Diastereomers of the Brominated Flame Retardant 1,2-Dibromo-4-(1,2 dibromoethyl)cyclohexane Induce Androgen Receptor Activation in the HepG2 Hepatocellular Carcinoma Cell Line and the LNCaP Prostate Cancer Cell Line

Background Reported incidences of prostate cancer and masculinization of animals indicate a release of compounds with androgenic properties into the environment. Large numbers of environmental pollutants have been screened to identify such compounds; however, not until recently was 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) identified as the first potent activator of the human androgen receptor (hAR). TBECH has been found in beluga whales and bird eggs and has also been found to be maternally transferred in zebrafish. Objectives In the present study we investigated interaction energies between TBECH diastereomers (α, β, γ, and δ) and the hAR, and their ability to activate the receptor and induce prostate-specific antigen (PSA) expression in vitro. Methods We performed computational modeling to determine interaction energies between the ligand and the AR ligand-binding site, and measured in vitro competitive binding assays for AR by polarization fluorometry analysis. We used enzyme-linked immunosorbent assays to determine PSA activity in LNCaP and HepG2 cells. Results We found the γ and δ diastereomers to be more potent activators of hAR than the α and β diastereomers, which was confirmed in receptor binding studies. All TBECH diastereomers induced PSA expression in LNCaP cells even though the AR present in these cells is mutated (T877A). Modeling studies of LNCaP AR revealed that TBECH diastereomers bound to the receptor with a closer distance to the key amino acids in the ligand-binding domain, indicating stronger binding to the mutated receptor. Conclusions The present study demonstrates the ability of TBECH to activate the hAR, indicating that it is a potential endocrine disruptor.

During the last several years, the incidences of prostate and testicular cancer have increased significantly. By 50 years of age, about 50% of all men will suffer from prostatic hyper plasia (Berry et al. 1984). It has been demon strated that exposure to androgens, such as dihydrotestosterone (DHT), increases the risk for the development of prostate cancer (Carson and Rittmaster 2003). There have also been reports of masculinization of animals and wild life populations (Ellis et al. 2003). The increase in prostate cancer and the recorded masculini zation of animals indicate that there are com pounds in the environment with androgenic properties. This has led to research focused on the identification of substances with poten tial AR agonistic properties by screening large numbers of environmental compounds (Fang et al. 2003;Kojima et al. 2004;Sohoni and Sumpter 1998). These studies led to the iden tification of several estrogen receptor (ER) ago nists as being androgen antagonists. However, although an earlier study demonstrated that 2tertbutylanthraquinone and benzoanthrone may act as weak agonists to the human andro gen receptor (hAR) at high concentrations (Araki et al. 2005), only recently did our group identify the brominated flame retardant (BFR) 1,2dibromo4(1,2 dibromo ethyl)cyclohexane (TBECH) as the first environmental chemi cal to bind to and activate the hAR with high potency (Larsson et al. 2006). AR is activated through binding of a ligand, such as testos terone or DHT, to its ligandbinding domain (LBD), followed by dis sociation of inhibi tory heatshock proteins. After activation, the AR-ligand complex migrates into the nucleus and binds to its response element, which together with coactivators initiates transcrip tion and cellular responses (Brinkmann et al. 1999;Veldscholte et al. 1992).
Because of the four chiral carbons pres ent in its structure, TBECH can exist in four diastereo isomeric forms (α, β, γ, and δ). The α and β forms are found in the commercial flame retardant Saytex BCL 462 (Albemarle Corp., Baton Rouge, LA, USA), whereas the γ and δ forms are converted from α and β at temperatures > 120°C ). In 2002, TBECH was reportedly produced at volumes between 4 and 225 metric tons [U.S. Environmental Protection Agency (EPA) 2002]. TBECH was reported to be mutagenic in a study that tested 27 different chemicals using a L5178Y tk + /tkmouse lymphoma cell forwardmutation assay (McGregor et al. 1991). In 1995, TBECH was found to be present in industrial waste water near Haifa, Israel (Santillo et al. 1997), and more recent studies have reported the presence of TBECH in beluga whales in the Canadian Arctic (Tomy et al. 2008). In a recent study using zebrafish, Nyholm et al. (2008) showed that TBECH can be maternally transferred; they speculated that higher levels of TBECH would be found in the offspring of species that invest more lipids in their eggs, which is in line with the recent discovery of TBECH in eggs from herring gulls and doublecrested cormorants (Gauthier et al. 2009).
In the present study, we analyzed the inter action energies between the different TBECH diastereomers and the hAR in silico and deter mined their potential to bind and activate the receptor and its downstream target, prostate specific antigen (PSA) in vitro.

