protein kinase C in the regulation of gap junctional intercellular communication.

The effects of TPA and/or DDT and oleic acid and/or DDT on gap junction-mediated intercellular communication (i.e. metabolic cooperation) between Chinese hamster V79 cells was examined. Addition of TPA, DDT or oleic acid alone to cocultures of 6-thioguanine-resistant (6-TGR) and 6-thioguanine-sensitive (6-TGS) V79 cells significantly increased the recovery of 6-TGR cells indicating inhibition of metabolic cooperation. In the presence of TPA and DDT or oleic acid and DDT the observed recovery of 6-TGR cells was significantly greater than the expected (calculated) additive 6-TGR cell recovery. No synergistic increases in 6-TGR cell recovery were observed when co-cultures of V79 cells were exposed to dieldrin and DDT. These results indicate that TPA and DDT or oleic acid and DDT can act synergistically to inhibit metabolic cooperation. These data suggest a role for protein kinase C in the regulation of gap junction-mediated intercellular communication.

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, 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.

Interaction between quercetin, TPA and DDT in the V79 metabolic cooperation assay.

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.

Inhibition of metabolic cooperation by glycyrrhetinic acid in cultured Chinese hamster cells

Inhibition of metabolic cooperation by glycyrrhetinic acid in cultured Chinese hamster cells

Inhibition of metabolic cooperation in Chinese hamster V79 cells by 1α,25-dihydroxycholecalciferol, the active metabolite of vitamin D 3

The biologically active metabolite of vitamin D 3, 1α,25-dihydroxycholecalciferol (1,25-(OH) 2D 3), inhibited the metabolic cooperation between cocultivated Chinese hamster V79 cells that are either susceptible or resistant to the cytotoxic effect of 6-thioguanine (TG). This inhibition was characterized by an increased survival of TG-resistant cells when they are cocultured with susceptible cells in the presence of TG. TG-resistant cells in the absence of susceptible cells and after treatment with TG yielded a cloning efficiency of about 90%. Cocultivation of these cells with the susceptible cells reduced this cloning efficiency to about 30%. At 0.5 μg/ml, 1,25-(OH) 2D 3 restored the cloning efficiency of the TG-resistant cells in the cocultures to 62%. Phorbol 12-myristate 13-acetate (PMA), a known inhibitor of metabolic cooperation, at 0.5 μg/ml restored the cloning efficiency to a similar extent. Vitamin D 3, 24,25-dihydroxycholecalciferol (24,25-(OH) 2D 3, and phorbol were ineffective in inhibiting this metabolic cooperation. 1,25-(OH) 2D 3, unlike PMA, did not inhibit the binding of [ 3H]phorbol 12,13-dibutyrate (PDBu) to its cellular receptors in either the TG-susceptible or resistant cells. These results indicate that 1,25-(OH) 2D 3 resembles tumor-promoting agents such as PMA in its capacity to inhibit metabolic cooperation, but this capacity is not mediated through phorbol diester receptors.

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.

Genotoxicity testing of arsenobetaine, the predominant form of arsenic in marine fishery products

Marine fishery products may contain high levels of arsenic, mainly in the form of organic arsenic compounds. Arsenobetaine has been identified as the predominant form occurring in marine fishery products. The potential initiating and promoting capacities of this compound were therefore investigated in vitro. In the Salmonella typhimurium assay, no mutagenicity was observed in strains TA97, TA98 and TA100 without activation or after addition of a liver-enzyme fraction or gut-flora extract. The compound was also negative in the forward mutation assay of the HGPRT gene and in the test for sister chromatid exchanges in V79 Chinese hamster cells. No inhibition of metabolic co-operation between V79 Chinese hamster cells was observed at arsenobetaine concentrations up to 10 mg/ml. In addition, arsenobetaine had no synergistic or antagonistic effects on the action of the positive controls benzo[ a]pyrene and tetradecanoylphorbol-13-acetate.

Genotoxicity testing of cigarette-smoke condensate in the SCE and HGPRT assays with V79 Chinese hamster cells

The genotoxicity of cigarette-smoke condensate (CSC) was investigated using V79 Chinese hamster fibroblasts in the sister-chromatid exchange (SCE) and HGPRT (hypoxanthine-guanine-phosphoribosyl-transferase) tests. CSC was negative in the SCE test both with and without metabolic activation (co-cultivation with chick-embryo primary hepatocytes). In the HGPRT test no direct mutagenicity of CSC was observed but after metabolic activation there was a considerable increase in the number of mutants. Comparison of different metabolizing systems showed that non-induced chick hepatocytes, liver homogenate from non-induced chick embryos and liver homogenate from rats pretreated with Aroclor 1254 generated similar numbers of mutants in cells treated with CSC. In addition the capacity of CSC to inhibit metabolic co-operation between V79 cells was studied. A dose-related increase in the inhibition of metabolic co-operation was observed.

Further characterization of the in vitro assay for inhibitors of metabolic cooperation in the Chinese hamster V79 cell line.

12-O-Tetradecanoylphorbol-13-acetate (TPA) has been previously shown to inhibit metabolic cooperation in Chinese hamster V79 cells. An in vitro assay, based on this phenomenon, has been developed to study tumor promoters. Several parameters concerning the metabolic cooperation assay using V79 Chinese hamster cells were further investigated in this report. Pretreatment of the cells with TPA in situ for different periods of time did not result in any detectable change in the inhibition of metabolic cooperation. If cells were replated after TPA treatment, a different result was obtained. There was an apparent decrease in the ability of TPA to inhibit metabolic cooperation when TPA was added back to the TPA-pretreated cultures. However, when TPA was omitted from the TPA pretreated cultures after replating, the inhibition of metabolic cooperation remained high. It was also found that pretreatment of the cells with another chemical, aldrin, exhibited the same pattern as the in situ TPA pretreatment effect on inhibition of metabolic cooperation. In order to obtain a high level of inhibition of metabolic cooperation when using aldrin in this assay, it was determined that the chemical needed to be present for more than one day. Our studies also showed that a 24 h treatment with 6-thioguanine did not kill 6-thioguanine-sensitive cells quickly, nor did it prevent them from performing metabolic cooperation. The relationship of cell density and TPA concentration was also studied. It was observed that a higher cell density required higher TPA concentration to inhibit, maximally, metabolic cooperation. A 'down regulation' type effect was noted when culture was challenged with different concentrations of TPA. These results were interpreted to be consistent with the hypothesis that inhibited gap-junctional intercellular communication is one of the components of tumor promotion.