Abstract:
Objective Much research related to phthalates has focused on parent chemicals; however, dibutyl phthalate and diethylhexyl phthalate may exert their toxicities via metabolites monobutyl phthalate (MBP) and mono-ethylhexyl phthalate (MEHP). This study aims to investgate the effects of MBP and MEHP on the autophagy of mouse testcular mesenchymal cells (TM-3) and phosphatase and tensin homology deleted on chromosome ten (PTEN).
Methods In vitro cultured TM-3 cells in logarithmic growth phase were treated with either MBP or MEHP at concentration gradients of 0 (control group), 50, 100, 200, 400, and 800μmol/L; afer 24h of treatment, respectve median lethal concentratons (IC50) were calculated by improved Karber's method, and a set of concentratons of 0 (control group), 200, 400, and 800 μmol/L were determined appropriate for subsequent experiment. The effects of MBP or MEHP exposure at designed concentratons for different tme on cell survival rate were assessed by CCK-8 method; autophagosomes with double membranes induced by 400 μmol/L MBP or MEHP for 24 h were observed under transmission electron microscope; the fluorescence signal intensity of autophagic vesicles was detected by monodansyl cadaverine (MDC) immunofluorescence staining; the expression levels of autophagy marker proteins (LC3 and p62) and autophagy regulaton protein (PTEN) were assessed by Western blot.
Results The CCK-8 results showed that compared with the control group, the cell survival rate decreased as the dose of MBP or MEHP increased (P < 0.05). Under transmission electron microscopy, the autophagosomes with double membranes were observed in the cells infected with 400 μmol/L MBP or MEHP for 24 h. The MDC staining results showed that compared with the control group, the fluorescence intensites of the 200 μmol/L MBP group and the 200 μmol/L MEHP group had no statstcal difference (P>0.05), but the fluorescence signal intensites of the 400 and 800 μmol/L MBP groups increased by 9.0 tmes and 16.8 tmes respectvely, and those of the 400 and 800 μmol/L MEHP groups increased by 16.3 tmes and 26.4 tmes, respectvely (P < 0.01). The Western blot results showed that the 200 μmol/L MBP and MEHP treatments did not change the LC3Ⅱ/LC3Ⅰ and PTEN protein expression levels compared with the control group (P>0.05); the protein expression levels of LC3Ⅱ/LC3Ⅰ in the 400 and 800 μmol/L MBP groups increased by 7.5 tmes and 13.6 tmes respectvely, and the PTEN expression levels increased by 4.8 tmes and 15.4 tmes respectvely; the protein expression levels of LC3Ⅱ/LC3Ⅰ in the 400 and 800 μmol/L MEHP groups increased by 4.5 tmes and 9.8 tmes respectvely, and the PTEN expression levels increased by 5.7 tmes and 14.4 tmes respectvely (P < 0.05); except the 200 μmol/L MBP group (P>0.05), the expression levels of p62 protein in the 400 and 800μmol/L MBP groups decreased by 43% and 72% respectvely; the expression levels of p62 protein in the 200, 400, and 800 μmol/L MEHP groups reduced by 55%, 80%, and 88%, respectvely (P < 0.01).
Conclusion A certain dose of MBP or MEHP can increase the autophagy of TM-3 cells, and the expression level of PTEN is positvely correlated with the autophagy level.
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