Growth modulation of hepatocytes and rat liver epithelial cells (WB-F344) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Modulation of DNA synthesis by 2,3,7,8-tetrachlorodi-benzo-p-dioxin(TCDD) was studied in primary cultures of hepatocytes and inrat liver epithelial cells (WB-F344) to develop models for studieson the interactions between the activated Ah receptor and cellulargrowth control. In hepatocytes TCDD either positively or negativelymodulated EGF-stimulated DNA synthesis. In the presence of ethlnylestradiol10^–12 M TCDD moderately increased EGF-stimulated DNA synthyesis(     

Inhibition of high-density growth arrest in human squamous carcinoma cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The highly toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent carcinogen and tumor promoter, and affects cellular proliferation and differentiation both in vivo and in vitro. This report presents data showing that TCDD enhances the proliferation of two human squamous carcinoma cell lines in monolayer culture by inhibiting growth arrest at high cell density. SCC-15G and SCC-25 cells were treated with 0-100 nM TCDD in culture medium, and examined for changes in proliferation and differentiation. TCDD stimulated increases in cell number and DNA synthesis of both cell lines, and inhibited differentiation of SCC-15G cells in a dose-dependent manner. The minimum effective concentrations for increases in proliferation were 0.1 nM in SCC-15G cells and 1 nM in SCC-25G cells. The saturation density of SCC-15G cells grown in 10 nM TCDD was approximately double that of untreated controls, while the saturation density of SCC-25 cells was 50% above controls. TCDD-induced increases in proliferation were detectable only in cells exposed at subconfluent density, then assayed after control cultures had reached high-density growth arrest. There was no difference in cell number or DNA synthesis between control and TCDD-treated cultures when cells were both treated and assayed during the logarithmic phase of growth, nor in cultures treated after the cells had reached high density growth arrest. Therefore, TCDD-induced proliferation resulted from failure of treated cells to undergo normal density-dependent growth arrest rather than from direct mitogenic stimulation of the cells. Differentiation (envelope competence and keratin staining) of SCC-15G cells was inhibited by TCDD, despite the fact that in these cultures cell density was twice that of the controls. The sensitivity of SCC-15G cells to modulation of growth and differentiation by TCDD provides the basis for a model to examine the biological mechanisms of TCDD-induced alterations in proliferation and differentiation of epidermal cells.     

Inhibition of hepatocyte gap junctional intercellular communication by endosulfan, chlordane and heptachlor

The cyclodiene pesticides endosulfan, chlordane and heptachlor have been reported to be non-genotoxic rodent hepatocarcinogens. These three compounds and several metabolites of endosulfan (endosulfan sulfate, endosulfan ether and endosulfan lactone) were examined for their effects on gap junctional intercellular communication (GJIC) in primary cultured male F344 rat hepatocytes and B6C3F1 mouse hepatocytes. GJIC was evaluated by Lucifer Yellow CH dye-coupling. Endosulfan and endosulfan sulfate inhibited rat and mouse hepatocyte GJIC in a dose-responsive manner (50-200 microM) after 4 h treatment. Endosulfan ether inhibited rat hepatocyte GJIC only at 200 microM and had no effect on mouse hepatocytes. Endosulfan lactone did not affect rat or mouse hepatocyte GJIC. Chlordane and heptachlor inhibited both mouse and rat hepatocyte GJIC at concentrations of 50-200 microM. The inhibition of GJIC by the cyclodienes showed similar dose-response relationships and kinetics of onset of inhibition and reversibility for both mouse and rat hepatocytes. Concomitant treatment of the cells with inhibitors of cytochrome P450 monooxygenases (SKF-525A, piperonyl butoxide or carbon monoxide) did not alter the inhibition of GJIC by the cyclodienes, suggesting that cytochrome P450 metabolism was not involved in the inhibitory mechanism. Dibutyryl cyclic AMP (0.5 mM), however, decreased the inhibition of GJIC by the cyclodienes and may indicate that these compounds inhibit intercellular communication through a cAMP-dependent process. The inhibition of mouse and rat hepatocyte GJIC by the cyclodienes correlated with previous reports indicating that these compounds are non-genotoxic rodent liver carcinogens.     

