Endocrine Disruption: Computational Perspectives on Human Sex Hormone-Binding Globulin and Phthalate Plasticizers.

Endocrine disrupting chemicals (EDCs) are man-made industrial compounds contaminating our ecosystem that have been associated with adverse effects on the endocrine homeostasis leading to abnormal developmental patterns, immunological problems, cancers, neurodevelopmental delays, and reproductive problems in the human population [ 1 – 3 ]. Thousands of chemicals are manufactured annually in the world and about a 1000 of them are estimated to have potential endocrine disrupting properties [ 4 ]. Phthalates are a family of environmentally pervasive and high volume production plasticizer chemicals, many of which have endocrine disrupting activity with harmful effects on different systems of the human body including the reproductive system (reviewed in [ 5 ]). Global plasticizer consumption was about 14 billion pounds in 2011 and phthalate contribution to this was 87% [ 6 ]. Because of the large scale production and wide commercial use, phthalates are ubiquitously distributed in the environment. Phthalate plasticizers are esters of phthalic acid and are short- or long-chain compounds based on their alkyl chains [ 7 ]. Both types of phthalate esters are linked non-covalently to industrial materials and readily leach into the environment. Several phthalate esters have been the focus of evaluation for the risks to human reproduction by National Toxicology Program of the United States Environmental Protection Agency and the European Union [ 8 – 9 ]. These important phthalate EDCs are, dimethyl phthalate (DMP), dibutyl phthalate (DBP), di-iso-butyl phthalate (DIBP), butylbenzyl phthalate (BBP), di- n -hexyl phthalate (DNHP), di-2-ethylhexyl phthalate (DEHP), di- n -octyl phthalate (DNOP), di-iso-nonyl phthalate (DINP), and di-iso-decyl phthalate (DIDP). From these DMP and DBP are short-chain phthalates and are used in aerosols, perfumes, creams, cosmetics, nail polishes, house fragrances, baby lotions, excipients for medications etc. [ 10 ]. Long chain phthalates such as BBP, DNHP, DEHP, DNOP, DINP, DIDP are frequently used as plasticizers in polyvinyl chloride plastics, adhesives, food packaging, medical equipments and several commercial and household products such as dolls, toys, shoes, tablecloths, floor tiles, furniture upholstery, etc. Thus, phthalate contamination is a global and ubiquitous public health problem and the human population is exposed at home, office, farm, and everywhere else through food, water, air, and skin absorption on a continuous basis. A study on 163 children of 2–36 months age revealed that all children had detectable levels of one or more phthalates, with 80% of children having mixture of seven or more phthalate compounds in their body [ 11 ]. A recent study [ 12 ] on 72 commonly used food samples from the market in Albany, New York revealed that every food sample had detectable levels of one or more phthalate compounds of the nine phthalates assayed, including the seven from above indicated phthalates. The frequencies of presence in food samples were DMP (37%), DBP (31%), DIBP (55%), BBP (54%), DNHP (15%), DEHP (74%) and DNOP (12%). This is of immense significance in view`of a report from the Centers for Disease Control, Atlanta, USA that shows about 100% of the population in the United States has detectable levels of one or more of the phthalate compounds [ 13 ]. Epidemiological and experimental studies on the adverse effects of phthalate chemicals on development and function of human and animal systems especially the reproductive system have been recently reviewed [ 5 , 14 – 15 ]. Phthalate exposure in men has been associated with cryptorchidism, hypospadias, gynecomastia, abnormal spermiogram and sperm DNA damage, and abnormal levels of prolactin, LH, FSH, testosterone, free androgen index, estradiol, and sex hormone binding globulin (reviewed in [ 15 ]). Phthalate exposure in women has been related with subfertility, endometriosis, leiomyomas, breast cancer, high rates of miscarriage, delayed or preterm gestation, and pregnancy complications such as anemia, toxemia, and preeclampsia (reviewed in [ 5 , 14 , 16 ]). Early pregnancy exposure may lead to shorter anogenital distance (AGD) in male infants [ 17 , 18 ] and less masculine play behaviors in boys [ 19 ] besides lower mental and physical development scores [ 20 ] and attention deficit hyperactivity disorder [ 21 ] in children. Experimental studies on phthalates in rodent models have shown a multitude of symptoms called the