Effect of Di-2-ethylhexylphthalate on alkalinephospatase activity was due to down regulation of osteogenic related genes

Di-2-ethylhexyl phthalate (DEHP) is used as plasticizer to produce flexible polyvinyl chloride (PVC) which is used in food and medical industries. Due to temperature change and contact with biological fluids and other liquids, DEHP leaches out from PVC. Humans get exposed to DEHP in different way including food consumption and medical utilities such as blood bags, blood transfusion tubes, syringes. At 2002, 120000 tons of DEHP has been produced in United States of America and this production was raised up to 230000 tons at 2006. In Iran, only in one of the industries called as Farabi petrochemical industries, 55000 tons of phthalate per year is produced. It has been reported that the DEHP concentration in human blood might reach to 52 to 55 µg/ml when blood is stored in polyvinyl chloride bags for two weeks. Previously, the effect of different concentration of DEHP on viability, proliferation and differentiation of rat bone marrow mesenchymal stem cells was investigated. In the present study, effect of 100 µM (39 µg/ml) of DEHP on the expression of genes related to osteogenic differentiation of rat bone marrow mesenchymal stem cells was investigated.

In this experimental study, rat bone marrow mesenchymal stem cells were extracted from Wistar rats and after 3rd passage the cells cultured was performed in osteogenic media in presence of 100 µM of DEHP for 21 days. The viability of the cells was studied using 3-[4, 5- dimethylthiazol-2yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay. In addition, the osteogenic differentiation of the rat bone marrow mesenchymal stem cells was investigated using quantitative alizarin red test and calcium concentration determination. In addition, alkaline phosphatase enzyme activity as a marker of osteogenic differentiation was measured. Expression of the osteogenic related genes (osteonectin, SMAD1 ،BMP2 ،BMP7 and RUNX2) were studied using reverse transcriptase-PCR. Data was analyzed and the minimum level of significant was considered as p<0.05.

Data analysis revealed, rat bone marrow mesenchymal stem cells viability reduced significantly (p<0.05) following treatment with 100 µM of DEHP when compared to control one. Also, we observed a significant (p<0.05) reduction in matrix production based on significant reduction in alizarin red concentration as well as calcium content and alkaline phosphatase enzyme activity. In addition, a significant (p<0.05) reduction in total protein also was observed in the samples extracted from BMSCs differentiated to osteoblasts in presence of DEHP. Meanwhile, a significant (p<0.05) down regulation of osteogenic related genes was confirmed while no change (p>0.05) was observed in the expression of GAPDH.

based on this study, long term exposure to DEHP caused reduction in matrix production which strongly showed the osteogenic differentiation of rat bone marrow mesenchymal stem cells is affected by low concentration of this environmental pollutant. As it was observed, the concentration used in this study was lower than the concentration of this pollutant in blood bags. Therefore, if a patient gets exposed to the biological or nonbiological fluids in a treatment procedure, large amount of DEHP would enter the blood circulation system. As DEHP is used in production of food containers and medical utilities such as blood tubing, blood bags, dialysis tubes in dialysis machines, we strongly suggest a legal restriction to be forced on the industrial which using this chemical as plasticizer.