Rat Liver Epithelial Cell Line Research Articles

Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing.

Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework.

Chlorination and ozonation reduce microcystin content and tumour promoting activity of complex cyanobacterial extract

<p>Despite intensive research and management efforts in the past decades, cyanobacterial blooms and their toxins, such as microcystins (MC), continue to represent a major ecological and health problem in fresh waters throughout the world. Our objective was to compare the efficacy of two commonly used drinking water treatment technologies, chlorination and ozonation, in removing MCs and in reducing tumour promotion-related effects of cyanobacteria<em>,</em> such as inhibition of gap junctional intercellular communication (GJIC) and activation of mitogen activated protein kinases (MAPKs) in a pluripotent rat liver epithelial cell line (WB-F344). This combined chemical and bioassay approach demonstrated that ozone effectively removed all MCs from an extract of globally important bloom-forming cyanobacterium, <em>Microcystis</em> sp. Ozone also significantly reduced the overall tumour promotional potency of the cyanobacterial extract, as indicated by a substantial reduction in the ability of the extract to inhibit GJIC and activate extracellular receptor kinase 1/2 (ERK1/2). Although comparable reduction of total organic carbon was achieved by ozone and chlorine treatment, chlorination was much less effective in removing MCs and reducing the effects on GJIC. Chlorination had a biphasic effect with reduction of extract-induced activation of ERK1/2 at lower doses of chlorine; at high doses of chlorine, however, the by-products of chlorination actually induced the activation of ERK1/2. The extracts induced p38 activation and chlorination was not effective in reversing this effect, while ozone did reverse this effect, but not as much as the activation of ERK1/2. Thus, ozone was effective in reducing the toxicity of cyanobacterial extracts while chlorination was not only lacking efficacy but at high doses further produced by-products that were equally as toxic as the untreated samples. Our study points to advances of ozonation approach and brings evidences supporting the need of effect-based monitoring implementation.</p>

Cadmium-induced malignant transformation of rat liver cells: Potential key role and regulatory mechanism of altered apolipoprotein E expression in enhanced invasiveness

Cadmium is a transition metal that is classified as human carcinogen by the International Agency for Research on Cancer (IARC) with multiple target sites. Many studies using various model systems provide evidence of cadmium-induced malignancy formation in vivo or malignant cell transformation in vitro. Nonetheless, further studies are needed to completely understand the mechanisms of cadmium carcinogenicity. Our prior studies have utilized a rat liver epithelial cell line (TRL 1215) as a model for cadmium-induced malignant transformation. In the present study, we focused on the molecular mechanisms of this malignant transformation, especially with regard to hyper-invasiveness stimulated by cadmium transformation. By performing a series of biochemical analyses on cadmium transformed cells, it was determined that cadmium had significantly down-regulated the expression of apolipoprotein E (ApoE). ApoE was recently established as a suppressor of cell invasion. A key factor in the suppression of ApoE by cadmium appeared to be that the metal evoked a 5-aza-2'-deoxycytidine-sensitive hypermethylation of the regulatory region of ApoE, coupled with interference of the action of liver X receptor α (LXRα), a transcriptional regulator for ApoE. Furthermore, the expression of LXRα itself was suppressed by cadmium-mediated epigenetic modification. Re-expression of ApoE clearly abrogated the cell invasion stimulated by cadmium-induced malignant transformation. Together, the current results suggest that the cadmium-mediated enhanced cell invasion is linked to down-regulation of ApoE during malignant transformation these liver cells.

Curcumin and Turmeric Modulate the Tumor-Promoting Effects of Iron In Vitro

ABSTRACTFree or loosely chelated iron has tumor-promoting properties in vitro. Curcumin, a polyphenol derived from the food spice turmeric (Curcuma longa), is a potent antioxidant that binds iron. The primary aim of this study was to investigate whether curcuminoids prevent tumor-promoting effects of iron in T51B cells, a non-neoplastic rat liver epithelial cell line. Purified curcuminoids (curcumin) or a standardized turmeric extract similarly reduced oxidative stress and cytotoxicity associated with iron overload (IC50 values near 10 μM, P < 0.05). Inhibition of iron-induced tumor promotion (seen upon treatment with 200 μM ferric ammonium citrate ± curcumin/turmeric for 16 wk in culture; subsequently assayed by soft agar colony formation) was nearly complete at 20 μM of total curcuminoids (P < 0.05), a concentration predicted to only partially chelate the added iron. Surprisingly, lower curcumin concentrations (10 μM) increased tumor promotion (P < 0.01). Curcuminoids delivered as a standardized turmeric extract were taken up better by cells, had a longer half-life, and appeared more effective in blocking tumor promotion (P < 0.01), suggesting enhanced curcuminoid delivery to cells in culture. The primary finding that curcuminoids can inhibit tumor promotion caused by iron in T51B cells is tempered by evidence for an underlying increase in neoplastic transformation at lower concentrations.

