The use of initiated cells as a test system for the detection of inhibitors of gap junctional intercellular communication.
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The effects of five non-mutagenic carcinogens--Aroclor 1260, benzoyl peroxide (BP), phenobarbital (PB), 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 1,1'-(2,2,2-trichloroethylidene)bis[4-chlorobenzene] (DDT)--on gap junctional intercellular communication (GJIC) were tested in a cell line consisting of initiated cells (3PC). Four agents suspected of tumor promotion activity--o-anisidine, clofibrate, L-ethionone and d-limonene--were also tested for their effects on GJIC. Finally sodium fluoride (NaF), whose carcinogenic property is still unclear, was tested for its effects on GJIC in the 3PC cell line. Four of the five selected tumor promoters (Aroclor 1260, BP, DDT and TPA) decreased GJIC between these initiated epidermal cells. The four non-mutagenic carcinogens with tumor-promoting activity in vivo (o-anisidine, clofibrate, L-ethionine and d-limonene) all inhibited GJIC, whereas NaF had no effect. Seven compounds (o-anisidine, Aroclor 1260, BP, DDT, L-ethionine, d-limonene and TPA) had a dose-dependent as well as time-dependent inhibitory effect on GJIC. Under the experimental conditions used, clofibrate showed only a dose-related inhibition of GJIC. PB showed no inhibitory effect on GJIC in the 3PC cell line. In order to determine the role of biotransformation in the tumor-promoting activity of PB, its effect on GJIC was also examined in the presence of an Aroclor 1254-induced rat liver homogenate (S9 mix) and in the hepatoma cell line HepG2. In the presence of rat liver homogenate PB decreased GJIC in the 3PC cell line, whereas in the HepG2 cells PB showed a time- and dose-dependent inhibitory effect. To study the potential differences in susceptibility of cells representing different stages in the process of tumor formation, the effect of the selected tumor promoters on GJIC was also investigated in primary mouse keratinocytes and in a mouse skin carcinoma-derived cell line (CA3/7). Primary keratinocytes were sometimes more (BP and clofibrate) and sometimes less sensitive (ethionine and limonene) for inhibitory effects on GJIC compared to the effects in the cell line 3PC. Except for TPA and anisidin, GJIC between the CA3/7 cells was less affected by the selected agents compared to the 3PC cell line. These results show that, during the process of tumor formation the susceptibility of cells to inhibition of GJIC by tumor promoters is variable. Overall the CA3/7 cells are less sensitive compared to 3PC cells. The susceptibility of primary keratinocytes is variable compared to 3PC cells, depending on the agent used. These results also show that GJIC is a valid parameter for testing the tumor-promoting activity of compounds. Finally, this study demonstrates that mouse keratinocyte cell lines could serve as an in vitro model for the detection of non-mutagenic carcinogens with diverse target organs in vivo. For this use the cell line consisting of initiated cells (3PC) is more sensitive than the carcinoma-derived cell line CA3/7. (+info)
Regression and progression characteristics of papillomas induced by chrysarobin in SENCAR mice.
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The present study was designed to test the effects of a free radical generating tumor promoter, chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone), on the growth and progression of papillomas generated in the skin of SENCAR mice. In the first set of experiments, papillomas were generated by initiation with 6.4 microg of 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with once-weekly applications of 52.8 microg chrysarobin for 10 weeks. The fate of individual papillomas was then monitored for a 20 week interval following cessation of promoter treatment. Five weeks after the cessation of chrysarobin treatment, the papilloma response reached a maximum of 13.2 papillomas/mouse. By the end of the 20 week interval 19% and 18% of the papillomas had regressed or coalesced respectively. A three-stage treatment protocol was also utilized to test the ability of chrysarobin to enhance the progression of pre-existing papillomas to squamous cell carcinomas (SCCs). In stage I, mice were initiated with 0.5 microg of DMBA. In stage II, mice were promoted with twice-weekly applications of 1 or 2 microg of 12-0-tetradecanoylphorbol-13-acetate (TPA) for 15 weeks. Then, in stage III, mice were treated with acetone, TPA (1 or 2 microg), chrysarobin (52.8 microg) or benzoyl peroxide (BzPo; 20 mg) for the next 45 weeks. The mean number of papillomas per mouse at plateau was very similar for all groups. The carcinoma incidence was also similar for all groups regardless of the treatment protocol used, as was the mean number of carcinomas per mouse. The ratio of papillomas that converted to SCCs in mice treated with chrysarobin during stage III was not significantly different from the acetone controls or any of the other treatment groups (P > 0.05, Kruskal-Wallis analysis). In addition, BzPo did not enhance the progression of papillomas to SCCs under the current experimental conditions. Collectively, the results indicate that papillomas promoted by chrysarobin have growth properties similar to those promoted by TPA under similar experimental conditions. Furthermore, despite its ability to generate free radical intermediates, chrysarobin does not enhance the malignant progression of pre-existing papillomas induced by TPA treatment. (+info)
Inhibition of gap junctional intercellular communication and delocalization of the cell adhesion molecule E-cadherin by tumor promoters.