Inhibition of gap junctional intercellular communication and malignant transformation of rat liver epithelial cells by neu oncogene

A retrovirus containing a neu oncogene was introduced into aFischer F344 rat liver epithelial cell line (WB-F344) to studythe effect of the expression of neu oncoprotein on gap junctionalintercellular communication (GJIC), the ability to form coloniesin soft agar and the ability to form tumors in rat liver bythese cells. After viral infection, five different neu-transducedepithelial clones were randomly selected for further analysis.Southern blot analysis of HindIII-digested genomic DNA hybridizedwith a neu specific probe indicated that the neu oncogene carriedby the retrovirus was integrated into different chromosomallocations in the five different neu-transduced WB cell lines.Using the fluorescence recovery after photobleaching (FRAP)assay, we found that GJIC was significantly reduced in neu-transducedWB clones, compared with control virus-infected and parentalWB cells. Western blot analysis of connexin 43 in the neu-transducedcell lines showed altered phosphorylation patterns comparedwith the normal WB-rat liver cell line. Confocal image analysisof the neu-transduced cells showed that the connexin 43 protein,as detected by fluorescent immunostaining, was localized inthe cell nucleus. The neu-transduced WB cell lines also acquiredthe ability to grow in soft agar. Furthermore, cells from threeof the five neu-transduced cell lines, when injected into theliver of Fischer F344 rats through the portal vein, were highlytumorigenic (multiple focal hepatic tumors developed within2 weeks). Cells derived from the tumor were shown to be G-418resistant, demonstrating that the tumor was derived from theinjected WB-neu cells. The results of this study demonstratethat the expression of the neu oncogene is able to block GJICand to induce tumorigenicity in the rat liver WB-F344 cell line.     

ACCELERATED PAPER: Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity

Rodent liver tumor formation can be promoted by certain barbituratesand this may involve their ability to inhibit hepatocyte gapjunctional intercellular communication (GJIC). In order to addressthe mechanisms and specificity of action of barbiturates onhepatocyte gap junctions, we have compared the effects of livertumor-promoting barbiturates (phenobarbital, sodium barbitaland amobar-bital: PB, SB and AB, respectively) and a non-livertumor-promoting barbiturate (barbituric acid: BA) on primarycultured rat hepatocyte GJIC and connexin32 (Cx32) expressionafter short (1–24 h) and long (2–14 days) treatmentGJIC was evaluated by fluorescent dye microin-jection (dye-coupling);Cx32 expression was monitored by Northern blot, Western blotand immunohistochemistry. Both parameters were maintained athigh levels over 14 days by coculture of the cells with WB-F344rat liver epithelial cells in the presence of dexamethasone.Treatment with PB (2 mM) for 1 h sharply reduced dye-couplingfrom     

Altered gap junctional intercellular communication in neoplastic rat esophageal epithelial cells

Gap junctional intercellular communication (GJIC) is reduced in many neoplastic cells, but few data exist for esophageal neoplasms. GJIC was examined by fluorescent dye microinjection in two nontumorigenic and two highly tumorigenic rat esophageal epithelial cell lines. All lines expressed high levels of dye coupling in homologous cell culture. In cocultures of nontumorigenic and tumorigenic cells, however, only one of six cell combinations displayed significant heterologous GJIC. Northern, Western, and immunohistochemical analyses indicated that all four cell lines expressed comparable levels of connexin43 (Cx43), but not connexin32 or connexin26, and formed Cx43-containing gap junction plaques at cell-cell interfaces. Immunostaining of rat esophageal frozen sections demonstrated that esophageal epithelial cells expressed Cx43 in vivo. In normal epithelium, the highest expression was seen in the basal cells and little suprabasal staining was evident. In preneoplastic and neoplastic lesions of the esophageal epithelium which were induced by treating rats with N-nitrosomethylbenzylamine, Cx43 staining of the basal layer was also seen but appeared to be more diffuse compared to normal epithelium. In addition, suprabasal Cx43 staining was apparent in dysplastic and papillomatous lesions. These results indicate that Cx43 is expressed in normal and neoplastic rat esophageal cells and that the cells exhibit extensive homologous GJIC, but little heterologous GJIC. This lack of heterologous GJIC may be due to differences in cell adhesion proteins or other factors.      

