Inhibition Of Intercellular Communication Research Articles

Inhibition of Intercellular Communication by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Dioxin-Like PCBs in Mouse Hepatoma Cells (Hepa1c1c7): Involvement of the Ah Receptor

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and the coplanar 3,3′,4,4′-tetrachlorobiphenyl (3,3′,4,4′-TCB) and 3,3′,4,4′,5,5′-hexachlorobiphenyl (3,3′,4,4′,5,5′-HCB) on intercellular communication (IC) were determined in order to investigate the in vitro tumor promoting potency of these compounds. 2,3,7,8-TCDD and the two coplanar PCBs tested caused a rapid (2 hr) and a sustained inhibition (48 hr) of IC in the mouse hepatoma cell line (Hepa1c1c7) to 20 and 50% of the unexposed control, respectively. Inhibition of IC was dose dependent with an EC50 range of about 50-100 pM for 2,3,7,8-TCDD, 2-5 nM for 3,3′,4,4′-TCB, and 10-15 nM for 3,3′,4,4′,5,5′-HCB, respectively. A comparison of the IC inhibitory effects of 2,3,7,8-TCDD and PCBs with a well-known aryl hydrocarbon receptor (AhR)-mediated response, the induction of ethoxyresorufin O-deethylase (EROD) activity, in the same cells revealed EROD induction by 2,3,7,8-TCDD, 3,3′,4,4′-TCB, and 3,3′,4,4′,5,5′-HCB with EC50 ranges of 100-200 pM, 20-70 nM, and 5-10 nM, respectively. The time course of IC inhibition was paralleled by EROD induction, although the time of onset of the response was earlier for IC (1 hr) than for EROD (2.5 hr). A role of the AhR in the inhibition of IC by 2,3,7,8-TCDD and PCBs was demonstrated by the lack of inhibition in AhR-defective Hepa1c1c7 cells. Transient inhibition of IC was observed in the mutant cells only at early time points (within 2 hr of exposure). These results and the observation that α-naphtoflavone (an AhR antagonist) greatly reduced the 2,3,7,8-TCDD-dependent sustained inhibition of IC strongly support a role of the AhR in the sustained inhibition of IC by these compounds. Furthermore, these data suggest that the mouse Hepa1c1c7 cells may be a good model in which to study in vitro tumor promoting capacity of dioxins, PCBs, and related compounds.

Inhibition of intercellular communication: An in vitro assay for detecting potential tumour promoters

The effects of non-cytotoxic concentrations of 13 chemicals on gap junctional intercellular communication (GJIC) in human keratinocytes and WB 344 cells were determined using a scrape-labelling method. The chemicals were selected according to their reported tumour-promoting effects in either the skin or liver, or their known skin irritancy potential. The in vitro assay was able to discriminate between genotoxic and non-genotoxic chemicals and, with keratinocyte cultures, ranked correctly a series of phorbol esters with regard to their reported tumour-promoting potencies in vivo. The data obtained support the suggestion that measurement of the inhibition of GJIC in vitro may provide a means to distinguish substances that have different tumour-promoting potencies.

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.

Enhancement of altered hepatic foci in rat liver and inhibition of intercellular communication in vitro by the pyrethroid insecticides fenvalerate, flucythrinate and cypermethrin.

Male Sprague-Dawley rats dosed with N-nitrosodiethylamine (NDEA) 24 h after two-thirds partial hepatectomy were treated with the pyrethroid insecticides fenvalerate, flucythrinate or cypermethrin in the diet for 20 weeks. Altered hepatic foci were analyzed by quantitative stereology from paraffin-embedded sections stained for gamma-glutamyltranspeptidase (GGT) or glutathione S-transferase P (GST-P). The present results demonstrate that the pyrethroids tested all enhance the development of NDEA-initiated, GGT-positive foci in rat liver at non-hepatotoxic doses. On the contrary, the volume fractions of GST-P-positive foci were not elevated as compared to the control group. The three pyrethroids tested all inhibited the transfer of Lucifer Yellow CH between WB-F344 rat liver epithelial cells in culture, supporting the increase of GGT-positive foci and suggesting that these substances can act as tumour promoters. The discrepancy between the results from analyses using GGT or GST-P as markers emphasizes the importance of understanding the mechanism underlying the expression of different markers for preneoplastic lesions and the importance of such effects in tumour promotion.