phthalate syndrome which in several aspects resembled the effects of phthalate exposure in human males (reviewed in [ 5 , 15 , 22 ]). The phthalate syndrome in rats is characterized by malformations in male organs (epididymis, vas deferens, seminal vesicles, prostate, external genitalia), cryptorchidism, retention of nipples/areolae, and reduced AGD [ 22 ]. The mechanisms by which EDCs, including phthalates, exert their actions are not clear and are thought to be through the nuclear hormone receptor signaling [ 1 ]. However, nonnuclear steroid receptors, orphan receptors, nonsteroid receptors, enzymatic pathways, and other mechanisms regulating the endocrine and reproductive functions have also been proposed to mediate the endocrine disrupting activity [ 1 ]. Sex hormone-binding globulin (SHBG) is a high molecular weight plasma protein that binds androgens and estrogens and plays a key role in maintaining the balance between unbound and bound sex steroids [ 23 ]. Owing to the high ligand-binding affinity, SHBG acts as a major carrier protein for steroids in the blood, and any changes in SHBG levels effects the distribution and access of these molecules to target tissues. Besides natural steroid hormones such as dihydrotestosterone, testosterone, and estradiol, SHBG has also been shown to bind several EDCs including phthalates esters [ 24 – 26 ]. Binding of the EDCs such as phthalate esters to SHBG in the body represents a potential way of interfering in the natural ligand-protein interactions and thus leading to harmful ramification for the normal functioning of the steroid target organs. Molecular modelling of zebra fish homolog of SHBG with several EDCs has been reported [ 27 – 29 ]. Recently, docking of many phthalates with androgen, progesterone, estrogen and peroxisome proliferating-activated receptors (PPARs) has also been reported [ 30 – 31 ]. However, molecular modelling studies of phthalate esters with human SHBG are apparently not available. The objective of the present study was to compare the structural binding characteristics of the above mentioned nine phthalates with SHBG using computational approaches. The binding mechanism of the nine EDCs with human SHBG was delineated by molecular docking simulation, and the comparisons of the distinctive binding pattern and the interacting residues was done.

The crystal structure of human SHBG with native ligand, DHT, is shown ( Fig 2 ). The nine phthalate compounds i.e. DMP, DBP, DIBP, BBP, DNHP, DEHP, DNOP, DINP, DIDP chosen for docking simulation achieved successful execution of IFD against steroid binding pocket of SHBG which resulted into multiple poses for each of these compounds. The best pose for each of the phthalates was chosen for analyses. Docking complexes of the nine phthalates displayed interactions with 15–31 residues of SHBG (Figs 3 – 5 , Table 2 ). For the co-complex ligand, DHT, interactions with 22 residues of SHBG were displayed by the docking complex ( Fig 5 ; Table 2 ). Overall, 12–21 SHBG interacting residues were common between the natural ligand, DHT, and the nine phthalate compounds (commonality of 55–95%; Table 2 ). Fifteen interacting residues of SHBG (Asn-82, Asp-65, Ile-141, Leu-171, Met-107, Met-139, Phe-56, Phe-67, Ser-41, Ser-42, Ser-128, Thr-40, Thr-60, Val-105, Val-112) were common among the native ligand, DHT, and all the nine phthalate compounds (the exceptions were four residues each that were not common for DMP and DBP; Table 3 ). Further, there were five interacting residues of SHBG (Asp-59, Gly-58, Leu-80, Lys-106, Trp-66) which were common among DHT and the majority (6–7) of the nine phthalate compounds (not shown). One interaction displayed by residue, Leu-34, was common for all the nine phthalate compounds but not for DHT. Conversely, one interacting residue, His-81, was displayed by the DHT docking complex but not by any of the nine phthalate compounds (not shown). The Dock score, Glide score, and binding affinity values (MM-GBSA values) for the nine phthalate compounds and the bound ligand, DHT, are presented ( Table 2 ). Among the nine phthalate compounds, DEHP, DNOP, DINP, and DIDP have the highest Dock score, and the highest Glide score which are, however, slightly lower than the native ligand, DHT. In addition, the binding affinity values (MM-GBSA values) for these four phthalates were highest among the nine phthalates and were higher than the native ligand, DHT ( Table 2 ). Four amino-acid residues indicated by hatch mark (#) were not shared by DMP and four residues indicated by star (*) were not shared by DBP.

