Reproductive Toxicology

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Ishfaq A. Sheikh

^⁎

, Mohd A. Beg

King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia

A R T I C L E I N F O

Keywords:

Endocrine disruption Molecular docking Sex hormone binding globulin Non-Phthalate alternate plasticizers Steroid homeostasis

A B S T R A C T

Plasticizers are chemical compounds used to increase the softness andfluidity of polymer materials. Phthalate compounds constitute the most common class of compounds used as plasticizers. However, phthalate plasticizers, especially the predominant di(2-ethylhexyl)phthalate, have been shown to have adverse effects on environment and human health. Hence, efforts have been made to use safer and environmentally friendly alternate non-phthalate plasticizers in industrial applications. The present study involves structural binding studies on endocrine disrupting potential of three high volume alternate plasticizer compounds,i.e.,di-(2-ethylhexyl) adipate (DEHA), acetyl tributyl citrate (ATBC) and 2,2,4-trimethyl 1,3-pentanediol diisobutyrate (TPIB) with sex hormone binding globulin (SHBG). This study showed that DEHA, ATBC and TPIB bind in the ligand binding pocket of SHBG and the structural binding results suggested that the three alternate plasticizers may interfere in the steroid binding of SHBG and thus may cause dysfunction in sex steroid homeostasis.

1. Introduction

Plasticizers are chemical compounds used to increase theﬂexibility and softness of polymers by lowering the polymer glass transition temperature, melt viscosity and elastic modulus [1]. Until recently, phthalate plasticizers constituted about 80–85% of the global plasticizer market for use in polyvinyl chloride plastics [2,3]. However, serious health concerns such as reprotoxicity, carcinogenesis, cardio- toxicity, hepatotoxicity and nephrotoxicity associated with phthalate compounds [4–6] prompted many governments including the United States, European Union, Japan,etc.,to put in place stringent regulations and prohibitions on the use of these plasticizers [7,8]. This has stimu- lated an increased demand for safer and environmentally friendly non- phthalate based alternate plasticizers. As of now, any new industries catering to the plasticizer production in the developed countries are almost exclusively dedicated to the production of phthalate-free plasticizers. The global plasticizer consumption was about 16 billion pounds in 2017 and the country/area-wise distribution is illustrated in Fig. 1[9]. The plasticizers contributed 15 billion dollars to the world economy in 2014 and consumption is expected to reach a market volume of 24 billion pounds in 2024, increasing the share in global

economy to 24 billion dollars [10]. The non-phthalate plasticizers ac- counted for about 35% of the world-wide consumption of plasticizers in 2017 compared to 12% in 2005 and are projected to increase to 40% by 2022 [9,11,12].

Several alternative plasticizers belonging to chemical groups such as citrates, benzoates, terephthalates, trimellitates, adipates, sebacates, phosphate esters, cyclohexane dicarboxylic acids, glycerol acetylated esters, epoxidized vegetable oils, etc., have been used to replace phthalate plasticizers [13]. However, the commonly used non-phthalate alternate plasticizers are di(2-ethylhexyl)terephthalate (DEHT), tris (2-ethylhexyl)trimellitate (TOTM), and diisononyl hexahydrophthalate (DINCH), di-(2-ethylhexyl) adipate (DEHA), acetyl tributyl citrate (ATBC) and 2,2,4-trimethyl 1,3-pentanediol diisobutyrate (TPIB) [14,15].Very limited studies are available on exposure of non-phthalate alternate plasticizers and their health eﬀects on adults and young children [16]. Hence, the Chronic Hazard Advisory Panel (CHAP) on phthalates and non-phthalate alternatives under the United States Consumer Product Safety Commission has recommended more exposure and hazard data to assess the potential health risks of non- phthalates alternate plasticizers [15]. Recently, we have reported a structural binding study [17] on molecular interactions of DEHT, TOTM

https://doi.org/10.1016/j.reprotox.2018.11.003

Received 21 May 2018; Received in revised form 20 September 2018; Accepted 19 November 2018

⁎Corresponding author.