Inhibition of intercellular communication in rat hepatocytes by phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and gamma-hexachlorocyclohexane (lindane): modification by antioxidants and inhibitors of cyclo-oxygenase.

Several tumour promoting chemicals have been shown to inhibit intercellular communication (IC) through gap junctions in cell cultures. In the present investigation we studied the effect of the hepatic tumour promoters phenobarbital (PB), 1,1,1-trichloro-2,2-(p-chlorophenyl)ethane (DDT) and gamma-hexachlorocyclohexane (lindane) on IC in rat hepatocyte cultures. IC was evaluated by microinjection of fluorescent Lucifer Yellow CH dye and visualization of dye spread to adjacent hepatocytes. Incubation of hepatocytes with PB (2 mM), DDT (30 microM) and lindane (25 microM) decreased dye-coupling of the cells by about 30%, 42% and 35%, respectively; dye-coupling in untreated cultures was 88.1 +/- 0.7%. Inhibition of IC was reversible when the xenobiotics were removed from the medium. The antioxidant vitamin E (100 microM) prevented inhibition of dye-coupling by PB and lindane and partially that by DDT. Superoxide dismutase (100 units/microliters) counteracted the effect on dye-coupling by PB, but not that by the insecticides. Similarly, the cyclo-oxygenase inhibitors indomethacin and aspirin only reversed the effect of PB on IC, but not that of DDT or lindane. As indicated by further experiments, prevention by non-steroidal anti-inflammatory agents of PB-induced inhibition of IC is most likely not mediated by inhibition of cyclo-oxygenase. The results indicate significant differences in the action of PB, DDT and lindane on IC in hepatocyte cultures. This is suggested by the differential effects of superoxide dismutase and non-steroidal anti-inflammatory agents on the action of the three tumour promoting chemicals. Whereas superoxide radicals may be involved in the inhibition of dye-coupling by PB, radical intermediates of the insecticides may be responsible for the decrease in dye-coupling by DDT and lindane.

Regulation of the 12‐o‐tetradecanoyl‐phorbol‐13‐acetate‐induced inhibition of intercellular communication

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and protein kinase C (PKC) inhibitors on cell-cell communication were studied in a normal rat liver cell line, clone 9. Communication was observed and quantitated with microspectofluorometric and image analysis techniques following scrape-loading of the cells with lucifer yellow. Lucifer yellow migrated as far as ten cells away from the scraped edge in control populations. Two minute TPA (25-50 micrograms/ml) treatment inhibited dye movement such that the dye remained mainly in the cells at the cut edge. The TPA-induced inhibition of cell-cell communication could be partially blocked by 15 min treatment of the cell populations with the PKC inhibitors trifluoperazine (30 micrograms/ml), staurosporine (2 x 10(-8) or 2 x 10(-6) M), sangivamycin (15 or 200 microM), or a PKC inhibitor peptide (20 micrograms/ml) scraped in at the same time as lucifer yellow. Normal communication was observed in cultures treated only with PKC inhibitors. Lower concentrations of TPA (50 ng/ml-1 micrograms/ml) used for 2 min did not inhibit dye communication. Our results demonstrate the phorbol ester-induced interruption of cell-cell communication. The inhibition of PKC by inhibitors eliminates the effect of TPA on communication. Our data are consistent with a role of PKC in the control of junctional communication.

Inhibition of intercellular communication in cells in culture by polychlorinated compounds

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.

Evaluation of the V79 cell metabolic co-operation assay as a screen in vitro for developmental toxicants

Inhibition of intercellular communication is proposed to be one of several possible mechanisms of teratogenesis. 38 coded compounds were tested for their effect on intercellular communication in the V79 cell metabolic co-operation assay. Test chemicals were selected from a list of 47 agents recommended for the evaluation of assays in vitro for developmental toxicants. In addition to testing the effects of chemicals on intercellular communication, a separate cytotoxicity assay determined the concentration of each chemical that inhibited clonal expansion of V79 cells. Seven of the 29 designated teratogens were positive for inhibition of intercellular communication in the V79 assay. Additionally, four teratogens and one non-teratogen inhibited intercellular communication at only a single concentration or at cytotoxic concentrations and were scored as equivocal. Therefore, the sensitivity of the V79 assay for teratogens was 24% (seven of 29 teratogens tested positive), or 38% if the four equivocal chemicals are considered positive. None of the nine non-teratogens unequivocally inhibited intercellular communication, resulting in a specificity of 100%, which decreased to 89% when the single equivocal score was considered positive. The overall accuracy for correctly identifying teratogens and non-teratogens was 42% when equivocal chemicals were considered negative, and 50% if they were considered positive in the V79 assay. The results demonstrate that despite relatively low accuracy regarding a diverse group of developmental toxicants, chemicals that did inhibit intercellular communication under the present conditions had a high probability of being a teratogen. The low accuracy reported here contrasts with earlier reports on the assay and possible reasons for this are discussed.

