Effect of cholesterol epoxides on the inhibition of intercellular communication and on mutation induction in Chinese hamster V79 cells

Cholesterol, cholesterol-5 alpha, 6 alpha-epoxide, cholesterol-5 beta, 6 beta-epoxide and cholestane-3 beta,5 alpha,6 beta-triol were tested for their ability to induce mutations at the Na+/K+-ATPase loci of the Chinese hamster V79 cells. None of these compounds induced ouabain-resistant mutations compared to the background mutation frequency in the control cells. These compounds were further tested for their ability to inhibit intercellular communication, using the Chinese hamster V79 cell metabolic cooperation assay. The diastereomeric epoxides and cholestane-triol, but not cholesterol, were found to be inhibitors of intercellular communication in a manner similar to other known tumor promoters.     

The effects of several croton oil constituents on two types of DNA repair and cyclic nucleotide levels in mammalian cells in vitro.

The purpose of the present study was to determine the effects of two potent tumor-promoting agents on two DNA repair mechanisms and cyclic nucleotide levels in mammalian cells. Human amnion (AV3) cells were treated with low dose levels of either UV of N-acetoxy-acetylaminofluorene. Subsequently, DNA excision repair as measured by unscheduled DNA synthesis was followed in the absence or presence of non-toxic levels of either 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibenzoate (PDB), both potent tumor promoters, or phorbol, a non-promoter. Neither of these compounds inhibited DNA repair synthesis occurring in response to low doses of the carcinogenic agents. In addition, TPA did not inhibit "post-replication repair" in response to UV irradiation of growing Chinese hamster (V79-4) cells. However, both TPA and PDB did cause rapid dramatic increases in cyclic guanosine monophosphate levels in human amnion cells; phorbol had no effect. Neither of these compounds affected cyclic adenosine monophosphate levels. These results are discussed in the light of a possible mechanism of the action of tumor promoters involving "post-replication repair".     

The non-phorbol ester tumor promoter okadaic acid does not promote morphological transformation or inhibit junctional communication in hamster embryo cells

Okadaic acid is a potent non-phorbol ester mouse skin tumor promoter. Unlike the phorbol ester tumor promoters, okadaic acid is unable to promote the induction of morphological transformation in Syrian hamster embryo cell colonies. On the contrary, okadaic acid seems to counteract the effect of phorbol esters on transformation. Also unlike phorbol ester tumor promoters, okadaic acid does not inhibit intercellular communication, neither in primary hamster embryo cells, nor in the phorbol ester sensitive cell line BPNi. Furthermore, okadaic acid has no effect on the reoccurrence of communication following removal of 12-O-tetradecanoylphorbol-13-acetate.     

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

Vanadium compounds were found to promote the induction of morphological transformation of hamster embryo cells. Exposure of the cells to Na−O-vanadate, vanadin (V) oxide or vanadin (IV) oxide sulfate following pre-exposure to a low concentration of benzo[a]pyrene, potentiated the induction of transformed colonies similar to 12-O-tetradecanoylphorbol-13-acetate. Unlike this phorbol ester, vanadium compounds did not inhibit intercellular communication, or activate protein kinase C. Nor did vanadate influence the reoccurrence of communication after removal of a communication blocking phorbol ester. On the other hand, vanadate showed strong synergism with the phorbol ester on induction of transformed morphology in the phorbol ester sensitive cell line BPNi. This suggests that vanadium and tumor promoting phorbol esters mediate their effect on the induction of morphological transformation of hamster embryo cells through different mechanisms.     

Phorbol ester-associated changes in ganglioside metabolism

The effect of phorbol esters on ganglioside metabolism in contact-inhibited Chinese hamster V79 cells was examined. Three phorbol esters of varying structure and tumor-promoting activity were used. Treatment of cells with tumor-promoting phorbol esters resulted in accumulation of gangliosides and increased incorporation of [1-¹⁴C]palmitate and [9-³H]sialic acid into gangliosides. Moreover, the phorbol esters were found to increase the activity of CMP-sialic acid: lactosylceramide sialyltransferase, the enzyme catalysing the first step in ganglioside biosynthesis. The magnitude of phorbol ester effects on V79 cell ganglioside metabolism correlated with the in vivo phorbol ester tumor-promoting activity.     