Liver tumor-promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in TCDD-sensitive and TCDD-resistant rat strains

Risk assessment of dioxins is currently based on induction of liver tumors in rats. The toxicity of dioxins is characterized by large sensitivity differences among animal species and even strains of the same species, which complicates the risk assessment. The significance of these differences in dioxin-induced carcinogenicity is not known. We therefore studied the liver tumor-promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the sensitive Long-Evans (L-E) and the resistant Han/Wistar (H/W) rats differing >1000-fold in their sensitivity to the acute lethaity of TCDD. Female rats were partially hepatectomized, initiated with nitrosodiethylamine, and treated with TCDD for 20 weeks. Altered hepatic foci (AHF) were stereologically quantitated using glutathione S-transferase P as a marker. AHF were significantly (P < 0.001) and dose dependently increased in L-E rats at 10 and 100 ng/kg/day, but in H/W rats only at 1000 ng/kg/day and above, indicating a remarkable (approximately 100-fold) sensitivity difference between L-E and H/W rats. The same sensitivity difference but 10-fold less foci were observed between nonhepatectomized/noninitiated L-E and H/W rats. Induction of AHF was related to hepatotoxicity but not to cytochrome P4501A1 activity in the liver. Liver TCDD concentrations were similar in both strains. H/W rats are exceptionally resistant to induction of AHF by TCDD, and the resistance is associated with an altered transactivation domain of the aryl hydrocarbon receptor. Genetic differences may account for significant interindividual/intraspecies sensitivity differences in dioxin-induced carcinogenesis. Understanding the role of transactivation domain of the aryl hydrocarbon receptor in carcinogenesis is therefore likely to improve dioxin risk assessment.     

Inhibitory effects of endosulfan on gap junctional intercellular communication in WB-F344 rat liver cells and primary rat hepatocytes.

The chlorinated cyclodiene insecticide endosulfan is a potent inhibitor of gap junctional intercellular communication (GJIC) in vitro, a property shared by many tumour promoters and suggested to indicate an intrinsic tumour-promoting potential. However, endosulfan did not act as a tumour promoter in an altered hepatic foci assay in the rat in vivo, and ambiguous results regarding the carcinogenic potential of endosulfan have emerged from long-term studies in rodents. In the present study the GJIC-inhibitory potentials of the two isomers of endosulfan were investigated in WB-F344 rat liver epithelial cells and primary rat hepatocytes. The results show that both isomers are inhibitors of GJIC. However, beta-endosulfan (ENDO beta) is a more potent inhibitor of GJIC in primary rat hepatocytes than alpha-endosulfan (ENDO alpha), whereas the two isomers were equally potent as inhibitors of GJIC in WB-F344 rat liver cells. In primary rat hepatocytes membrane-permeant dibutyryl cyclic AMP (dB-cAMP) counteracts the inhibitory effect of ENDO beta without affecting the effect of ENDO alpha. However, in WB-F344 rat liver cells dB-cAMP failed to prevent the inhibitory effects of either ENDO alpha or ENDO beta. In addition, studies in WB-F344 rat liver cells show that ENDO alpha beta does not decrease the intracellular cAMP concentration. Thus, it is unlikely that ENDO alpha beta or its isomers and metabolites inhibit GJIC by lowering the intracellular cAMP concentration. Furthermore, comparison of the effective doses and recovery times imply that GJIC in WB-F344 rat liver cells is more sensitive to treatment by ENDO alpha beta, its isomers and metabolites than GJIC in primary rat hepatocytes. Thus, the present results demonstrate significant differences between primary rat hepatocytes and WB-F344 rat liver cells in the response of their GJIC to endosulfan.     

Inhibition of apoptosis by pentachlorophenol in v-myc-transfected rat liver epithelial cells: relation to down-regulation of gap junctional intercellular communication

Pentachlorophenol (PCP), a promoter of murine hepatocarcinogenesis, inhibits gap junctional intercellular communication (GJIC) in rat liver epithelial cells in vitro. To test the hypothesis that both inhibition of GJIC and apoptosis contribute to tumor promotion, we investigated the effect of PCP on both GJIC and serum deprivation-induced apoptosis in v-myc-transfected rat liver epithelial cells. The results showed that PCP inhibited apoptosis, as measured by the TUNEL assay and DNA ladder formation. Inhibition of apoptosis was associated with a decrease in GJIC. The study demonstrated that PCP has a potential for inhibiting apoptosis and GJIC, supporting the hypothesis.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) does not inhibit intercellular communication in Chinese hamster V79 cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmentalpollutant which has been shown to be both a potent teratogenand carcinogen and also to have tumorpromoting activity. Wehave compared TCDD with the prototype phorbol ester tumor promoter,12-O-tetradecanoylphor-bol-13-acetate (TPA) using the metaboliccooperation assay as a measure of tumor promotional competence.Unlike TPA, TCDD was found to be ineffective in inhibiting metaboliccooperation at concentrations which elicit many of TCDD's biologicalresponses. These results suggest that TPA and TCDD elicit tumorpromotion by different pathways. We discuss these results inthe context of cell-type-specific and TCDD receptor-mediatedbiological responses to tumor promoters.     

