Endocrine disruption: In silico perspectives of interactions of di-(2-ethylhexyl)phthalate and its five major metabolites with progesterone receptor

The chemical industry contributes significantly to the prosperity and economic development of modern society. However, many chemical compounds that are discharged into the environment due to industrial activity and leaching from consumer products interfere with the physiological functions of the exposed human and animal populations and are referred to as endocrine disrupting compounds (EDCs) [1–3].

Di-(2-ethylhexyl)phthalate (DEHP) is a high volume plasticizer used as a softener in polyvinyl chloride industry with a 54 % market share (2010 data) and is considered as one of the most common EDCs present in the environment [4]. DEHP is frequently used in the manufacture of medical devices, blood storage bags, surgical gloves, dialysis equipment, cosmetics, household and personal items such as soap, shampoo, detergents, adhesives, vinyl flooring, shower curtains, plastic bags, garden hoses, children’s toys, and many other plastic products [4]. Exposure of human population to DEHP occurs continuously through inhalation, ingestion, and skin contact [5]. Recently [6], DEHP was detected in 74 % of 72 common food items including infant foods, chicken, pork and other food items in a market in Albany, New York. DEHP is metabolized in the body by hydrolysis to a primary metabolite, mono-(2-ethylhexyl)phthalate (MEHP), which is then further metabolized into multiple hydroxylative and oxidative secondary metabolites [7, 8]. The four major secondary metabolites of DEHP are mono(2-ethyl-5-hydroxyhexyl)phthalate (5-OH-MEHP), mono(2-ethyl-5-oxyhexyl)phthalate (5-oxo-MEHP), mono(2-ethyl-5-carboxypentyl)phthalate (5-cx-MEPP) and mono[2-(carboxymethyl)hexyl]phthalate (2-cx-MMHP) [7, 8]. A simplified metabolic pathway of 5 major metabolites of DEHP is illustrated (Fig. 1).

DEHP and its metabolites have been detected in various human body fluids such as blood and breast milk [9], follicular fluid [10], amniotic fluid [11], cord blood of newborns [12] and urine [5] indicating immense potential for adverse health effects. Monoester metabolites rather than native DEHP are thought to be responsible for toxicity of DEHP [13] with secondary metabolites displaying a 100 fold increase in embryo-toxicity compared to MEHP [14]. In a recent study [15], positive associations were reported between total DEHP metabolites, MEHP, 5-OH-MEHP, and 5-oxo-MEHP levels in urine and plasma estradiol and ratio of estradiol to testosterone. Higher levels of MEHP, 5-OH-MEHP, and 5-oxo-MEHP were associated with lower sperm concentration, lower sperm motility, higher sperm apoptosis, and ROS generation [16]. Prenatal exposure with DEHP and its metabolites has been associated with reduced gestational age for pregnancies bearing male fetus [17], anogenital distance problems in male babies [18–20], cryptorchidism [21], altered reproductive hormone levels [22], hypospadias [23], intellectual and motor development in children [24], and preterm birth [25, 26]. Retrospective analyses of DEHP metabolites in pregnancy serum of mothers [27] indicated that prenatal exposure of children to DEHP was associated with reproductive problems during adolescence; higher 5-OH-MEHP level in prenatal maternal serum was related with lower semen volume and lower sperm concentrations and higher 5-cx-MEPP was associated with lower free testosterone concentrations.

Studies in rats and mice have also shown that exposure to DEHP can induce deleterious reproductive and endocrine effects [28–31]. In rats, prenatal DEHP treatment was associated with developmental abnormalities in male pups such as cryptorchidism, anogenital problems, and malformations of epididymis, vas deferens, seminal vesicles, prostate, and external genitalia collectively called as the phthalate syndrome, which is similar to effects of DEHP exposure in men [32, 33]. In vitro, MEHP and 5-OH-MEHP decreased gonocyte number and increased gonocyte apoptosis in rat testis organ culture [34].

In general, EDCs have been proposed to exert their toxic effects through interactions with nuclear steroid receptors, sex steroid binding proteins, and steroid enzymatic pathways regulating reproductive and endocrine functions [1]. Progesterone receptor (PR) belongs to the family of nuclear receptors and binds to progesterone, which is an important hormone involved in female reproductive function and maintenance of pregnancy [35, 36] as well as an important modulator of male reproductive function [37]. Interference in PR signaling leads to reproductive dysfunction and pregnancy failure [38]. Recently [39], docking studies of PR with three stereoisomers of DEHP have been reported. Docking of DEHP and its primary metabolite, MEHP, with PR have also been reported [40], however, the important secondary metabolites were not included in the study.

