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.
Highlights
Phthalates or phthalate esters are a family of lipophilic chemicals that are primarily used as plasticizers and additives to improve flexibility and durability of a product
Star promoter fused to luciferase was transiently transfected in MA-10 Leydig cells which were exposed to increasing doses of MEHP (0.5, 10, 30, 75, and 150 μM)
Experiments performed in triplicate (±SEM). (C) MA-10 Leydig cells were treated with either dimethylsulfoxide (DMSO) or 100 μM MEHP with or without 0.1 mM 8Br-cAMP and whole cell extracts were prepared for immunodetection of C/EBPβ and SF1. αTUBULIN was used as a loading control
Summary
Phthalates or phthalate esters are a family of lipophilic chemicals that are primarily used as plasticizers and additives to improve flexibility and durability of a product. Leydig cell steroidogenesis is stimulated by the binding of the pituitary luteinizing hormone (LH) to its receptor, leading to a rise in cAMP production and activation of multiple signaling pathways and kinases including PKA, ERK, and CAMKI ([44], reviewed in [45,46]). These kinases phosphorylate transcription factors which upregulate the expression of several genes involved in steroidogenesis (reviewed in [45,46]), including the Star gene which codes for the STAR protein (reviewed in [47,48,49,50]). Sci. 2021, 22, 11456 as phthalate replacements and found that two had no significant impact on Leydig cell steroidogenesis
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