Induction of hepatic DNA single strand breaks in rats by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces lipid peroxidation in hepatic and extrahepatic tissues. DNA single strand breaks as well as other forms of DNA damage are believed to occur in conjunction with lipid peroxidation. We have therefore examined the effect of TCDD on hepatic DNA single strand breaks. Ten days after the administration of 100 micrograms TCDD/kg to female rats, a 7.5-fold increase in the DNA elution constant (single strand breaks) occurred. Similar changes were observed in the content of thiobarbituric acid reactive substances (TBARS) in the nuclei as well as the NADPH-dependent production of TBARS. The accumulation of TBARS appeared to precede the accumulation of DNA single strand breaks. The tumor promoting effects of TCDD may be associated with the enhanced formation of DNA single strand breaks.     

Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells

Two flavones, apigenin and tangeretin, were studied for theirability to modulate gap junctional intercellular communication(GJIC) in the rat liver epithelial cell line REL. Their cytotoxicitywas first determined by cell density and neutral red uptakeassays: neither apigenin nor tangeretin are cytotoxic at 10and 25 µM, the concentrations used in our experiments.We then studied GJIC using the dye transfer assay and we observedthat both apigenin and tangeretin enhance it, the maximum stimulation(x 1.7–1.8) being achieved at 25 µM for 24 h. Whenthe dye transfer was enhanced, the amount of connexin 43 increased,which was demonstrated by Western blot and immunofluorescenceanalysis. For apigenin only, Northern blot analysis showed anaccumulation of connexin 43 mRNA. In addition, the incubationof REL cells with the two compounds, for 1 or 24 h, preventedthe inhibition of dye transfer by 12-O-tetradecanoylphorbol-13-acetate(1or 10 ng/ml). The enhancement of GJIC by apigenin could be oneof the major mechanisms responsible for apigenin's anti-tumourpromoting action in vivo. As for tangeretin, its capacity toenhance GJIC completes its potential protective properties towardsthe post-initiation process.     

Suppressed gap junctional intercellular communication in carcinogenesis of endometrium.

To examine whether and at which stage of endometrial carcinogenesis decreased connexin expression occurs, we investigated changes in the expression of the gap junction proteins, connexin 26 (Cx26), Cx32 and Cx43, in human endometrial hyperplasia and cancer samples. Forty-eight endometrial tissue samples (15 endometrial hyperplasias and 33 endometrial cancers) were subjected to immunofluorescence and RT-PCR analysis. In endometrial hyperplasia, Cx26 was aberrantly expressed in all samples as revealed immunohistochemically. There was weak or negative expression in 12 samples (80.0%) and diffuse expression in cytoplasm in 3 samples (20.0%). Cx32 expression in those samples was similar to that of Cx26; there was weak or negative expression in 11 samples (73.3%) and diffuse expression in 4 samples (26.7%). In endometrial cancer, Cx26 was expressed weakly or negatively in 25 samples (75.8%), diffusely in 6 samples (18.2%) and normally in 2 samples (6.1%), while Cx32 was expressed weakly or negatively in 26 samples (78.8%), diffusely in 5 samples (15.2%) and normally in 2 samples (6.1%). It was confirmed that weak staining of Cx26 and Cx32 was due to poor expression of their mRNA. All samples showed weak Cx43 protein expression as revealed by immunohistochemical analysis. In the majority of samples, concomitant expression levels of Cx26 and Cx32 protein were observed, confirming our long-term hypothesis that Cx26 and Cx32 are both abnormally regulated in a coordinated fashion in the endometrium. Our results indicate that during endometrial carcinogenesis, loss of gap junctional intercellular communication (GJIC) may occur due to the suppressed expression and the aberrant localization of connexin at relatively early stages.     

