Disruption of Retinol (Vitamin A) Signaling by Phthalate Esters: SAR and Mechanism Studies

A spectrum of reproductive system anomalies (cryptorchidism, hypospadias, dysgenesis of Wolffian duct-derived tissues and prostate, and reduced sperm production) in male rats exposed in utero to phthalate esters (PEs) are thought to be caused by PE inhibition of fetal testosterone production. Recently, dibutyl and dipentyl phthalate (DBuP, DPnP) were shown to disrupt the retinol signaling pathway (RSP) in mouse pluripotent P19 embryonal carcinoma cells in vitro. The RSP regulates the synthesis and cellular levels of retinoic acid (RA), the active metabolite of retinol (vitamin A). In this new study, a total of 26 di- and mono-esters were screened to identify additional phthalate structures that disrupt the RSP and explore their mechanisms of action. The most potent PEs, those causing > 50% inhibition, contained aryl and cycloalkane groups or C4-C6 alkyl ester chains and were the same PEs reported to cause malformations in utero. They shared similar lipid solubility; logP values were between 4 and 6 and, except for PEs with butyl and phenyl groups, were stable for prolonged periods in culture. Mono- and cognate di-esters varied in ability to disrupt the RSP; e.g., DEHP was inactive but its monoester was active while DBuP was active yet its monoester was inactive. DBuP and dibenzyl phthalate both disrupted the synthesis of RA from retinol but not the ability of RA to activate gene transcription. Both PEs also disrupted the RSP in C3H10T1/2 multipotent mesenchymal stem cells. Based on this in vitro study showing that some PEs disrupt retinol signaling and previous in vivo studies that vitamin A/RA deficiency and PEs both cause strikingly similar anomalies in the male rat reproductive system, we propose that PE-mediated inhibition of testosterone and RA synthesis in utero are both causes of malformations in male rat offspring.