Functional consequences of co-expressing connexin40 or connexin45 with connexin43 on intercellular electrical coupling

The functional characteristics of the co-expression of connexin43, connexin40, and connexin45 proteins in human myocardium are thought to play an important role in governing normal propagation of the cardiac electrical impulse and in generating the myocardial substrate for some arrhythmias and conduction disturbances.A rat liver epithelial cell line, that endogenously expresses connexin43, was used to induce also expression of connexin40 or connexin45 after stable transfection using an inducible ecdysone system. Electrical coupling was estimated from measurement of the input resistance of transfected cells using an intracellular microelectrode to inject current and record changes to membrane potential.However, varied expression of the transfected connexin40 or connexin45 did not change electrical coupling, although connexin43/40 co-expression led to better coupling than connexin43/45 co-expression. Quantification of endogenous connexin43 expression, at both mRNA and protein levels, showed that it was altered in a manner dependent on the transfected connexin isotype.The data using rat liver epithelial cells indicate an increased electrical coupling upon expression of connexin40 and connexin43 but decreased coupling with connexin45 and connexin43 co-expression.

Gap Junctional Intercellular Communication Increases Cytotoxicity and Reduces Resistance to Hydroxyurea

Background: Gap junctions enable small molecules to diffuse between adjacent cells and have been associated with greater cytotoxicity of radiation and anti-cancer drugs. We investigated whether this gap junctional intercellular communication (GJIC) affected the cytotoxicity of the classic ribonucleotide reductase (RR) inhibitor and anti-cancer agent, hydroxyurea (HU). Materials and Methods: We used GJIC-proficient and deficient, connexin 43-expressing WB rat liver epithelial cell lines. We compared HU toxicity by crystal violet assay, effects of the drug on deoxynucleotide pools by HPLC, and ability of GJIC to increase toxicity of HU-resistant cells through a bystander effect in co-culture experiments. Results: GJIC-proficient cells were three- to five-fold more sensitive (IC50 0.1 mM) to HU than GJIC-deficient derivatives (IC50 0.3 - 0.5 mM). This sensitivity depended upon GJIC because treatment of GJIC-proficient cells with the GJIC blocker oleamide decreased HU toxicity by approximately 60% - 80% and restoration of GJIC in GJIC-deficient cells by stable transduction of connexin 32-encoding Gjb1 increased HU toxicity (IC500.1 mM). The effects were not due to connexin expression per se or its localization since all cell lines expressed comparable quantities of connexin 43 that was localized to the plasma membrane. Also HU sensitivity was not related to differential effects on nucleotide metabolism in the cells. Thymidine triphosphate levels increased and deoxyadenosine triphosphate levels decreased similarly (15% - 20%) in GJIC-proficient and deficient cells over 24 h of HU treatment. More importantly, when HU-resistant cells were co-cultured with sensitive cells, the resistant cells were killed only when GJIC was present. Conclusion: The data suggest that GJIC enhances cytotoxicity and decreases resistance to HU. These results may be important clinically if GJIC can be enhanced in drug-resistant cells.

2-Methoxyestradiol binding of GPR30 down-regulates angiotensin AT1 receptor

Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line. Cell membranes were isolated and used to determine 2ME2 specific binding. Cell membranes exposed to [3H]2ME2 showed specific saturable binding, which was found to be pertussis toxin (PTx) sensitive. Under similar conditions, G-protein coupled receptor 30 (GPR30) agonist (G1) and antagonist (G15) inhibited 2ME2 specific binding. In these cells GPR30 was found localized to endoplasmic reticulum (ER) membranes. In intact cells, G1 down-regulated angiotensin AT1 receptor expression and this effect was reversed by G15. Furthermore, 2ME2 mediated activation of epidermal growth factor receptor (EGFR) followed by ERK1/2 phosphorylation, an essential signaling step in angiotensin AT1 receptor down-regulation, was abrogated by G15, suggesting that this signal is GPR30 dependent. Additionally, EGF was found to independently down-regulate angiotensin AT1 receptor in an ERK1/2-dependent manner. In summary, our results demonstrate for the first time that 2ME2 down-regulation of angiotensin AT1 receptor is dependent on ER membrane-associated GRP30. Moreover, this effect is facilitated by GPR30 dependent transactivation of EGFR and ERK1/2 phosphorylation. This study provides further understanding of the physiological significance of 2ME2 and its role in modulating angiotensin AT1 receptor expression.