Decreased gap junctional intercellular communication in hexachlorobenzene-induced gender-specific hepatic tumor formation in the rat

Hexachlorobenzene (HCB), an epigenetic carcinogen, HCB induces the formation of liver tumors in female rats, whereas only a small percentage of males are responsive. Intercellular communication via gap junctions is decreased in carcinogenesis. Gap junctions are composed of proteins termed connexins (Cxs). The objectives of this study were (i) to determine if HCB-induced tumor development is associated with a loss of gap junctional communication; (ii) to assess if HCB causes a gender-specific decrease in the expression of Cx32 and Cx26; and (iii) to establish if these effects result from gender differences in the constitutive expression of these Cxs. Rats were given HCB by gavage for five consecutive days. In the first experiment, control and HCB-treated female rats were sampled on day 100. Intercellular communication was significantly decreased in HCB-treated females compared to controls. To investigate if changes in Cx levels occur prior to day 100, experiments done using male and female rats sampled on day 50. Hepatic mRNA levels for Cx26 and Cx32 were significantly lower only in HCB-treated females as compared to controls. Cx26 mRNA levels were 3-fold higher and Cx32 mRNA levels were 8-fold lower in females compared with males. In a third experiment, ovariectomy abolished any differences between male and female controls for both Cxs, while estradiol had a partial role in the regulation of Cx32. This suggests that the sexual dimorphism in hepatic Cx levels is determined by the ovarian hormones. However, the HCB-induced decrease in Cx32 and Cx26 mRNA levels was maintained in ovariectomized rats, suggesting that the HCB effects are not mediated via an ovary-dependent pathway. Overall results show that HCB exposure induces gender-specific long-term alterations in intercellular gap junctional communication in female rat liver. This effect appears to be a critical mechanism of HCB-induced liver carcinogenesis and tumor promotion.     

Changes in gap junctional intercellular communication in mouse skin carcinogenesis

Gap junction intercellular communication (GJIC) has been measured in cell lines that represent different stages of chemically induced mouse skin carcinogenesis. No significant difference in GJIC, as measured by dye spread, was found in cultures of normal keratinocyte, papilloma or squamous carcinoma cell lines. There was no correlation, in this system, between the presence of a mutant Ha-ras gene and down- regulation of communication. There was, however, a marked decrease in GJIC (80-90%) on progression from squamous to spindle carcinoma cells. Measurement of GJIC in somatic cell hybrids shows that the genetic defect responsible for this down-regulation is recessive and is common to two independently isolated spindle cell lines. No abnormalities were found in the spindle cells in expression of connexin 43, a cell component involved in gap junction formation and permeability. However, expression of E-cadherin, a cell-cell adhesion molecule implicated in the process of gap junction formation, was missing in the spindle carcinoma cells. Introduction of an E-cadherin cDNA into the spindle cells partially restored junctional communication without causing any noticeable alterations in cell morphology. During the study a non- tumourigenic keratinocyte line, a sub-clone of a normal keratinocyte line, was also found to have a low level of GJIC. However, the defect in this line was shown, by genetic complementation in somatic cell hybrids, to be different from that in the spindle carcinoma cell lines. Consistent with these data, analysis by immunofluorescence shows an abnormal distribution of connexin 43 in these cells.      

Modulation of gap junctional intercellular communication by EGF in human kidney epithelial cells

Modulation of gap junctional intercellular communication (GJIC)was studied in a multistep model of human renal epithelial carcinogenesis.We report that the majority of primary human kidney epithelialcells (NHKE) grown from fetal kidney explants did not communicatethrough gap junctions. Communication could, however, be observedwithin a subpopulation of the cells. Ni(II)-immortalized cells(IHKE) showed GJIC at a level of 10–20 communicating cells,but with heterogeneous regions on the dish, with regard to bothcommunication and distribution of connexin43. The heterogeneitywas less pronounced in a ras-transfected tumourigenic cell line(THKE), which also showed communication of     

