| Objective The adverse effects of Phthalate (Phthalic Acid Esters, PAEs), especially on the reproductive system, has become an important public health problem that threatens to people’s health and future generations to survive and multiply, PAEs may affect Steroids synthesis by influencing the levels of gene expression of P450scc,3β-HSD, P450c17and changing the distribution of interstitial cells, and thus impact the hypothalamus-pituitary-testicular axis, meanwhile, PAEs reduce the levels of gene expression of insulin-like factor3,therefore, impact the testis decline normally. A series of changes can induce developmental disorders. Dibutyl phthalate (DBP), di (2-ethylhexyl) ester (DEHP) are the most dominant PAEs. In this study, the testosterone synthesis pathway as the starting point to explore the effects of DBP and its major metabolite monobutyl phthalate (MBP), DEHP and its major metabolite mono (2-ethylhexyl) phthalate,(MEHP) on mechanism of testosterone synthesis in interstitial cells, The aim of this study was to explore the molecular mechanisms of the reproductive toxicity of PAEs, provide a scientific basis to evaluate the potential effects of endocrine disruptors on human. The aim of this study was to evaluate the effects of DBP/MBP, DEHP/MEHP on steroidogenesis in a murine Leydig tumor cell line (MLTC-1) in vitro.Methods1. Cell viability and mitochondrial integrity in MLTC-1cells affected by DBP/MBP, DEHP/MEHP were analysed using the method of CCK-8. The MLTC-1cells were treated with different concentrations of DBP/MBP, DEHP/MEHP for24h, then, cell viability was tested by CCK-8assay.2. Testosterone measurements. At the end of exposure, testosterone concentrations were measured in the Laboratory of the General Hospital of Tianjin Medical University by direct chemiluminescence assay.3. P450scc,3β-HSD, P450c17, INSL3mRNA expression levels measurements. After treated with different concentrations of DBP/MBP, DEHP/MEHP, P450scc,3β-HSD, P450c17, INSL3mRNA expression levels was measured by real-time PCR.4. Protein expression of INSL3in MLTC-1cells measurements. After treated with different concentrations of DBP/MBP, DEHP/MEHP for24h, protein expression of INSL3in MLTC-1cells was assessed by Western Blot.5. Flow cytometry. After treated with different concentrations of DEHP/MEHP, ApoScreen Annexin V Apoptosis Kit was used to quantitatively determine the percentage of cells undergoing apoptosis.Results1. Compared with the control group, DBP and MBP at the doses of2000μmol/1reduced the cell viability significantly (p<0.05), and cell viability was increased significantly by MBP at the concentrations of0.001,0.01μmol/L (p<0.05). When the dose was increased to100,1000μmol/1DEHP or MEHP, the growth of MLTC-1cells was significantly inhibited (p<0.05). Thus exposure doses were1000,10,0.1and0μmol/1for DBP and MBP, and10,0.1,0.001μmol/1for DEHP and MEHP.2. The testosterone secretion of MLTC-1cells cultured with hCG was stimulated at the lowest doses of MBP (0.1μmol/1), and reduced at1000μmol/L of MBP. Testosterone production was inhibited at the highest treatment doses of DBP (1000μmol/L). Testosterone productions were inhibited by DEHP at the dose of10μmol/L. Testosterone productions were increased at0.001μmol/L of MEHP.3. Compared to the controls, DBP and MBP significantly reduced the mRNA levels of P450scc,3β-HSD and P450c17in treatment groups except the group of MBP at the concentration of0.1μmol/L. The mRNA levels of P450scc,3β-HSD and P450c17were increased significantly by DEHP at the dose of O.OOlμmol/L, and the mRNA levels of3β-HSD and P450c17were increased significantly by DEHP at the dose of0.1μmol/L. the mRNA levels of3β-HSD were increased significantly by MEHP at the dose of0.001μmol/L.4. INSL3mRNA expression levels were inhibited in MLTC-1cells exposed to DBP and MBP, and with doses increasing, INSL3mRNA expression levels showed a trend of dose-dependent. INSL3protein levels at the lowest dose group of MBP (0.1μmol/L) are significantly higher than that of the control group (p<0.05); For DEHP and MEHP group, results of Western blot are nearly the same with RT-PCR results, compared with control group, expression levels of INSL3mRNA and protein were significantly increased by DEHP at concentration of0.001μmol/L and0.1μmol/L (p<0.05); Expression levels of INSL3mRNA and protein in group of MEHP at concentration of0.001mol/L were higher than that of the control group (p<0.05).5. The result of flow cytometry analysis revealed that the proportion of apoptotic cells was significantly increased after the exposure of DEHP. Although the proportion of apoptotic cells in MEHP treatment groups had a increasing trend, there was no statistical difference until the dose of0.1μmol/1treatment.Conclusions1. Low concentrations of DBP, MBP, DEHP and MEHP can stimulate the proliferation activity of MLTC-1cells; the cell activity was gradually reduced with concentration of chemical substances increasing.2. Effects of DBP, MBP, DEHP and MEHP on testosterone levels are the low-dose stimulation and high-dose inhibition in MLTC-1cell.3. DBP, MBP can reduce the expression levels of key enzyme genes related to testosterone synthesis and INSL3mRNA and protein; Exposed to relatively low doses of DEHP and MEHP, expression levels of mRNA and protein related to testosterone synthesis pathway were increased. And there may be certain causal relationship between gene expression and testosterone synthesis changes.4. The proportion of apoptotic cells was increased after the exposure of DBP, MBP, DEHP and MEHP. |
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