Inhibition of intercellular communication in human keratinocytes by fractionated asphalt fume condensates.

Asphalt fume condensate (AFC) and chromatographically separated fractions have been shown to cause cancer in mouse skin. The levels of known carcinogenic initiators in these complex mixtures, however, are considered too low to account for their carcinogenic potency. It has been proposed that AFC may contain co-carcinogenic or tumor-promoting agents in addition to carcinogenic initiators. Modulation of gap junctional intercellular communication (GJIC) has been implicated as an important effect of tumor promoters. In this study, we examined the effect of five chromatographically generated fractions of AFC on GJIC in cultured human epidermal keratinocytes (HEK). HEK cells were exposed overnight to medium containing DMSO extracts of AFC fractions. GJIC was evaluated by dye-coupling of microinjected Lucifer Yellow CH. All AFC fractions produced a concentration-dependent inhibition of GJIC. The apparent potency of each fraction correlated with its relative polarity based on HPLC elution characteristics. Cells with reduced GJIC as a result of AFC fraction exposure were found to exclude propidium iodide, suggesting that inhibition of GJIC occurred in the absence of cell killing. However, significantly reduced culture DNA content was found following the overnight exposure to the highest concentrations of AFC fractions C, D and E.

Reversible inhibition of intercellular communication among cardiac myocytes by halogenated hydrocarbons

We examined the effect of 11 aliphatic halogenated hydrocarbons on the transfer of microinjected dye among cardiac myocytes from neonatal rats. Myocytes were suffused with increasing concentrations of halocarbon added as a 0.2% solution of dimethyl sulfoxide to M199 containing 1.8 m m Ca and 5% serum. Single cells were microinjected with the fluorescent probe Lucifer yellow (5% in 0.1 m m LiCl) and dye coupling to adjacent cells was monitored. All of the halocarbons tested exhibited a concentration-dependent inhibitory effect on intercellular communication that was reversible following washout of the compounds. Intercellular communication was blocked within 1 min of exposure to an effective concentration, and recovery of communication occured even after 2 hr of exposure. Pretreatment of cells with SKF 525-A (25 μM) did not prevent the inhibition of intercellular communication by carbon tetrachloride suggesting an absence of P-450 involvement. EC50s were calculated for each chemical using probit analysis. A log-log comparison of the EC50s and the physicochemical properties of the chemicals demonstrated a high correlation ( R 2 = 0.933) between the EC50s and the octanol/water partition coefficients of the halocarbons. This suggests that incorporation of halocarbons in the membrane may block intercellular communication though modification of the immediate environment of the gap junctions. The results are in agreement with the hypothesis that inhibition of gap junctional communication is a factor in the arrhythmogenic effects of acute halocarbon exposure.

Reversible Inhibition of Intercellular Communication among Cardiac Myocytes by Halogenated Hydrocarbons

Reversible Inhibition of Intercellular Communication among Cardiac Myocytes by Halogenated Hydrocarbons

Inhibition of Dye Transfer in Rat Liver WB Cell Culture by Polychlorinated Biphenyls

In the present investigation the scrape loading/dye transfer assay and microinjection technique are used in order to investigate inhibition of cell-cell communication induced by different polychlorinated biphenyl (PCB) congeners. In these in vitro assays, inhibition of intercellular communication is directly measured as decreased transfer of a fluorescent dye (Lucifer Yellow CH) from donor cells loaded with the dye to surrounding recipient cells. The results show that substitution in the ortho position from the carbon bridge is essential and at least one chloro substituent in ortho position is necessary for the ability to inhibit intercellular communication. The results also suggest that an increase in the number of ortho substituted chlorine atoms in the PCB molecule enhances the ability to inhibit intercellular communication. On the other hand, the total number of substitutions may not be crucial for the ability to inhibit intercellular communication. Our results suggest that PCB-induced down-regulation of intercellular communication is a result of a specific mechanism and not due to unspecific membrane perturbation.