Effect of DDT on hepatic gap junctional intercellular communication in rats

The effects of in vivo exposure to DDT on hepatic gap junctionalintercellular communication (GJIC) and connexin gene/proteinexpression in Sprague-Dawley rats were examined by in vivo/in vitro dye-transfer assay, immunohistochemical staining, andby Western and Northern blot analyses. In the dose-responsestudy, three dose levels of DDT (5, 25 and 50 mg/kg/day) wereadministered orally to rats once a day for 2 weeks. The averagesize of the dye spread after injection of Lucifer Yellow andthe area of Cx32 spots per hepatocyte decreased in a dose-dependentmanner, but there was no effect on the number of Cx32 spotsper hepatocyte. In the time-course study, DDT (50 mg/kg/day)was administered orally once a day for up to 6 weeks. HepaticGJIC decreased at week 1 but recovered at week 6. The averagearea of Cx32 spots per hepatocyte gradually decreased at weeks2 and 4, and remained at the same level at week 6, correlatingwith the decreased Cx32 protein level in plasma membranes. Theaverage area of Cx26 spots per hepatocyte in the peripheralzones clearly decreased at week 1, but quickly recovered atweek 2 and increased at week 6; however, no clear change ofthe Cx26 protein level in plasma membranes was observed. Nochanges of Cx32 and Cx26 mRNA levels were observed in DDT groups.These results suggest that DDT, a liver tumor-promoting agent,inhibits hepatic GJIC in vivo dose-dependently in rats and thataberrant Cx32 and Cx26 protein expression and/or localizationmay be responsible for this effect.     

Inhibitory effect of pentachlorophenol on gap junctional intercellular communication in rat liver epithelial cells in vitro

To understand the initiating/promoting actions of pentachlorophenol (PCP), a non-mutagenic hepatocarcinogen, and its metabolite, tetrachlorohydroquinone (TCHQ), we investigated the effects of each chemical on gap junctional intercellular communication (GJIC) in rat liver epithelial cells (WB cells) by the scrape-loading and dye transfer method. After treatment with PCP, the GJIC was initially inhibited at 4 h but was restored in 6–8 h, followed by a second phase of inhibition between 16 and 24 h. Both the first and second inhibitions were concentration-dependent and were restored by 2–4 h after removal of PCP. The phosphorylation state of connexin 43 (CX43) and its localization on the plasma membrane were unchanged up to 24 h after treatment; however, this was accompanied by a decrease in the CX43 protein level. No inhibitory effect was apparent on the GJIC of cells treated with TCHQ. These results suggest that PCP may play a critical role of promoting activity via non-mutagenic mechanisms.     

Indolo[3,2-b]carbazole inhibits gap junctional intercellular communication in rat primary hepatocytes and acts as a potential tumor promoter

Indole-3-carbinol (I3C) is a naturally occurring substance that shows anti-carcinogenic properties in animal models. Besides its clear anti-carcinogenic effects, some studies indicate that I3C may sometimes act as a tumor promoter. Indolo[3,2-b]carbazole (ICZ), which is formed in the acidic environment of the stomach after intake of I3C, has a similar structure to, and shares biological effects with, the well-known tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Therefore, we hypothesized that ICZ could be responsible for the potential tumor-promoting activity of I3C. The aim of the present study was to investigate the effect of ICZ on gap junctional intercellular communication (GJIC) in primary cultured rat hepatocytes co-cultured with the rat liver epithelial cell line WB-F344. Indolo[3,2-b]carbazole inhibited GJIC in the rat hepatocytes in a dose- and time-dependent manner. Significant inhibition was observed after 8 and 12 h of treatment with 1 and 0.1 micro M ICZ, respectively. Maximum GJIC inhibition (cell-cell communication only 5% of control values) was observed after 24-48 h of ICZ treatment. Continued exposure to 1 micro M ICZ suppressed GJIC until approximately 120 h. Both ICZ and TCDD treatment reduced the Cx32 mRNA level as well as the plasma membrane Cx32 staining. Indolo[3,2-b]carbazole increased the Cyp1a1, Cyp1a2 and Cyp1b1 mRNA levels concurrently with an increase in 7-ethoxyresorufin O-deethylase (EROD) activities. Maximum EROD activity and Cyp1a1 mRNA levels were observed after approximately 12 h, whereas Cyp1a2 and Cyp1b1 mRNA levels peaked after 48 h. This study shows that ICZ may possess tumor promoter activity down-regulating GJIC by mechanisms, which seem to include activation of the Ah receptor and/or Cyp1 activity. Further studies are needed in order to clarify the anticarcinogenic/carcinogenic effects of I3C and ICZ before high doses of I3C may be recommended as a dietary supplement.