Distinct gene expression signatures during development of distant metastasis

Using cDNA microarrays, we have conducted a systematic characterization of global gene expression in v-raf or v-raf/v-myc transformed rat liver epithelial (RLE) cell lines exhibiting both non-metastatic and metastatic phenotypes. Seven transformed cell lines were compared with the non-transformed parental RLE cell. The hierarchical clustering analysis of gene expression profiles revealed two groups reflecting the in vivo metastatic potential of the cells. Surprisingly, one non-metastatic cell line T1 was co-clustered with metastatic cell lines, suggesting that T1 underwent significant genetic changes. The T1 cell line was further compared against all the metastatic cell lines in order to reveal the critical genes required for metastatic conversion but not expressed in the T1. These data demonstrated that expression of genes involved in apoptosis and immune cell homing were altered in all metastatic cell lines. Survival of both intravasated cells in circulation systems and extravasated cells in a new tissue environment might be critical for the final step in the metastatic process. Our study provides gene expression signatures consistent with two critical events in the metastatic process, namely, the acquisition of early homing capacity and increased survival potential of the tumor cells.

Tumor promoting effects of cyanobacterial extracts are potentiated by anthropogenic contaminants – Evidence from in vitro study

Inhibition of gap junctional intercellular communication (GJIC) is affiliated with tumor promotion process and it has been employed as an in vitro biomarker for evaluation of tumor promoting effects of chemicals. In the present study we investigated combined effects of anthropogenic environmental contaminants 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) and fluoranthene, cyanotoxins microcystin-LR and cylindrospermopsin, and extracts of laboratory cultures of cyanobacteria Aphanizomenon gracile and Cylindrospermopsis raciborskii, on GJIC in the rat liver epithelial cell line WB-F344. Binary mixtures of PCB 153 with fluoranthene and the mixtures of the two cyanobacterial strains elicited simple additive effects on GJIC after 30min exposure, whereas microcystin-LR and cylindrospermopsin neither inhibited GJIC nor altered effects of PCB 153 or fluoranthene. However, synergistic effects were observed in the cells exposed to binary mixtures of anthropogenic contaminants (PCB 153 or fluoranthene) and cyanobacterial extracts. The synergistic effects were especially pronounced after prolonged (6–24h) co-exposure to fluoranthene and A. gracile extract, when mixture caused nearly complete GJIC inhibition, while none of the individual components caused any downregulation of GJIC at the same concentration and exposure time. The effects of cyanobacterial extracts were independent of microcystin-LR or cylindrospermopsin, which were not detected in cyanobacterial biomass. It provides further evidence on the presence of unknown tumor promoting metabolites in cyanobacteria. Clear potentiation of the GJIC inhibition observed in the mixtures of two anthropogenic contaminants and cyanobacteria highlight the importance of combined toxic effects of chemicals in complex environmental mixtures.

Toxic Effects of Methylated Benzo[a]pyrenes in Rat Liver Stem-Like Cells

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.

Modulation of gap-junctional intercellular communication by a series of cyanobacterial samples from nature and laboratory cultures

Cyanobacterial extracts have been recently shown to alter two in vitro biomarkers of tumor promotion, namely to cause inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (Blaha et al., 2010a). In the present study, we investigated GJIC-inhibitory potencies of 10 laboratory strains representing common water bloom-forming cyanobacteria (Anabaena, Aphanizomenon, Cylindrospermopsis, Microcystis and Planktothrix) and six natural water bloom samples (dominated by Aphanizomenon sp. or Microcystis). The most pronounced inhibitions of GJIC in a model rat liver epithelial cell line WB-F344 were caused by methanolic extracts of Anabaena flos-aquae UTEX 1444, Aphanizomenon flos-aquae SAG 31.87, Aphanizomenon gracile RCX 06, Microcystis aeruginosa PCC 7806, Cylindrospermopsis raciborskii SAG 1.97, Planktothrix agardhii CCALA 159 and SAG 32.79, whereas weaker effects were induced by Aphanizomenon klebahnii CCALA 009 and no inhibition was induced by extracts of Aph. flos-aquae PCC 7905 and Aph. gracile SAG 31.79. Exudates of the laboratory cultured strains concentrated by solid phase extraction also induced species-specific inhibitory effects, but they did not necessarily correlate with the inhibitory potencies of extracts from the corresponding species. Interestingly, the GJIC-inhibitory effects may not be restricted to cyanobacteria, since exudates of two green alga species also affected GJIC, although their extracts caused no effects. The extracts from different natural water blooms inhibited GJIC with different potencies without apparent relation to bloom-species composition. Since the observed effects on GJIC did not correlate with the content of cyanotoxins microcystins and cylindrospermopsin in the tested samples, they were most likely induced by unknown compound(s). Our results indicate that putative tumor promoting compound(s) could be associated with different species of bloom-forming cyanobacteria, but their production is probably species- and strain-specific.