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 (TG^r) and 6-thioguanine-sensitive (TG^s) V79 cells significantly increased the recovery of TG^r cells. This finding indicates an inhibition of metabolic cooperation of V79 cells by theophylline. Theophylline was tested at concentrations <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.Abbreviations TG 6, thioguanine     

Scrape-loading and dye transfer : A rapid and simple technique to study gap junctional intercellular communication

Gap junction-mediated intercellular communication has been recognized in cells from different tissues of various organisms and has been implicated in a variety of cellular functions and dysfunctions. Here we describe a new, direct and rapid technique with which to study this cellular phenomenon. It employs scrape-loading to introduce a low molecular weight (MW) fluorescent dye, Lucifer yellow CH (MW 457.2) into cells in culture and allows the monitoring of its transfer into contiguous cells. In communication-competent cells the dye transmission occurred within minutes after loading. The involvement of membrane junctions in Lucifer yellow transfer was verified by the concurrent loading of a high MW marker dye conjugate, rhodamine dextran (MW 10,000). Once introduced intracellularly the rhodamine dextran is unable to cross the relatively narrow membrane junctions. Chemicals of variable potency known to block junctional communication were tested in Chinese hamster V79 cells and other mammalian cells. The results showed effective blockage of the dye transfer at non-cytotoxic doses. This new technique can be applied to a wide variety of mammalian (including human) cells. In addition, it has the potential to be utilized as a rapid screening assay to detect chemicals that can modulate intercellular communication and to study their mechanism of action.     

Effects of pyrethroid insecticides on gap junctional intercellular communications in Balb/c3T3 cells by dye-transfer assay

The effects of fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin, p-chlorophenylisovaleric acid (CPIA, major metabolite of fenvalerate) and DDT, a liver tumor promoter, on gap junctional intercellular communication (GJIC) were examined in Balb/c3T3 cells by dye-transfer assay. Separate groups of Balb/c3T3 cells were exposed to the chemicals for 1 day. On the following day, GJIC was measured by counting the number of dye-transferring cells per injection of Lucifer Yellow under a fluorescent microscope. Fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin and DDT inhibited GJIC at noncytotoxic concentrations, while CPIA did not inhibit GJIC even, at a cytotoxic concentration. It is concluded that the examined pyrethroid insecticides, but not a metabolite, have inhibitory effects on GJIC in Balb/c3T3 cells.Abbreviations DDT 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane - DMSO dimethyl sulfoxide     

Metabolic cooperation in CHO and V79 cells following treatment with a tumor promoter

Tumor promoters are a class of chemicals which, when given to cells in vitro or to organisms that have been previously exposed to physical or chemical carcinogens, decrease the latency period for the appearance of transformed colonies or tumors. 12-O-tetradecanoyl phorbol-13-acetate (TPA), a powerful tumor promoter, has been shown to inhibit metabolic cooperation in V79 Chinese hamster cells and rat hepatocytes as well as between mouse epidermal and 3T3 cells. We report comparative studies utilizing V79 and CHO cells indicating that metabolic cooperation is inhibited by TPA in V79 cells while CHO cells show the opposite response with a slight enhancement of metabolic cooperation following promoter treatment. We speculate that these observations are the result of membrane differences between these cell lines.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Inhibition of gap-junctional intercellular communication between Chinese hamster lung fibroblasts by di(2-ethylhexyl) phthalate (DEHP) and trisodium nitrilotriacetate monohydrate (NTA)

Di(2-ethylhexyl)phthalate and trisodium nitrilotriacetate monohydrate, two apparently nongenotoxic carcinogens, were tested for effects on gap-junctional communication between Chinese hamster V79 lung fibroblasts. Both compounds inhibited gap-junctional communication in a concentration-dependent manner. The inhibiting effects of these chemicals on gap-junctional communication in vitro correlate with their tumor-promoting activity. Such results further support the hypothesis that inhibition of gap-junctional communication is an in vitro biomarker for some tumor-promoting chemicals.Abbreviations CAS Chemical Abstracts Service - DEHP di(2-ethylhexyl)phthalate - GJIC gap-junctional intercellular communication - NTA trisodium nitrilotriacetate monohydrate     

Decreased incidence of gap junctions between Chinese hamster V-79 cells upon exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate

Previous studies have shown that metabolic cooperation between Chinese hamster V-79 cells is inhibited by the tumor-promoting phorbol esters, but not by those phorbol esters inactive in tumor promotion. Metabolic cooperation is believed to be mediated by gap junctions. Therefore, in the present study we have used freeze-fracture and quantitative morphological techniques to examine the effect of the most potent tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of the inactive analog, 4α-phorbol-12,13-didecanoate (4α-PDD) on the frequency of gap-junctional contacts between V-79 cells. In both control and in 4α-PDD treated cultures the junctions were frequent, though rather small in size. In contrast, in the TPA-treated cultures, gap junctions were few, and the area of membrane occupied by gap junctions was reduced more than 20-fold from that found for controls. These results suggest that the TPA-induced inhibition of metabolic cooperation between V-79 cells is a consequence of a decrease in the number of gap junctions.     