E-mail address:[email protected](I.A. Sheikh).

Available online 20 November 2018

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and DINCH with sex hormone binding globulin (SHBG). However, structural aspects of endocrine disrupting potential of DEHA, ATBC, TPIB against SHBG are not available.

DEHA is a diester of 2-ethylhexanol and adipic acid and is preferred as a low temperature plasticizer good for cold solution storage such as in blood banks,etc.[14,18]. It is a high production volume chemical that is also commonly used in toys, vinylflooring, cables, wood veneer, coated fabrics, artificial leather, household plastic food contact materials and is becoming more popular for use in medical products and packaging [13,19–22]. It is more lipophilic and has almost three times greater leachability compared to di(2-ethylhexyl)phthalate (DEHP) [18,19,23]. Extensive use of DEHA in food packing, plastic and other commercial and household industries has become a source of exposure to human population [24]. Animal studies have shown that DEHA is slightly toxic if administered by intravenous route and is also slightly irritant to rabbit skin [19]. It does not have genotoxic properties but a number of studies revealed that repeated dose of DEHA and some of its metabolites cause reduced body weight, increased liver weight, increased liver tumors and induced peroxisome proliferation in mice [18,19,22,25]. DEHA has been reported to be toxic to crustaceans and fish [21]. DEHA has not been shown to induce testicular toxicity or antiandrogenic effects [26], but was reported to cause disturbed estrous cyclicity and increased atresia of ovarian follicles at high doses [27] and developmental problems including prolonged gestation, smaller pup size, and high postnatal mortality [28].

ATBC is an ester of citric acid having three ester bonded butyl groups and one acetyl group bonded to tertiary hydroxyl group [3,20].

It is highly lipophilic, sparingly soluble in water and is 10 times more leachable compared to DEHP in feeding solutions eliciting concern for repeated use over a long period of time [19]. Fire and explosive properties of ATBC especially in the presence of nitrates and oxidizing agents are also of major concern for its use [29]. ATBC is a high production volume chemical and is used in pharmaceutical tablet coatings, food packaging, cosmetic products,ﬂavoring in foods, adhesives, inks and as surface lubricant for metal articles in food industry [13,18,29,30]. The anticoagulant properties of ATBC also render it increasingly useful in production of blood bags and medical tubing [18,19]. Exposure of general population may occur through skin

contact or by mouth either through children’s toys or ingestion of contaminated food. Preliminary toxicological assessment of ATBC did not indicate any toxicological effects in animals [31]. Parenteral administration may affect the central nervous system and hematological parameters in laboratory animals [18] and a recent study showed that ATBC inhibits the proliferation of lymph node T cells [32]. Local sciatic nerve application of ATBC in rats showed some neurotoxicity and also blocked neural transmission when placed near the neural trunk in rabbits and rats [20]. However, oral treatment of ATBC in rats showed low acute toxicity [29], and repeated dose treatment from 14 days to 2 years showed low subchronic toxicity in rats and cats. In addition, ATBC was shown to have no genotoxicity, reproductive or developmental toxicity [29,31]. Besides, No Adverse Effect Level (NOAEL) values for ATBC were shown to be 20 times higher than the phthalate plasticizer DEHP [19]. Interestingly, in a new study [33], a lower dose of ATBC was shown to have detrimental effects on ovarian folliculo- genesis in mice and suggestions were made for further thorough studies.

Tests in human volunteers (21–60 years age) for dermal irritation and sensitization with ATBC revealed uneventful results during initial or subsequent patch tests [29].

TPIB, also abbreviated as TXIB, is an ester of the branched alkane trimethyl pentanyl with two butyrate groups. It is insoluble in water, highly lipophilic and has very low viscosity [20]. TPIB is a high production volume chemical and is used in soft surface products such as PVC leather, shoes, toys and other child care articles,flooring, wall- paper, paint, inks, packaging, fillers, traffic cones, vinyl gloves, etc.