Chloral hydrate decreases gap junction communication in rat liver epithelial cells

Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involved are unknown. Chloral hydrate (CH), a by-product of chlorine disinfection of water, is carcinogenic in mice, and we demonstrated that CH reduced GJC in a rat liver epithelial cell line (Clone 9). To examine the mechanism(s) by which CH inhibits GJC, Clone 9 cells treated with CH were examined using Western blot, real-time polymerase chain reaction, immunocytochemical, and dye-communication techniques. Treatment with CH (0.1–5 mM for 24 h) resulted in a dose-dependent inhibition of GJC as measured by Lucifer yellow dye transfer. Western blot analysis demonstrated expression of connexin (Cx) 43 and 26 in control cells and reduced expression of Cx 43 but not Cx 26 protein from 0.1 to 1 mM CH. CH treatment from 2.5 to 5 mM caused an apparent increase in expression of both connexins that was concomitant with a reduction in mRNA expression for both connexins. Similarly, with immunocytochemistry, a dose-dependent decrease in Cx 43 staining at sites of cell–cell contact was apparent in CH (0.5–5 mM)-treated cultures, whereas no Cx 26 staining was observed. Thus, Clone 9 cells contain two types of connexins but only one type of plasma membrane channel. Understanding of the regulation of connexin may shed light on mechanisms responsible for inhibition of GJC by chemical carcinogens.

Rapamycin Response in Tumorigenic and Non-Tumorigenic Hepatic Cell Lines

BackgroundThe mTOR inhibitor rapamycin has anti-tumor activity across a variety of human cancers, including hepatocellular carcinoma. However, resistance to its growth inhibitory effects is common. We hypothesized that hepatic cell lines with varying rapamycin responsiveness would show common characteristics accounting for resistance to the drug.Methodology/Principal FindingsWe profiled a total of 13 cell lines for rapamycin-induced growth inhibition. The non-tumorigenic rat liver epithelial cell line WB-F344 was highly sensitive while the tumorigenic WB311 cell line, originally derived from the WB-F344 line, was highly resistant. The other 11 cell lines showed a wide range of sensitivities. Rapamycin induced inhibition of cyclin E–dependent kinase activity in some cell lines, but the ability to do so did not correlate with sensitivity. Inhibition of cyclin E–dependent kinase activity was related to incorporation of p27Kip1 into cyclin E–containing complexes in some but not all cell lines. Similarly, sensitivity of global protein synthesis to rapamycin did not correlate with its anti-proliferative effect. However, rapamycin potently inhibited phosphorylation of two key substrates, ribosomal protein S6 and 4E-BP1, in all cases, indicating that the locus of rapamycin resistance was downstream from inhibition of mTOR Complex 1. Microarray analysis did not disclose a unifying mechanism for rapamycin resistance, although the glycolytic pathway was downregulated in all four cell lines studied.Conclusions/SignificanceWe conclude that the mechanisms of rapamycin resistance in hepatic cells involve alterations of signaling downstream from mTOR and that the mechanisms are highly heterogeneous, thus predicting that maintaining or promoting sensitivity will be highly challenging.

Crystal structure of human retinoblastoma binding protein 9

Crystal structure of human retinoblastoma binding protein 9

Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts Scientific Opinion of the Panel on Contaminants in the Food chain.

Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts Scientific Opinion of the Panel on Contaminants in the Food chain.

The concentration-dependent induction of cell death by trimethyltin chloride in rat liver epithelial IAR20 cells

In this study we intended to evaluate the cytotoxic and genotoxic effects of trimethyltin chloride (TMT), one of the most widely used organometallic compounds, on liver cells with the rat liver epithelial cell line IAR20. The results showed that TMT significantly inhibited cell growth in a concentration-dependent manner and caused an increase in DNA damage. Additionally, Hoechst 33342 staining and flow cytometry detected increases in apoptotic and necrotic cells. Western blots demonstrated that the Bcl-2 family of proteins was involved in the apoptotic process, but p53 was not concerned.