Nucleotide changes in mitochondrial 16S rRNA gene from different mammalian cell lines

The partial nucleotide sequences of mitochondrial 16S rRNA gene were analyzed in five rodent cell lines, prior to the analysis of mutation spectrum in the gene. Total DNA was isolated from V79 and CHO-K1 cell lines from Chinese hamster and murine cell lines, Balb Y SV and PCC4 AG Cap, and the 3' terminal regions including the peptidyl transferase domain which is the target for chloramphenicol, a selective inhibitor of mitochondrial protein synthesis, were amplified by polymerase chain reaction (PCR) using two sets of primers and directly sequenced. In Chinese hamster cells, C to T transition at one site was observed in CHO-K1, and either A was deleted at the sequence of AA in all three cell lines, relative to the V79-cell sequence registered in GenBank. One G to A transition mutation in heteroplasmic state was observed in mouse PCC4 AG Cap cells which have chloramphenicol resistant phenotype, whereas there was no change in the Balb Y SV cell line, relative to the L-cell sequence. These mutation sites were located outside the peptidyl transferase domain.     

In vitro testing and the carcinogenic potential of several nitrosated indole compounds

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.Abbreviations BrdU 5-bromo-2-deoxyuridine - 4Cl 4-chloroindole - 4C6MI 4-chloro-6-methoxy-indole - DMSO dimethyl sulfoxide - EBSS Earle's balanced salt solution - EMS ethyl methanesulfonate - GJIC gap junctional intercellular communication - HBSS Hanks balanced salt solution - HGPRT hypoxanthine guaninephosphoribosyl transferase - I₃A indole-3-acetonitrile - MNNG 1-methyl-1-nitroso-3-nitroguanidine - NOC N-nitroso compounds - NQO 4-nitroquinolone-N-oxide - SCE sister chromatid exchange - 6TG 6-thioguanine - TPA 12-O-tetradecanoylphorbol-13-acetate     

Inhibition of benzo[a]pyrene-induced mutagenesis in Chinese hamster V79 cells by hemin and related compounds

The inhibitory effects of hemin and related compounds on the mutagenicity of benzo[a]pyrene (BP) were investigated in Chinese hamster V79 cells co-cultivated with X-irradiated hamster embryo cells. Mutant V79 cells were selected by their resistance to ouabain. The mutation frequency induced by BP was substantially inhibited dose dependently by hemin. The mutagenicity of BP (1 microgram/ml) on V79 cells was reduced to 6.5% by hemin, 52% by biliverdin, 73% by protoporphyrin and 85% by chlorophyllin at the highest concentration of the compounds tested (15 microM).     

Effects of phorbol myristate acetate,phorbol dibutyrate,ethanol, dimethylsulfoxide,phenol, and seven metabolities of phenol on metabolic cooperation between chinese hamster v79 lung fibroblasts

The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT–) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol diputyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generallyAbbreviations CAS Chemical Abstracts Service - DMSO dimethylsulfoxide - ETOH ethanol - HGPRT hypoxanthine-guanine phosphoribosyl transferase - HGPRT+ HGPRT-competent - HGPRT– HGPRT-te]deficient - MC metabolic cooperation - MC+ metabolic cooperation-competent - MC– metabolic cooperation-deficient - MEM minimum essential medium - PDBu phorbol dibutyrate - PMA phorbol myristate acetate - 6TG 6-thioguanine - 6TG^r 6-thioguanine-resistant - 6TG^s 6-thioguanine-sensitive - V79/MC assay Chinese hamster V79 lung fibroblast assay for metabolic cooperation     

Phorbol esters and diacylglycerols amplify bradykinin-stimulated prostaglandin synthesis in Swiss 3T3 fibroblasts. Possible independence from protein kinase C

When Swiss 3T3 fibroblasts were incubated with bradykinin, prostaglandin E2 (PGE2) synthesis was stimulated. Phorbol esters or the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG), by themselves, did not acutely stimulate PGE2 synthesis. However, when cells were preincubated with phorbol esters or OAG, bradykinin-stimulated PGE2 synthesis was potentiated markedly. When phorbol esters and OAG were added together, bradykinin-stimulated PGE2 synthesis was potentiated in an additive manner. When cells were preincubated for 48 h with phorbol esters, then bradykinin added, amplification of bradykinin-stimulated PGE2 synthesis by phorbol ester or OAG was still apparent, even though prolonged pretreatment with phorbol esters abolished protein kinase C (Ca2+/phospholipid-dependent enzyme) activity in cell-free preparations. Further, the protein kinase C antagonist, H-7, only slightly inhibited phorbol ester or OAG amplification of bradykinin-stimulated PGE2 synthesis. The possibility is raised that diacylglycerol, formed in response to many receptors, may serve as a transducer of receptor-receptor interactions. Since desensitization or inhibition of protein kinase C only partially reduced the amplification of bradykinin-stimulated PGE2 synthesis by phorbol esters or OAG, the possibility is raised that diacylglycerol mimetics may have actions in addition to activation of protein kinase C.     