[13,20,21]. In rats and rabbits, TPIB administration by oral route shows low acute toxicity, and in guinea pigs but not in rabbits, TPIB was slightly irritating to the skin [20]. Chronic exposure of rats with TPIB was found to induce reversible increase in liver weight [18]. Repeated doses of TPIB was found to have no reproductive toxicity; however, higher dose was shown to cause reduced number of implantation sites, reduced mean litter weights, reduced number of live pups and reduced epididymal and testicular sperm counts [20]. A 13 week treatment in dogs also resulted in no adverse eﬀects [20]. In human subjects, brief exposure to TPIB vapors was associated with slight irritation to the eyes and nasal mucosa [34]. In another study, no evidence of skin sensitization with repeated application of TPIB patches for 3 weeks was found Fig. 1.Global consumption of plasticizers illustrated by A) country/region and B) type of plasticizer (adapted from IHS Markit 2018). World consumption of plasticizers was about 16 billion pounds in 2017 contributing approximately 15 billion USD to the world economy. The ratio of phthalate and non-phthalate alternate plasticizer consumption was 65:35 in 2017 (down from 88% for phthalates in 2005) and alternate plasticizers are projected to increase to 40% by 2022.

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some proliferating-activated receptors (PPARs) did not show any sig- niﬁcant interactions [41,42]. However, DEHT, TOTM, DINCH have been shown to possess high binding aﬃnity with SHBG in a recent study [17].

The present study was carried out for structural binding characterization of DEHA, ATBC and TPIB with SHBG usingin silicoapproaches.

The structural binding information was expected, on a preliminary basis, to help in identifying potential adverse eﬀects of the three alternate plasticizers (DEHA, ATBC and TPIB) and also could assist in designing furtherin vitroandin vivoexperimental studies.

2. Methods

Three commonly used alternate plasticizers DEHA, ATBC and TPIB were considered for this study. The binding interactions of the three compounds with SHBG was performed using Schrodinger 2015 suite with Maestro 10.3 as user interface (Schrodinger, LLC, New York, NY, 2015). The procedure for ligand preparation was followed as previously reported [43]. The chemical structures of the three alternate plasticizer compounds are shown (Fig. 2) and the PubChem compound identities (CIDs) and Chemical Abstracts Service Registry Number (CASRN) are presented (Table 1).

2.1. Protein selection and preparation

An online search was conducted in the Protein Data Bank (PDB;

http://www.rcsb.org/) and the crystal 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 preparation of crystal structure was conducted using

2.2. Ligand preparation and conformational search

Maestro 10.3 (Maestro, version 10.3, Schrodinger, LLC) was used to draw structures of the three alternate plasticizer ligands DEHA, ATBC and TPIB (Fig. 2). Preparation of the ligands was carried out utilizing LigPrep module (Schrodinger 2015: LigPrep, version 3.1, Schrodinger, LLC) as described [17].

2.3. Inducedﬁt docking

Schrodinger’s Induced Fit Docking (IFD) protocol in Prime Program was used for structural binding analyses of the three alternate plasticizers DEHA, ATBC and TPIB and procedure has been described in detail [40]. A softened-potential docking was done during theﬁrst IFD stage in which docking of the ligands took place in an ensemble of SHBG conformations. Afterwards, adjustments were performed for the highest ranked pose of the docking display as described [17].

2.4. Binding aﬃnity calculations

The binding aﬃnity calculations between ligands and SHBG were done using Prime module of Schrodinger 2015 with MMGB-SA function as described [17].

3. Results

Upon IFD, the alternate plasticizer compounds DEHA, ATBC and TPIB docked successfully in the hormone binding pocket of SHBG.

Many poses of the docking interaction were generated and the computational analyses was carried out on the best pose for each of three

Fig. 2.Two dimensional representation of three alternate phthalate ligands, (A) di-(2-ethylhexyl) adipate (DEHA), (B) acetyl tributyl citrate (ATBC), (C) 2,2,4- trimethyl-1,3 pentanediol diisobutyrate (TPIB), and natural ligand (D) dihydrotestosterone (DHT).