This study aimed at analyzing and comparing the structural binding characteristics of DEHP and its five major metabolites, MEHP, 5-OH-MEHP, 5-oxo-MEHP, 5-cx-MEPP, and 2-cx-MMHP with PR using in silico approaches. The study involved the delineation of the binding mechanism of all the six xeno-ligands with PR by molecular docking simulation and comparing the distinctive binding pattern and the interacting residues.

Di-(2-ethylhexyl)phthalate (DEHP) is a widely used phthalate compound representing more than half of all phthalate compounds manufactured worldwide for use in the industry as a plasticizer. Several reviews showed that DEHP is a universally prevalent environmental contaminant and behaves as a reproductive and developmental toxin [5, 28, 29, 32, 42]. Several epidemiological reports have identified DEHP and its metabolites as the cause of adverse effects on various systems of the body including endocrine and reproductive system [28, 29, 32]. Many studies have reported developmental problems during prenatal period and postnatal period in unborn and new born children as a result of gestational exposure of mothers to DEHP [18–20, 24, 42, 43]. In women, higher urinary or serum levels of DEHP and its metabolites were associated with problems in conception, endometriosis, and high rates of miscarriage, delayed or preterm gestation, and pregnancy associated toxemia and preeclampsia [25, 26, 28, 29]. In men, higher urinary or serum levels of DEHP and its metabolites were linked with lower semen volume, lower sperm concentrations, lower sperm motility, higher sperm apoptosis, and lower testosterone concentrations [5, 16, 27, 32, 44, 45]. Due to side effects of DEHP mentioned above, it has been banned since 2009 in the United States for use in children’s toys and the European Union has also classified DEHP as a reproductive toxicant. However, DEHP continues to be manufactured and used in many countries across the world.

DEHP is metabolized in the body by hydroxylative and oxidative reactions to many metabolic products which include five major metabolites: MEHP, 5-OH-MEHP, 5-oxo-MEHP, 5-cx-MEPP, and 2-cx-MMHP ([7, 8]; see Introduction section). The toxicity of DEHP in the body is attributed mainly to the actions of its secondary metabolites [13, 14]. Progesterone receptor signaling is an essential pathway controlling reproductive function and is involved in reproductive periodicity and establishment and maintenance of pregnancy [35, 36]. DEHP and the indicated five major metabolites can act as potential xeno-ligands for PR and disrupt the normal progesterone signaling pathway and this could be one of the important mechanisms which lead to adverse effects in the human population. In the present study, docking simulations of DEHP and its five major metabolites namely, MEHP, 5-OH-MEHP, 5-oxo-MEHP, 5-cx-MEPP, and 2-cx-MMHP were performed with PR and comparison of docking displays and interacting residues was performed among the ligands and the co-complex bound native ligand, norethindrone (NET) of PR crystal structure.

Induced Fit Docking of DEHP and its five metabolites with PR showed that all the six xeno-ligands fitted well into the steroid binding pocket of the receptor. The high binding affinity values, IFD scores, and dock scores indicated that the docking complexes formed by DEHP, MEHP, 5-OH-MEHP, 5-oxo-MEHP, 5-cx-MEPP, and 2-cx-MMHP with PR were in their most favorable conformation. A number of important PR amino acid residues interacted through hydrophobic and hydrogen-bonding interfaces with each of the six xeno-ligands during docking simulation contributing to the ligand-PR docking complex stability. A consistent and high overlapping (82-95 % commonality) of the interacting residues of PR among native bound ligand, NET, and DEHP and its metabolites suggested a common platform of action. This was further supported by the fact that 16 of the 22 PR residues interacting with bound native ligand, NET, also interacted with DEHP and each of the five metabolites. In addition, DEHP and each of its five metabolites, and bound native ligand, NET, formed a hydrogen bonding interaction against residue Gln-725 of PR altogether pointing to the common structural binding characteristics of the native bound ligand and the six xeno-ligands. Commonality of structural binding characteristics of bound native ligand, NET, and DEHP and its metabolites with PR suggest, on a preliminary basis, potential disruption of PR function by DEHP and its metabolites.