Intercellular communication of transformed and non-transformed rat liver epithelial cells : Modulation by TPA

Gap-junctional intercellular communication of transformed and non-transformed rat liver epithelial cell lines was compared using a dye transfer method in the presence and absence of 12-O-tetradecanoylphorbol 13-acetate (TPA). Whereas non-transformed cells (IAR 20, non-tumorigenic in newborn rats and in nude mice) showed very high communication capacity throughout a culture period of 3 weeks, transformed cells (IAR 6-1, tumorigenic in newborn rats and in nude mice) were less able to communicate. Similar correlation between intercellular communication and expression of transformed phenotypes were also found in newly cloned epithelial cell lines, IAR 27 E and IAR 27F. When TPA was added to culture medium at 100 ng/ml, intercellular communication in all lines tested was reduced within 60 min. However, communication recovered completely from the effect within 10 h after addition of TPA. Further addition of TPA to the cultures every 24 h for 3 weeks had no effect on intercellular communication (measured 30 min after each TPA addition), suggesting that a single application of TPA made these cells refractory to further doses. A known stimulator of gap-junctional communication, db-cAMP, also increased dye transfer in IAR 20 and IAR 6-1 cells. TPA added to db-cAMP-treated cultures of IAR 20 and IAR 6-1 cells inhibited intercellular communication, suggesting that cAMP is not an antagonist of the effect of TPA on intercellular communication in these cell lines. These results are in sharp contrast to those obtained with the fibroblast cell line BALB/c 3T3, in which db-cAMP antagonized TPA effect [1] and inhibition by TPA of intercellular communication was transient only when administered during their growth phase, and was stable and continuous when TPA was applied at confluence [2], and suggest that TPA may not be an effective tumour promoter in rat liver.     

Inhibition of metabolic cooperation in Chinese hamster V79 cells by various organic solvents and simple compounds

Gap-junctional intercellular communication is a biological process implicated in the regulation of cell proliferation and differentiation. Metabolic cooperation between 6-thioguanine-sensitive and resistant Chinese hamster cells, in vitro, has been used as a means to detect chemicals which can inhibit this form of intercellular communication. To further characterize this in vitro system as a potential screening assay for potential teratogens, tumor promoters and reproductive toxicants, a series of common solvents as well as other chemicals representing eight different functional groups, i.e., alcohols with straight or side chains, glycols, ketones, esters, ethers, phenols, aldehydes, amines and amino compounds and oxygen-heterocyclic compounds, were tested for their ability to inhibit colony-formation and to inhibit metabolic cooperation. A wide range of effects were observed which suggested a structure/activity relationship between a chemical's ability to inhibit gap junction-mediated intercellular communication and the cytotoxicity of a chemical. Possible mechanisms affecting the modulation of gap junctional communication by these chemicals are discussed.Abbreviations: Hypoxanthine guanine, phosphoribosyltranferase, HG-PRT; 6-thioguanine, 6-TG.On leave from: Beijing Municipal Research Institute of Environmental Protection, Beijing, People's Republic of China     

Retinaldehyde, a potent inhibitor of gap junctional intercellular communication

Retinaldehyde and retinoic acid are derivatives of vitamin A, and retinaldehyde is the precursor for the synthesis of retinoic acid, a well-known inhibitor of gap junctional intercellular communication. In this investigation, we asked the question if retinaldehyde has similar effects on gap junctions. Gap junctional intercellular communication was measured by scrape-loading and preloading dye-transfer methods, and studies were carried out mainly on cultured liver epithelial cells. Retinaldehyde was found to be a more potent inhibitor (dye transfer reduced by 50% at 2.8 μM) than retinoic acid (dye transfer reduced by 50% at 30 μM) and glycyrrhetinic acid (dye transfer reduced by 50% at 65 μM). Both the 11-cis and all-trans forms of retinaldehyde were equally effective. Retinaldehyde inhibited dye transfer of both anionic Lucifer yellow and cationic Neurobiotin. Inhibition by retinaldehyde developed in less than two minutes at 50 μM, but unlike the reported case with retinoic acid, recovery was slower, though full. In addition to liver epithelial cells, retinaldehyde inhibited gap junctional communication in lens epithelial cells, retinal pigment epithelial cells and retinal ganglion cells.