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Fig. 3.Ribbon form representation of docking complex of human sex hormone-binding globulin (SHBG) with di-(2-ethylhexyl) adipate (DEHA) (left panel). Amino- acid residues in the binding pocket of SHBG involved in interactions with DEHA (right panel).

Fig. 4.Ribbon form representation of docking complex of human sex hormone-binding globulin (SHBG) with acetyl tributyl citrate (ATBC) (left panel). Amino-acid residues in the binding pocket of SHBG involved in interactions with ATBC (right panel).

Fig. 5.Ribbon form representation of docking complex of human sex hormone-binding globulin (SHBG) with 2,2,4-trimethyl-1,3 pentanediol diisobutyrate (TPIB) (left panel). Amino-acid residues in the binding pocket of SHBG involved in interactions with TPIB (right panel).

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compounds (Figs. 3–5). For bound native ligand, DHT, the analysis was also done on the best docking pose following IFD (Fig. 6).

3.1. Molecular docking of DEHA with SHBG

The docking display of DEHA in the binding pocket of SHBG and the amino-acid residues involved in binding interactions are shown (Fig. 3).

The binding interactions involved 29 SHBG residues (Leu-34, Thr-35, Thr-40, Ser-41, Ser-42, Phe-56, Gly-58, Asp-59, Thr-60, Asp-65, Trp-66, Phe-67, Ile-78, Leu-80, Asn-82, Trp-84, Val-105, Lys-106, Met-107, Val- 112, Leu-124, Val-127, Ser-128, Gly-129, His-136, Pro-137, Met-139, Ile-141, Leu-171). The residues Asn-82 and Gly-129 of SHBG formed two hydrogen bonding interactions with DEHA (Fig. 3). The values for the IFD score, Dock score, Glide score, and the binding aﬃnity of DEHA were lower than those for the native ligand, DHT (Table 2).

The docking display of the native ligand, DHT, in the binding pocket of SHBG and the amino-acid residues involved in binding interactions are shown (Fig. 6). The binding interactions involved 22 SHBG residues (Thr-40, Ser-41, Ser-42, Phe-56, Gly-58, Asp-59, Thr-60, Asp-65, Trp- 66, Phe-67, Leu-80, His-81, Asn-82, His-83, Val-105, Lys-106, Met-107, Val-112, Ser-128, Met-139, ILe-141, Leu-171). The native ligand, DHT, formed three hydrogen bonding interactions with SHBG residues Ser- 42, Asp-65 and Asn-82 (Fig. 6). Comparisons between docking displays of DEHA and DHT showed that 18 of 22 SHBG residues interacting with DEHA were common between DEHA and native ligand, DHT (Table 2).

3.2. Molecular docking of ATBC with SHBG

The docking display of ATBC in the binding pocket of SHBG and the amino-acid residues involved in binding interactions are shown (Fig. 4).

The binding interactions involved 29 SHBG residues (Leu-34, Thr-40, Ser-41, Ser-42, Phe-56, Gly-58, Asp-59, Thr-60, Asn-61, Asp-65, Trp-66,

Phe-67, Leu-80, Asn-82, His-83, Trp-84, Val-105, Lys-106, Met-107, Ser-111, Val-112, Val-127, Ser-128, Gly-129, His-136, Met-139, Arg- 140, Ile-141, Leu-171). The residues Asn-82 and Trp-84 of SHBG formed two hydrogen bonding interactions with ATBC (Fig. 4). The values for the IFD score, Dock score, Glide score, and the binding af- ﬁnity of ATBC were lower than those for the native ligand, DHT (Table 2). Comparisons between docking displays of ATBC and DHT showed that 21 of 29 SHBG residues interacting with ATBC were common between ATBC and native ligand, DHT (Table 2).

3.3. Molecular docking of TPIB with SHBG

The docking display of TPIB in the binding pocket of SHBG and the amino-acid residues involved in binding interactions are shown (Fig. 5).