Inhibition of intercellular communication in Chinese hamster V79 cells by fractionated asphalt fume condensates

Asphalt fume condensate is a skin carcinogen in mice, yet this complex mixture contains relatively low levels of known carcinogenic initiators. Consequently, its biological activity has been attributed to the presence of cocarcinogenic or tumor-promoting agents. One of several proposed mechanisms of tumor promotion is inhibition of intercellular communication. In an attempt to determine if asphalt fume has tumor-promoting potential inhibition of intercellular communication was measured in V79 cells exposed to fractionated asphalt fume condensate. Fume from air-blown Arabian crude asphalt was trapped and separated into five fractions by preparative-scale high-pressure liquid chromatography. The parent fume condensate and the five fractions inhibited intercellular communication in a concentration-dependent fashion, with a minimum effective concentration of 2.5 microgram/ml for the most potent fraction. Cytotoxicity assays were performed at the same time and concentrations as the metabolic cooperation assays. Cytotoxic responses paralleled the inhibition of intercellular communication.

Inhibition of intercellular communication by airborne particulate matter

To investigate the inhibition of gap junction mediated intercellular communication (IC) by extracts of airborne particulate matter (APM), V79 cells were incubated with extracts of APM and subsequently microinjected with the fluorescent dye Lucifer Yellow, after which the number of fluorescent (= communicating) cells was determined. To compare inhibitory effects on IC with mutagenicity, APM was also tested in the Salmonella microsome assay. Six different extracts were tested, two outdoor extracts representing a heavily polluted and a relatively clean sample, and four indoor extracts, taken either in livingrooms with or without wood combustion in an open fire place, or in a room with or without cigarette smoking. Non-cytotoxic doses of outdoor and indoor APM inhibited IC in V79 cells in a dose- and time-dependent manner. Mutagenicity data and IC data were correlated. These results suggest that APM has tumor promoter activity in addition to mutagenic activity.

Modulation of low-density lipoprotein-induced inhibition of intercellular communication by antioxidants and high-density lipoproteins

In order to study the capacity of antioxidants and high-density lipoproteins (HDL) to modulate the effects of low-density lipoprotein (LDL) on intercellular communication, arterial smooth muscle cells and a dye transfer method were used. LDL, in contrast to HDL, inhibited the communication between arterial smooth muscle cells from human umbilical cord and thoracic aorta in a dose-dependent manner. LDL, which can be oxidized, as detected by the lipid-peroxidation assay and gel electrophoresis, did not influence cell-cell communication in the presence of the antioxidants butylated hydroxytoluene (BHT), α-tocopherol and glutathione. The results suggest that LDL must undergo oxidative modification before it can influence intercellular communication. Like antioxidants, HDL diminished the LDL-induced inhibition of cell-cell communication. This study suggests that the modulation of gap-junctional communication by the balance of HDL and LDL in plasma may influence atherogenesis.

Inhibition of intercellular communication in smooth muscle cells of humans and rats by low density lipoprotein, cigarette smoke condensate and TPA

Inhibition by tumor promoting chemicals of intercellular communication via gap junctions may be important in carcinogenesis. In order to investigate the possible role of gap junctional intercellular communication in atherogenesis, we examined the effect of known inhibitors of intercellular communication, 12- O-tetradecanoylphorbol-13-acetate (TPA) and cigarette smoke condensate (CSC), and low density lipoproteins (LDL) and high density lipoproteins (HDL) on cellular communication in smooth muscle cells of human and rat by the microinjection-dye transfer technique. When lucifer yellow CH solution is injected into a cell, the average numbers of human and rat smooth muscle cells that become fluorescent is about 22 and 6, respectively. The tumor promoter (TPA) almost completely blocked gapjunctional communication between smooth muscle cells at 100 ng/ml after 4 h exposure. LDL and CSC were able to inhibit intercellular communication in human and rat cells in a dose-dependent manner up to 60%. LDL-pretreatment of human smooth muscle cells did not affect inhibition of intercellular communication, which suggests that this effect is mainly non-receptor mediated. HDL did not influence junctional communication. The results indicate that inhibition of intercellular communication may also contribute to the pathogenesis of atherosclerotic lesions, such as plaques.