The present study was done to delineate the structural binding characteristics of nine phthalates i.e. DMP, DBP, DIBP, BBP, DNHP, DEHP, DNOP, DINP, DIDP with SHBG using computational approaches. Docking complexes of the nine phthalates displayed interactions with 15–31 amino acid residues of SHBG and 55–95% interacting residues of natural ligand of SHBG, DHT, were common with those for the nine phthalate compounds. The binding affinity values were more negative for long chain phthalates DEHP, DNOP, DINP, and DIDP compared to that for the short chain phthalates such as DMP and DBP. The Dock score and Glide score values were also higher for long chain phthalates compared to that for the short chain phthalates. This study shows that all the nine phthalate esters were able to engage important interacting residues of SHBG during molecular interactions and, hence, can potentially displace or compete with the natural SHBG ligands such as dihydrotestosterone, testosterone, and estradiol for the availability of SHBG binding sites resulting in altered androgen-estrogen homeostasis. However, long chain phthalates may be potentially more active endocrine disruptors. Current study enhances our understanding of underlying molecular mechanism of potential interfering mechanisms of phthalates in steroid homeostasis of the human body.

Schrodinger 2015 suite containing series of modules along with Maestro 10.3 (graphical user interface) software (Schrodinger, LLC, New York, NY, 2015) was used to perform the molecular modeling of nine phthalate compounds i.e. DMP, DBP, DIBP, BBP, DNHP, DEHP, DNOP, DINP, DIDP with SHBG. The two dimensional structures of the nine phthalate compounds are illustrated ( Fig 1 ) and the abbreviations and PubChem compound identities (CIDs) of the compounds are presented ( Table 1 ). The Protein Data Bank (PDB; http://www.rcsb.org/ ) was searched and the three-dimensional structure of human SHBG (PDB code: 1D2S) at 1.55 Å. resolution was retrieved. The SHBG crystal structure was co-complexed with its natural ligand, dihydrotestosterone (DHT). The crystal structure was prepared by utilizing the protein preparation wizard workflow of Schrodinger after importing the structure into the docking software Glide (Schrodinger suite 2015–3; Schrodinger, LLC). Water molecules were removed and hydrogen atoms were added during the preparation protocol of the crystal structure and loops and missing side chains were built using Prime 3.0 module. Optimization of H-bond particularly for Asp, Glu, and His hydroxyl containing residues was then done. This was followed by optimization of the hydrogen bonding network and finally the OPLS_2005 force field was used for a geometry optimization to a maximum root-mean-square deviation (rmsd) of 0.30 Å. The bound ligand, DHT in crystal complex was selected and used for docking of the indicated nine phthalate esters and grid boxes were generated. Maestro 10.3 (Maestro, version 10.3, Schrodinger, LLC) was used for drawing ligand structures as shown ( Fig 1 ). Ligands were prepared using LigPrep module (Schrodinger 2015: LigPrep, version 3.1, Schrodinger, LLC). Correct molecular geometries and ionize at biological pH 7.4 were obtained by using the OPLS-2005 force field software. Retention of specific chirality and generation of least energy conformations was done using OPLS-2005 software. Schrodinger’s Induced Fit Docking (IDF) module with Prime program was utilized for docking analyses of the nine phthalate esters i.e. DMP, DBP, DIBP, BBP, DNHP, DEHP, DNOP, DINP DIDP. After preparation using LigPrep module, the ligands were submitted as starting geometries to IFD. The IFD has been shown to have the ability of sampling the minor changes in the backbone structure as well as robust conformational changes in side chains [ 32 ]. A softened-potential docking is performed in the first IFD stage where docking of the ligand occurs into an ensemble of the binding protein conformations. Subsequently, complex minimization for highest ranked pose is performed where ligand as well as the binding sites are free to move. The calculation of binding affinity of ligands against the binding protein molecule was done using Prime module of Schrodinger 2015 with MMGB-SA function.