The binding interactions involved 23 SHBG residues (Ser-41, Ser-42, Phe-56, Gly-58, Asp-59, Thr-60, Asn-61, Asp-65, Trp-66, Phe-67, Leu- 80, His-81, Asn-82, Trp-84, Val-105, Lys-106, Met-107, Val-112, His- 136, Met-139, Ile-141, Trp-170, Leu-171). The residue Asn-82 of SHBG formed a hydrogen bonding interaction with TPIB (Fig. 5). The values for the IFD score, Dock score, Glide score, and the binding aﬃnity of TPIB were lower than those for the native ligand, DHT (Table 2).

Comparisons between docking displays of TPIB and DHT showed that 19 of 23 SHBG residues interacting with TPIB were common between TPIB and native ligand, DHT (Table 2).

3.4. Comparison among ligands

The binding interactions of alternate plasticizers, DEHA, ATBC and TPIB, in the binding pocket of SHBG involved 23–29 amino-acid residues of SHBG (Table 2). Comparison of the three ligands with native ligand, DHT, revealed that 19–21 SHBG interacting amino acid residues overlapped between DHT and the compounds showing commonality of Fig. 6.Overall all ribbon form representation of human sex hormone-binding globulin (SHBG) co-complex with natural ligand, dihydrotestosterone (DHT) (left panel). Amino-acid residues in the binding pocket of SHBG involved in interactions with DHT (right panel).

Table 2

Number of interacting residues, number and percentage of residues common with dihydrotestosterone (DHT), Induced Fit Docking (IFD) score, Dock score, Glide score and binding aﬃnity values (MMGB-SA values) of di-(2-ethylhexyl) adipate (DEHA), acetyl tributyl citrate (ATBC), 2,2,4-trimethyl-1,3 pentanediol diisobutyrate (TPIB), and natural ligand, DHT, after IFD with human sex hormone-binding globulin (SHBG).

S. No. Ligand Number of interacting residues Number of interacting residues common with DHT (%) IFD Score Docking score (Kcal/mol) Glide score (Kcal/mol)

MMGB-SA (Kcal/mol)

1 DEHA 29 20 (91%) −381.29 −9.02 −9.02 −111.01

2 ATBC 29 21 (95%) −380.48 −7.63 −7.63 −109.43

3 TPIB 23 19 (86%) −379.58 −5.90 −5.90 −78.50

4 DHT 22 22 (100%) −375.72 −12.02 −12.02 −129.89

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86–95% (Table 2). Eighteen SHBG interacting residues were common among DHT and all the three alternate plasticizer compounds (Table 3).

In addition, two SHBG amino acid residues were common among DHT, DEHA and ATBC but not TPIB (Table 3). Conversely, two interacting SHBG amino acid residues (Trp-84, His-136) were common among the three alternate plasticizer ligands but not for DHT. All the three alternate plasticizer ligands and the native ligand, DHT, formed a hydrogen bonding interaction with amino-acid residue, Asn-82, of SHBG. The IFD score, Dock score, Glide score and binding aﬃnity were lower in all the three alternate plasticizer ligands compared to those of DHT and the order of values was DHT < DEHA < ATBC < TPIB (Table 2).

4. Discussion

Human SHBG is a liver secretory protein which transports sex steroids, mainly DHT, testosterone, and estradiol in blood and regulates their metabolic clearance and availability to the target tissues [36,44].

Major portion of the steroids in the blood is in a bound form and only a small percentage 1–3% is free or“bioactive”, which is thought to be responsible for the steroidogenic action on the target organs [36]. The androgens (DHT, testosterone) and estradiol have been shown to bind competitively with the same steroid-binding sites of SHBG, but orient in opposite and inverted manner [45]. Dysfunctions and abnormal plasma concentrations of SHBG have been suggested to cause reproductive and other disorders such as infertility, ovarian dysfunction, endometrial cancer, cardiovascular problems and diabetes [46].