Comparative effects of phenobarbital, DDT, and lindane on mouse hepatocyte gap junctional intercellular communication

Gap junctional intercellular communication appears to be important in the regulation of cellular homeostasis, differentiated cell functions, and growth control in adult tissues. Interruption of intercellular communication by chemical compounds has been shown to be a sublethal response to a number of tumor promoters. The mechanism by which tumor promoters inhibit intercellular communication remains unresolved. In the present study the kinetics of inhibition of mouse hepatocyte gap junctional intercellular communication (measured by dye coupling) by three well-established hepatic tumor promoters [phenobarbital, 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT), and γ-hexachlorocyclohexane (lindane)] are compared. All three compounds inhibited intercellular communication in a time- and dose-dependent manner in both freshly plated and 24-hr-old hepatocyte cultures. Following removal of the tumor promoters from the culture medium, intercellular communication was reestablished within 0.5 hr (phenobarbital) to 1.5 hr (DDT and lindane). Prolonged treatment of hepatocytes for up to 48 hr with the three promoters resulted in the continued inhibition of intercellular communication by lindane and DDT, but the development of refractoriness to phenobarbital-induced inhibition of intercellular communication. Concomitant treatment with combinations of the three promoters showed an additive effect of the compounds on inhibition of intercellular communication. Inhibition of intercellular communication by phenobarbital was prevented by addition of the cytochrome P450 enzyme inhibitor SKF-525A. SKF-525A had no effect on the inhibition of intercellular communication induced by lindane or DDT. Coincubation of the three promoters with the cAMP analog 8-bromo-cAMP prevented the promoter-induced inhibition of intercellular communication.

Effects of trichloroethylene and its metabolites on rodent hepatocyte intercellular communication

Chronic exposure to trichloroethylene (TCE) results in hepatocellular cancer in mice but not rats. The induction of hepatic tumors by TCE appears to be mediated through nongenotoxic or tumor promotion mechanisms. One cellular effect exhibited by a number of nongenotoxic carcinogens and tumor promoters is the inhibition of gap junction mediated intercellular communication. In the present study, the effects of trichloroethylene (TCE) and its metabolites. trichloracetic acid (TCA), trichloroethanol (TCEth), and chloral hydrate (CH) on gap junction mediated intercellular communication in cultured B6C3F1 mouse and F344 rat hepatocytes were assessed. TCE and TCA inhibited intercellular communication in mouse hepatocytes but not in rat hepatocytes. TCEth and CH had no effect on hepatocyte intercellular communication in either rat or mouse cells. TCE and TCA inhibited intercellular communication in both 24-hr-old and freshly plated mouse hepatocytes. Both compounds produced greater inhibition of intercellular communication in freshly plated cells when compared to 24-hr-old cultures. TCE appeared to require cytochrome P450 metabolism by the mouse hepatocytes to exhibit its inhibitory effect on dye coupling since treatment with SKF-525A prevented the inhibition of intercellular communication by TCE. The inhibitory effect of TCA on intercellular communication was unaffected by treatment with SKF-525A. While the species dependent effect of TCE on intercellular communication may be correlated with different rates and extent of metabolism of TCE by rat and mouse hepatocytes, the inhibiting effect of TCA only on mouse hepatocytes suggests that other intrinsic factors in the male mouse make this species more susceptible to the effects of TCE and TCA on gap junction mediated intercellular communication. These findings may account, in part, for the observed species difference in susceptibility to TCE induced liver carcinogenesis.

Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea

An antioxidant fraction of Chinese green tea (green tea antioxidant; GTA), containing several catechins, has been previously shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. In the present study, GTA was shown to have antioxidative activity toward hydrogen peroxide (H2O2) and the superoxide radical (O2-). GTA also prevented oxygen radical and H2O2-induced cytotoxicity and inhibition of intercellular communication in cultured B6C3F1 mouse hepatocytes and human keratinocytes (NHEK cells). GTA (0.05-50 micrograms/ml) prevented the killing of hepatocytes (measured by lactate dehydrogenase release) by paraquat (1-10 mM) and glucose oxidase (0.8-40 micrograms/ml) in a concentration-dependent fashion. GTA (50 micrograms/ml) also prevented the inhibition of hepatocyte intercellular communication by paraquat (5 mM), glucose oxidase (0.8 micrograms/ml), and phenobarbital (500 micrograms/ml). In addition, GTA (50 micrograms/ml) prevented the inhibition of intercellular communication in human keratinocytes by TPA (100 ng/ml). Cytotoxicity and inhibition of intercellular communication, two possible mechanisms by which tumor promoters may produce their promoting effects were therefore prevented by GTA. The inhibition of these two effects of pro-oxidant compounds may suggest a mechanism by which GTA inhibits tumor promotion in vivo.