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Chronic Consumption of DNOP Induces an Epithelial-to-mesenchymal Transition State in Mouse LiverHepatocellular carcinoma is the cancer of the liver cells that is developed over time by the evolution of pre-neoplastic lesions. Di-n-octylphthalate (DNOP) is a plasticizer used to keep plastics flexible. If mice are exposed to DNOP, it causes an increase in pre-neoplastic hepatic lesions. Previously, our group found that DNOP increased the expression of transforming growth factor β (tgf-β) in AML-12 cells. Because tgf-β induces an epithelial-to-mesenchymal transition (EMT) state in mouse hepatocyte in vitro, our goal was to study the extent to which DNOP induces an EMT state in mouse liver. Two antibodies were used: anti-albumin antibody (a hepatocyte marker), and anti-vimentin (a mesenchymal cell maker). We first treated AML-12 cells with 0.1 % DNOP for 24, 48 and 72 h. No changes in the expression of albumin was seen. Because the limited time of 72 h may not have allowed sufficient time for a change in the phenotype, mice were fed diet containing 0.1 % DNOP for a month. We found that DNOP decreased the levels of albumin, whereas increased the levels of vimentin. In conclusion, chronic consumption of DNOP induces an EMT state in mouse liver. This mechanism may be involved in formation of hepatic pre-neoplastic lesions.
Di-N-Octylphthalate Acts as a Proliferative Agent in Murine Cell Hepatocytes by Regulating the Levels of Pro-Apoptotic ProteinsHepatocellular carcinoma (HCC) is the fifth most common cancer in the US. Its development is thought to be associated with inactivation of tumor suppressors by methylation. Di-n-octylphthalate (DNOP), a common plasticizer, is believed to cause hepatic pre-neoplastic lesions. Because a number of tumor suppressors are shown to be not expressed in HCC, our goal was to identify tumor suppressor genes methylated upon treatment with 0.1 % DNOP at 24, 48, 72 h in mouse hepatocytes cell line AML-12 and isolated primary cultured mouse hepatocytes. None of tumor suppressors experienced a change in the methylation status in presence of DNOP. Because we found that DNOP causes an increase in cell proliferation, we studied whether the effect is paralleled to a suppression of apoptosis. We found that DNOP causes a decrease in pro-apoptotic proteins and no change in anti-apoptotic proteins. We studied the physiological effects of DNOP in mouse liver. Mice were treated with 0.1 % DNOP for a month. DNOP caused a decrease in bile secretion and an increase in the hepatic levels of bile acids and glutathione. Avoiding the use of DNOP as a plasticizer in products for human consumption can reduce the incidence of diseases related to its hepatotoxicity.
Epidermal Growth Factor Mediates Di-N-Octyl Phthalate-Induced Hepatocyte ProliferationCertain chemicals used in manufacturing plastics are linked to severe, negative health effects such as cancer. Di-N-Octylphthalate (DNOP) is a phthalate found in many plastics and has been linked with hepatocellular carcinoma. The aim of this research project was to study the effect of DNOP on both proliferation and differentiation of normal mouse hepatocytes. The expression of several growth factors and their receptors (epidermal growth factor (egf), epidermal growth factor receptor (egfr), insulin-like growth factor 1 (igf1), insulin-like growth factor 2 (igf2), insulin-like growth factor 1 receptor (igf1r), hepatocyte growth factor (hgf), and transforming growth factor-β (tgf-β )) was assessed in the normal hepatocyte AML-12 cell line by RT-PCR and qPCR. The rate of cell proliferation of AML-12 cells was measured using an MTT cell proliferation assay kit. Changes in cytoskeletal reorganization were assessed by immunostaining filamentous-actin using phalloidin staining. We found that DNOP at 0.1% caused an increase in expression of egf at 48h and tgf-β at 72h. This result was confirmed by Western blot. DNOP did not cause changes in any of the other studied genes. The rate of cell proliferation increased in those cells treated with 0.1% DNOP at 24, 48, and 72h. DNOP induced reorganization of the filamentous actin from basolateral to perinuclear. Our observation suggests that DNOP, through an increase in the expression of egf, acts as a proliferative agent in normal mouse hepatocytes, and through the expression of tgf-β, induces a reorganization of the filamentous-actin. These observations could be a mechanism by which DNOP leads to the development of hepatocellular carcinoma.