Biomonitoring studies on human fluids have shown that the phthalate exposure occurs at every stage of human development including prenatal life, early childhood, adolescence and adult life [47,48]. Under natural conditions exposure occurs to a mixture of phthalate compounds simultaneously. Besides adverse effects on reproductive tissues and functions, exposure of phthalate plasticizers in children and adults may lead to gastrointestinal distress, respiratory problems, allergy and asthma, and thyroid problems [49,50]. Sig- nificant correlations have been reported between phthalate metabolites in humanfluids and obesity and insulin resistance [51]. Experimental exposure in rodents causes adverse effects on weight gain and food consumption, adverse effects on kidneys, increase in weight of liver and lungs, and hepatocellular carcinoma along with a variety of other liver related effects such as proliferation of peroxisomes and mitochondrial activities, tissue proliferation, suppression of apoptosis, etc.[47]. Some of these adverse effects may be through deregulation of metabolic pathways by phthalate compounds through interaction with peroxisome proliferator-activated receptors [51].

Recommended limits or thresholds for exposure are available for only four phthalate plasticizer compounds, i.e., dibutyl phthalate, die- thyl phthalate, DEHP and dimethyl phthalate, according to the latest

lists of chemicals from National Institute for Occupational Safety and Health (NIOSH) and the Occupational Safety and Health Administration (OSHA) in the United States [52]. However, the limits are not available for any of the six most common alternate plasticizer compounds including the three indicated in the current study. The recommended exposure limit by NIOSH and permissible exposure limit by OSHA for all the four phthalate compounds have a cutoﬀconcentration of 5 mg/

m³averaged over an 8 h work shift based on a time-weighted average [52]. In addition, DEHP being the most prevalent and most regulated phthalate, NIOSH has recommended a short term exposure limit of 10 mg/m³for any 15 min work period for DEHP.

Regarding the strategies to reduce the exposure, it is practically impossible to avoid phthalate exposure because of their ubiquitous presence in almost all of our daily use commercial and domestic ap- pliances and products. But adoption of few practices and changing personal and eating behavior may help in reducing the burden [53,54].

These include minimizing plastic use and increasing the use of stainless steel and glass and if you have to, choose plastics with codes of 1, 2, or 5; including more vegetarian and organic food stuﬀs in diets and avoiding processed foods; choosing low fat meat and dairy products;

routine use of waterﬁlters; completely avoiding synthetic fragrances;

using a respirator and/or changing ventilator in occupational setting, etc.

The current study involved structural binding characterization of three alternate plasticizer compounds, DEHA, ATBC and TPIB with SHBG using IFD. Comparable IFD scores between the native ligand DHT and the three alternate plasticizer compounds together with good docking scores indicated that the ligands positioned well into the steroid binding pocket of SHBG. Good binding affinity of the ligands also indicated that their docking complexes were in the most favorable conformation. A number of important amino acid residues of SHBG interacted with the alternate plasticizer ligands through hydrophobic, pi-pi interactions and hydrogen bonding that aid the stability of the docking complex. A commonality of 86–95% amino acid residues of SHBG in the docking complexes of the alternate plasticizer ligands and the DHT indicated similarity in the structure and positioning of the compounds in the steroid binding pocket of SHBG. A high majority of amino acid residues (18 of 22) together with a common hydrogen bonding interaction (Asn-82) were shared between the docking complex of DHT-SHBG and docking complexes of all the three alternate plasticizers. This suggested that under exposure conditions when suf- ficient quantities of the indicated alternate plasticizers compounds gain access into the body, a potential interference in the transport me- chanism of sex steroids may occur which may hinder the availability of these endogenous steroids to target tissues resulting in organ dysfunction. Dock score values and binding affinity of the three alternate plasticizer compounds reveal that the prediction for DEHA causing dysfunction is more compared to ATBC and TPIB.