To the best of our knowledge, the current study is the first structure based report for docking stimulation of secondary metabolites of DEHP with PR. In vitro competitive binding of DEHP and its metabolites with PR are seemingly unavailable. Docking studies of PR with three stereoisomers of DEHP have recently been reported [39]. Docking of DEHP and its primary metabolite, MEHP, with PR have also been reported [40]. The results of the current study with docking of DEHP and PR support the results of the reported study [40] showing residues Gln-725, Arg-766 and Phe-778 as the crucial interacting residues of PR interaction with DEHP. The importance of the current study lies in the fact that the secondary metabolites of DEHP viz. 5-OH-MEHP, 5-oxo-MEHP, 5-cx-MEPP, and 2-cx-MMHP are the best biomonitoring markers of DEHP in the urine or blood and are potentially more potent disruptors because of their long elimination half-life compared to the primary metabolite, MEHP [7, 8]. Approximately 75 % of a single dose of DEHP was excreted in urine within two days; 67 % was excreted within the first 24 h which included 6 % MEHP, 23 % 5-OH-MEHP, 15 % 5-oxo-MEHP, 19 % 5-cx-MEPP, and 4 % 2-cx-MMHP (Koch et al. [8]). Of the 3.8 % excreted in the next 24 h, more than 75 % included 5-cx-MEPP and 2-cx-MMHP and the rest included 5-OH-MEHP and 5-oxo-MEHP indicating long elimination half-lives of the former two secondary metabolites.

Although not related to the progesterone receptor, DEHP treatment inhibited progesterone secretion from human luteal cells in culture [46]. Furthermore, in vivo treatment of DEHP decreased secretion of progesterone in mice [47] and in vitro treatment of MA-10 mouse Leydig cells with MEHP resulted in inhibition of steroidogenesis including progesterone secretion [48]. Interestingly, in sheep, DEHP causes shortening of estrous cycles due to a reduction in the size and lifespan of CL, however, in contrast to mice, an increase in circulating concentrations of progesterone was noted [49]. Conversely, MEHP treatment was associated with an increase in steroidogenesis including progesterone concentrations in cultured rat ovarian follicles [50]. Apparently, direct studies involving treatments with secondary metabolite compounds namely 5-OH-MEHP, 5-oxo-MEHP, 5-cx-MEPP, and 2-cx-MMHP in laboratory animals or in in vitro cell cultures are not available. It goes without saying that no single mechanism or pathway can explain the endocrine disrupting effects of DEHP and its metabolites on reproductive and endocrine systems in the human body. As an example, PPAR alpha was thought to be a possible pathway of adverse effects of DEHP in mice, however, the toxic effects were observed despite the use of PPAR alpha null mice suggesting the involvement of additional pathways [51]. Besides the PR signaling pathway, multiple other pathways could mediate the adverse effects of DEHP and its metabolites in the body. Androgen receptor pathway could also be an important mechanism as agonistic (androgenic) and antagonistic (antiandrogenic) actions of DEHP and other phthalate compounds have been shown at the androgen receptor level [52]. This study showed that DEHP and all its five major metabolites were able to bind to PR with structural binding characteristics that were common with the bound native ligand, NET, of PR. Hence, DEHP and its five metabolites have potential to interfere with the binding of progesterone to its receptor resulting in adverse effects and the dysfunction of progesterone signaling.

This study was undertaken to understand the structural binding mechanisms of DEHP and its five major metabolites (MEHP, 5-OH-MEHP, 5-oxo-MEHP, 5-cx-MEPP, and 2-cx-MMHP) with PR in order to predict their potential adverse effects on progesterone signaling. The results indicated, a high percentage of overlap (82-95 %) among the interacting residues of PR for the native bound ligand, NET, and for DEHP and its metabolites. The structural binding similarities were further supported by a common hydrogen bonding interaction between Gln-725 residue of PR and DEHP,each of its five metabolites, and bound native ligand, NET. Therefore, on a preliminary basis, the six xeno-ligands have potential disruptive activities in the binding of progesterone to its receptor resulting in the dysfunction of progesterone signaling and adverse effects.