Mechanisms of MEHP Inhibitory Action and Analysis of Potential Replacement Plasticizers on Leydig Cell Steroidogenesis

Abstract

Steroid production in Leydig cells is stimulated mainly by the pituitary luteinizing hormone, which leads to increased expression of genes involved in steroidogenesis, including the gene encoding the steroidogenic acute regulatory (STAR) protein. Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of the widely used plasticizer DEHP, is known to disrupt Leydig steroidogenesis but its mechanisms of action remain poorly understood. We found that MEHP caused a significant reduction in hormone-induced steroid hormone production in two Leydig cell lines, MA-10 and MLTC-1. Consistent with disrupted cholesterol transport, we found that MEHP represses cAMP-induced Star promoter activity. MEHP responsiveness was mapped to the proximal Star promoter, which contains multiple binding sites for several transcription factors. In addition to STAR, we found that MEHP also reduced the levels of ferredoxin reductase, a protein essential for electron transport during steroidogenesis. Finally, we tested new plasticizers as alternatives to phthalates. Two plasticizers, dioctyl succinate and 1,6-hexanediol dibenzoate, had no significant effect on hormone-induced steroidogenesis. Our current findings reveal that MEHP represses steroidogenesis by affecting cholesterol transport and its conversion into pregnenolone. We also found that two novel molecules with desirable plasticizer properties have no impact on Leydig cell steroidogenesis and could be suitable phthalate replacements.

Mechanism of Action of an Environmentally Relevant Organochlorine Mixture in Repressing Steroid Hormone Biosynthesis in Leydig Cells

“…The OC mixture was designed to approximate levels of organochlorines found in Arctic ringed seal blubber [ 39 ], a main diet constituent of the Inuit population in Northern Québec, Canada. The pure mixture of organochlorines (15 components in total) was diluted in dimethylsulfoxyde (DMSO) purchased from Sigma-Aldrich Canada (Oakville, ON, Canada) as described previously [ 17 , 18 , 22 , 39 ] to obtain the proportions listed in Table 1 . This organochlorine mixture was used in whole animal studies and found to disrupt reproductive development and function in male rats over several generations [ 22 , 33 , 34 , 35 ].…”

“…ELISAs for progesterone quantification were performed as recommended by the manufacturer (Cayman Chemical, Ann Arbor, MI, USA) as described previously [ 17 , 18 , 45 , 46 ].…”

“…It is widely recognized that endocrine-disrupting chemicals affect Leydig cell function in several species, including humans, resulting in hormone insufficiency and incomplete male sex differentiation (reviewed in [ 15 ]). There is a growing body of evidence showing that a reduction in Star gene expression leading to decreased steroidogenesis is a result of exposure to several endocrine disrupting chemicals (reviewed in [ 16 ]), including phthalates [ 17 ] and organochlorines [ 18 , 19 ]. Organochlorines are a family of organic compounds containing at least one covalently bonded chlorine atom.…”

Differential Response of Transcription Factors to Activated Kinases in Steroidogenic and Non-Steroidogenic Cells

“…Penicillin and streptomycin sulphate were added to the cell culture media to a final concentration of 50 mg/L, and all cell lines were kept at 37 °C, 5% CO 2 in a humidified incubator. All cell lines were validated by morphology and Leydig cell lines by quantifying steroidogenic output (progesterone for MA-10) as previously described [ 25 , 27 , 39 , 73 , 74 , 75 , 76 , 77 ]. MA-10 (60,000 cells per well) and CV-1 (25,000 cells per well) were transiently transfected using polyethylenimine hydrochloride (PEI) (Sigma-Aldrich Canada, Oakville, ON, Canada) as previously described [ 39 , 78 ] or the calcium phosphate co-precipitation method as described in [ 23 , 24 ].…”

Recent Updates on the Effect of Endocrine Disruptors on Male Reproductive Functions

“…MEHP decreases the production of StAR protein and leads to a reduction in cholesterol transport. Apart from inducing oxidative stress in Leydig cells, exposure to a high level of MEHP inhibits the activities of steroidogenic enzymes such as 3β and 17β hydroxysteroid dehydrogenases, thereby altering testosterone biosynthesis [31,32]. MEHP affects spermatogenesis by decreasing the number of Sertoli cells and its interaction with gonocytes and triggers testicular apoptosis by increasing Fas ligand expression [33 -35].…”