Previous studies on the docking of DEHA, ATBC and TPIB with SHBG are not available. However, many steroid and nonsteroid compounds have been shown to bind with SHBG using comparative molecular field analysis (CoMFA) and molecular similarity indices in a comparative analysis (CoMSiA) [55–57]. But, DEHA, ATBC and TPIB were not part of any of these studies.In vitrostudies on competitive binding of alternate plasticizers with SHBG are also not apparently available. Docking of DEHA and ATBC but not TPIB with peroxisome proliferator-activated receptors (PPAR)-αand -γwas recently reported [42]. DEHA was shown to bind with PPAR-αand PPAR-γ with low affinity but ATBC did not show any binding. Even the low affinity of DEHA with PPAR-α was suggested to explain the peroxisome proliferation in rat liver as reported in an earlier study [25] and induction of liver tumors in male and female rats [14].

We have recently reported several structural binding studies on phthalate plasticizers, bisphenol A, octylphenol and nonylphenol with SHBG [17,40]. All these ligands showed a high binding aﬃnity with SHBG and about 80–90% of the interacting amino acid residues of Table 3

Human sex hormone-binding globulin (SHBG) interacting residues that were common among co-complex natural ligand, dihydrotestosterone (DHT), and alternate plasticizers, di-(2-ethylhexyl) adipate (DEHA), acetyl tributyl citrate (ATBC), 2,2,4-trimethyl-1,3 pentanediol diisobutyrate (TPIB).

S.No. Common interacting residues S.No. Common interacting residues

1 Thr-40^* 11 Leu-80

2 Ser-41 12 Asn-82

3 Ser-42 13 Val-105

4 Phe-56 14 Lys-106

5 Gly-58 15 Met-107

6 Asp-59 16 Val-112

7 Thr-60 17 Ser-128^*

8 Asp-65 18 Met-139

9 Trp-66 19 Ile-141

10 Phe-67 20 Leu-171

Amino-acid residues indicated by star.

* were not shared by TPIB.

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alter the steroid and drug metabolism [59]. Low levels of ATBC also altered folliculogentic parameters in ovaries of rats and increased apoptotic changes in isolated mouse follicles [33,60]. Even the low level exposures of the alternate plasticizer compounds may also be important during times of low gonadal hormone levels in prepubertal period [61,62] or when SHBG levels are higher such as in gestation [44]

and during contraceptives use [63]. The greater availability of the SHBG steroid binding sites will shift the equilibrium favorably towards the toxic compounds resulting in interference in estrogens and androgens homeostasis.

Human exposure data for alternate plasticizers is very limited.

However, few studies have recently reported the extent of indoor/

outdoor contamination and food contamination. In a study on dust contamination across USA [64], non-phthalate plasticizers were detected in majority of the dust samples from childcare facilities, homes and salons. ATBC and DEHA were dominant non-phthalate plasticizers in salon dust and were also present at equal levels to DEHP in childcare facilities and homes. Similarly, DEHA and ATBC were also found at high levels in indoor child care centers in Germany [30]. In Canada, a study showed that majority of the 26 food types including beef,ﬁsh, poultry pork, veal, lamb were found to have high levels of DEHA possibly present due to packaging in PVCﬁlms [65]. Further, DEHA was also recently detected in commercially available baby foods [66]. It is expected that regulatory prohibitions for many phthalate plasticizers in toys and other articles in the USA and Europe [7,8] will likely result in increased applications of non-phthalate alternate plasticizers and thus result in higher contamination of indoor environment.

5. Conclusions

Non-phthalate alternate plasticizers are increasingly having many applications in commercial and household products which become a source of eco-contamination leading to direct or indirect exposure of human population. The present study involved structural binding characterization of three alternate plasticizers, DEHA, ATBC and TPIB with human SHBG. Interactions of important amino acid residues of SHBG through hydrophobic, pi-pi and hydrogen bonding interactions with alternate plasticizer ligands and good binding aﬃnity of the ligands with SHBG was found. The interacting amino acid residues of SHBG among alternate plasticizer ligands and the DHT indicated similarity in structure and positioning of the compounds in the steroid binding pocket of SHBG. This suggested that alternate plasticizers have potential to interfere in estrogen and androgen homeostasis which may hinder the availability of these endogenous steroids to target tissues resulting in organ dysfunction.

Conﬂict of interest

The authors have declared that no competing interests exist.

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