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The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and benzoyl peroxide (BoP) on gap junctional intercellular communication (GJIC) and the amount and localization of E-cadherin was studied in initiated mouse epidermal cells (3PC) and in carcinoma cells (CA3/7) originating from the same cell type. In addition, the localization and phosphorylation of connexin43 was studied in both cell lines and in primary keratinocytes. GJIC inhibition by TPA and BoP was stronger in primary keratinocytes compared with both cell lines. BoP strongly decreased the amount of E-cadherin protein and the level occurring in the membranes in both cell lines, whereas TPA caused a translocation of E-cadherin from the membrane towards the cytosol, without decreasing the total amount of E-cadherin present. The effect of both tumor promoters on connexin43 phosphorylation and localization was agent as well as cell dependent. These results show for the first time that tumor promoters can decrease the quantity and membrane localization of E-cadherin in different cell types. (+info)
Bioavailability of components of resin-based materials which are applied to teeth.
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Chemical components of many materials used in dental practice can move into the local biophase, where they can have beneficial or adverse effects. The strongest indirect evidence that components of resin-based materials used in dentistry can move into the biophase are the many reports of allergic dermatitis in dental personnel. Direct measurement of component release has shown that triethylene glycol dimethacrylate (TEGDMA), hydroxyethyl methacrylate (HEMA), and, in the case of some orthodontic cements, bis-glycidyl methacrylate and benzoyl peroxide can move into an aqueous medium from a range of resin-based materials which are applied to teeth as part of oral care. In the case of resin composite restorations, HEMA and TEGDMA are available in microgram quantities via the salivary surface in the minutes and hours after clinical placement and via dentin and pulp in the hours and days after placement. Fortunately, moderate thickness of dentin protects pulp tissue against local toxicity. There are no data which suggest that systemic toxicity is a risk with any of these materials. There are some case reports of allergic responses to the monomers in patients, but the incidence of such responses appears at present to be much lower than that in dental personnel. (+info)
Benzoyl peroxide increases UVA-induced plasma membrane damage and lipid oxidation in murine leukemia L1210 cells.
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Ultraviolet A radiation induces oxidative stress and cell damage. The purpose of this investigation was to examine whether ultraviolet A-induced cell injury was amplified by the presence of a non-ultraviolet A absorbing molecule capable of generating free radicals. Benzoyl peroxide was used as a lipid soluble potential radical-generating agent. Plasma membrane permeability assessed by trypan blue uptake was used to measure cell damage in murine leukemia L1210 cells. Cells were irradiated with a pulsed Nd/YAG laser at 355 nm using 0-160 J per cm2. The ratio of the fluence-response slope in the presence of 40 microM benzoyl peroxide to that of irradiated controls was 4.3 +/- 2.6. Benzoyl peroxide alone or benzoyl peroxide added after irradiation did not cause increased trypan blue uptake. The ratio of the fluence-response slopes in the presence of 40 microM benzoyl peroxide to that of irradiated controls was 4.7 +/- 1.4 when cells were irradiated (0-43 J per cm2) with a xenon lamp, filtered to remove wavelengths <320 nm. The increased trypan blue uptake in 355 nm-irradiated cells in the presence of benzoyl peroxide was inhibited in a concentration-dependent manner by butylated hydroxytoluene, vitamin E, and trolox, a water-soluble vitamin E derivative. Lipid oxidation, assessed as thiobarbituric acid reactive substances, was significantly increased in samples irradiated with ultraviolet A in the presence of benzoyl peroxide at fluences >34 J per cm2. The increased trypan blue uptake and thiobarbituric acid reactive substances were inhibited by butylated hydroxytoluene. These results suggest that agents not absorbing ultraviolet A radiation may enhance ultraviolet A-initiated oxidative stress in cells. (+info)
Adhesion to dentin with resin using sulfinic acid initiator system.
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A study was made on the dentin bonding of MMA/PMMA resin using sulfinic acid/BPO/amine polymerization initiator, to examine: 1) the effect of ferric and copper chloride contained in phosphoric acid or citric acid dentin conditioners, and 2) the effect of the addition of acidic monomers to the resin. Tensile bond strength was significantly improved by conditioning dentin with 10% phosphoric acid containing 3% ferric chloride. The citric acid-based conditioners and the addition of the copper salt to the acidic conditioners were less effective in improving bond strength. The mechanism of the improved bond strength is discussed in terms of polymerization promoting action of ferric ion at the dentin-resin interface. For improving bond strength, the presence of a suitable acidic monomer was essential. Its effectiveness depended on the types of acids: Two phosphoric acid ester monomers were effective but methacrylic acid had little effect. (+info)