Malachite green(MG), an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in aquaculture. Malachite green is reduced to and persists as leucomalachite green(LMG) in the tisssues of fish. Concern over MG and LMG are due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Several hepatotxicants and liver carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercellular communication (GJIC).This study wanted to determine if disruption of cell-cell interactions occurs in rat liver epithelial cells in response to MG and LMG treatment. Rat liver epithelial cells(WB) were treated with LMG(0~6£gg/ml) or MG.(0~5£gg/ml) for one hour and gap junction was analyzed using the scrape- loading/dye transfer assay. The viability and proliferation of rat liver epithelial cells treated with LMG or MG were determined by MTT and colony forming efficiency. In addition, expression and intracellular localization of connexin43, E-cadherin,£]-catenin,£\-tubulin, ZO-1 and occludin were determined by immunoblot and immunostain analysis. A clear decrease in the distance of dye transfer was evident following treatment with MG(0~5£gg/ml) or LMG(0~6£gg/ml). Treatment with LMG and MG at different concentrations resulted in a decrease in cell viability and cell proliferation. Preincubation of cells with protein kinase C(PKC) inhibitor decreased the inhibition of GJIC by 5ug/ml MG and the specific MEK 1 inhibitor decreased substantially the inhibition of GJIC by 5£gg/ml MG and 5£gg/ml LMG. On the other hand, the specific PI3 kinase inhibitor decreased the inhibition of GJIC only by 3£g/mlLMG and we treated WB cells with EDTA to chelate extracellular calcium ion. The decrease of free calcium ion caused the expression of GJIC. At the transcriptional level, 10£gg/ml LMG and 10£gg/ml MG after treatment for one hour caused no change in the level of connexin43 mRNA. At the translational level, the different concentrations of MG or LMG after treatment for one hour or 24 hours caused a decrease in the level of the concentrations of connexin43 protein, E-cadherin protein,£]-catenin protein,£\-tubulin protein, ZO-1 protein and changed the distribution of occludin and ZO-1. Therefore, these data speculated the hypothesis that disruption of cell-cell signaling by interference with GJIC may contribute to LMG and MG toxicity, carcinogenicity and apoptosis.

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