Effect Of MEHP On The Steroidogenesis In Leydig Cells And Its Mechanism

Di(2-ethylhexyl)phthalate(DEHP) has been used as plasticizer in the manufacture of a wide variety of consumer products of polyvinyl chloride (PVC) for a long time. It is currently the most abundant phthalate and has been affirmed as one kind of environmental endocrine disruptors(EEDs). A large number of studies using in vivo and in vitro protocols have been shown that DEHP produced male reproductive toxicity. In a recent review of laboratory studies, the U.S. National Toxicology Program's Center (NTP) for the evaluation of risks to human reproduction expert panal concluded that DEHP has the potential to produce adverse reproductive effects on humans. It is necessary to explore the mechanisms of action from multiple viewpoints.Previous studies suggest that DEHP disrupted the regulatory pathways in the hypothalamo-pituitary-testicular axis (HPTA) and the alteration of steroidogenesis might be one of the mechanisms of action in its reproductive toxicity. Since Leydig cells are the main sites for steroidogenesis in the testis, we hypothesize that Leydig cells were thetarget cells of DEHP. However, the mechanisms of action remain unclear. Previous findings have affirmed that mono(2-ethylhexyl)phthalate (MEHP), the metabolite of DEHP achieved by lipases located mainly in the gut, is the active agent responsible for its testicular toxicity.In the present work, based on the models of both mouse Leydig Tumor cells (mLTC-1) and primary cultured Leydig cells, we investigated the alterations of hCG-stimulated steroidogenesis in Leydig cells induced by MEHP.The transport of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane is the rate-determining step in steroidogenesis. Two proteins, the peripheral-type benzodiazepine receptor (PBR) and the steroidogenesis acute regulatory protein (StAR), have been shown to be involved in the regulation of the movement of cholesterol across the mitochondrial membranes, Therefore, we observed the effect of MEHP on the mRNA and protein expression of these two factors, and further studied the relationship betwen these factors and steroidogenesis induced by MEHP, which would provide some reference indicators for human early biological monitorings.Part I Effects of MEHP on the Biosynthesis of Testosterone inPrimary cultured Leydig cellsObjective: This part was performed to evaluate the effect of MEHP on testosterone biosynthesis in primary cultured Leydig cells. Methods: 1. Testes were took out from pubertal SD rats (35d, 160~170g), and the primary culture of Leydig cells was used as the experimental model.2. The mitochondrial integrity and cell viability of Ley dig cells affected by MEHP were analysed using the method of MTT.3. RIA was employed to measured the level of testosterone in the medium.4. The level of mRNA expression of PBR, StAR and P450scc in Leydig cells was determined by RT-PCR.5. Protein expression of PBR, StAR and P450scc in Leydig cells was assessed by Western Blot.Results: The doses of MEHP were selected in virtue of the MTT result, that is, there is nontoxic effect of MEHP on Leydig cell viability and mitochondrial integrity, They are control (0.1% solvent), 62.5, 125, 250 and 500umol/L. Whether in basal or hCG-stimulated state, testosterone level showed a dual trend with the doses of MEHP exposure increasing, it is higher than the control in 125umol/L and lower in 500umol/L with statistical significance (PO.05). As to the duration of MEHP treated, the level of hCG-stimulated testosterone synthesis was higher than corresponding control in 125umol/L at all times, and the diversity was significant at 18 and 24 h, however, it was lower in 500umol/L with the statistical significance at 12, 18, and 24h CP<0.05). The alteration of testosterone level induced by MEHP disappeared when we added 22R-Hydroxycholesterol (22R-CHO) into the medium. RT-PCR results showed that the PBR mRNA expression increased markedly in 125umol/L (PO.05) then decreased significantly in 500umol/L (PO.01). Although the StAR mRNA expression was upregulated in 62.5, 125 and...