Non-dioxin-like polychlorinated biphenyls induce a release of arachidonic acid in liver epithelial cells: A partial role of cytosolic phospholipase A 2 and extracellular signal-regulated kinases 1/2 signalling

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) have been shown to act as tumor promoters in liver; however, the exact mechanisms of their action are still only partially understood. One of the interesting effects of NDL-PCBs is the acute inhibition of gap junctional intercellular communication (GJIC), an effect, which has been often found to be associated with tumor promotion. As previous studies have suggested that NDL-PCB-induced disruption of lipid signalling pathways might correspond with GJIC inhibition, we investigated effects of PCBs on the release of arachidonic acid (AA) in the rat liver epithelial WB-F344 cell line, a well-established model of liver progenitor cells. We found that both 2,2′,4,4′-tetrachlorobiphenyl (PCB 47) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153), but not the dioxin-like, non- ortho-substituted, 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), induce a massive release of AA. The AA release, induced by PCB 153, was partially inhibited by extracellular signal-regulated kinases 1/2 (ERK1/2) signalling inhibitor, U0126, and by cytosolic phospholipase A 2 (cPLA 2) inhibitor, AACOCF 3. Although PCB 153 induced both ERK1/2 and p38 activation, the specific p38 kinase inhibitor, SB203580, had no effect on AA release. Inhibitors of other phospholipases, including phosphatidylcholine-specific phospholipase C or phosphatidylinositol-specific phospholipase C, were also without effect. Taken together, our findings suggest that the AA release, induced by non-dioxin-like PCBs in liver progenitor cell line, is partially mediated by cytosolic PLA 2 and regulated by ERK1/2 kinases. Our results suggest that more attention should be paid to cell signalling pathways regulated by AA or eicosanoids after PCB exposure, which might be involved in their toxic effects.

Coexpression of Connexin45 with Connexin43 Decreases Gap Junction Size

In the human heart, ventricular myocytes express connexin 43 (Cx43) and traces of Cx45. In congestive heart failure, Cx43 levels decrease, Cx45 levels increase and gap junction size decreases. To determine whether alterations of connexin coexpression ratio influence gap junction size, we engineered a rat liver epithelial cell line that endogenously expresses Cx43 to coexpress inducible levels of Cx45 under stimulation of the insect hormone, ponasterone A. In cells induced to express Cx45, gap junction sizes are significantly reduced (by 15% to 20%; p < 0.001), an effect that occurs despite increased levels of junctional connexons made from both connexins. In contrast, coexpression of Cx40 with Cx43 does not lead to any change in gap junction size. These results are consistent with the idea that increased Cx45 expression in the failing ventricle contributes to decreased gap junction size.

Assessment of the genotoxicity of the rat carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rat liver epithelial cells in vitro

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a disinfection by-product in chlorinated drinking water, is a multisite carcinogen in rats. One main target organ is the liver. The mechanism of the tumorigenicity was evaluated by testing the genotoxicity of MX in rat liver epithelial cell line cells. In the studies, the single cell gel/Comet assay and the hypoxanthine phosphoribosyl transferase locus assay to 6-thioguanine resistance were used. MX induced a dose-related genotoxic response in the comet assay. The lowest effective concentration was 120 microM when the exposure was in medium plus supplements and 3.75 microM when the exposure was in phosphate-buffered salt solution. MX also increased the frequency of TG(r) mutants, when the cells were treated in phosphate-buffered salt solution, at a concentration range of 2.3-9.2 microM. The present results show for the first time that MX causes DNA damage and gene mutations in rat liver epithelial cells, the target cells of MX's tumorigenicity in rats. We have earlier shown that MX also inhibits gap junctional intercellular communication in the same cells. The genotoxic effects were induced starting at about 60 times higher concentration, in identical exposure conditions, compared with the lowest concentration of MX causing the tumor promoter effect.

The vitamin E analog tocopherol succinate strongly inhibits gap junctional intercellular communication in rat liver epithelial cells (IAR203)

Vitamin E is a scavenger molecule trapping free radicals in biological membranes. However, it has also been shown to elicit the formation of reactive oxygen species and apoptosis in cancer cells. In this study, we tested the ability of α-tocopherol, tocopherol acetate, tocopherol phosphate and tocopherol succinate (TS) to modulate gap junctional intercellular communication in the rat liver epithelial cell line IAR203, as measured by the transfer of Lucifer yellow. While α-tocopherol, tocopherol acetate and tocopherol phosphate moderately reduced the dye transfer, TS at 10 and 25 μM strongly inhibited it, probably via the induction of the hypophosphorylation of connexin 43. Our results show that, besides their interesting antioxidant properties, vitamin E analogs, especially TS, can exert adverse effects on gap junctional intercellular communication, which could explain their controversial effects in carcinogenesis.