Chemicals.
We synthesized TBECH diastere omers as previously described . DHT and testosterone were purchased from Sigma Aldrich (St. Louis, MO, USA). TBECHαβ and TBECHβ were dissolved with dichloromethane that was allowed to evaporate after addition of dimethyl sulfoxide (DMSO). All other ligands were dissolved in DMSO. Exposure of cells was performed in cell culture media with a maximum of 0.1% DMSO present.
Computational modeling. We determined the interaction energies between the ligand and the AR ligandbinding site using the Background: Reported incidences of prostate cancer and masculinization of animals indicate a release of compounds with androgenic properties into the environment. Large numbers of environmental pollutants have been screened to identify such compounds; however, not until recently was 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) identified as the first potent activator of the human androgen receptor (hAR). TBECH has been found in beluga whales and bird eggs and has also been found to be maternally transferred in zebrafish. oBjectives: In the present study we investigated interaction energies between TBECH diastereomers (α, β, γ, and δ) and the hAR, and their ability to activate the receptor and induce prostatespecific antigen (PSA) expression in vitro. Methods: We performed computational modeling to determine interaction energies between the ligand and the AR ligand-binding site, and measured in vitro competitive binding assays for AR by polarization fluorometry analysis. We used enzyme-linked immunosorbent assays to determine PSA activity in LNCaP and HepG2 cells. results: We found the γ and δ diastereomers to be more potent activators of hAR than the α and β diastereomers, which was confirmed in receptor binding studies. All TBECH diastereomers induced PSA expression in LNCaP cells even though the AR present in these cells is mutated (T877A). Modeling studies of LNCaP AR revealed that TBECH diastereomers bound to the receptor with a closer distance to the key amino acids in the ligand-binding domain, indicating stronger binding to the mutated receptor. conclusions: The present study demonstrates the ability of TBECH to activate the hAR, indicating that it is a potential endocrine disruptor.  (PDB 2009) was subjected to minimiza tion using the Amber99 force field embed ded in MOE, and the minimized structure was used as template for dockings with all ligands. Before docking, the ligandbinding site was determined using the MOE Alpha Site Finder. We performed the dockings as Monte Carlo-simulated annealingbased flex ible docking of the ligands into the receptor, using the automated docking as incorporated in MOE. Each evaluated system was set to a maximum of 500 confirmed dockings, where the docked structures were sorted based on the lowest Svalue (the objective function, based on evaluating the affinity ΔG scoring func tion, which is a combination of strain energy and mutual similarity score). Before calcula tion of interaction energies, the docked struc tures were subjected to relaxation, molecular dynamics simulations, and additional relax ation as previously described (Larsson et al. 2006). The AR from LNCaP cells (a prostate cancer cell line) harbors a mutation (T877A) in the ligandbinding pocket (LBP), so the LNCaP AR was modeled using the crystal structure of the hAR as a template, as pre viously described (Larsson et al. 2006). The model was generated as a Cartesian average of 10 models and minimized using the Amber99 force field. When this was done, the LNCaP AR model was used for docking simulations in the same manner as the hAR.
Competition assay. We performed com petitive binding assays for AR using the PolarScreen AR competition assay (PanVera, Madison, WI, USA) according to the manu facturer's instructions, using polarization fluo rometry analysis, with excitation at 485 nm and emission at 535 nm, on a GeniosPro instrument (Tecan Trading AG, Männedorf, Switzerland). The ARLBD used in the PolarScreen AR competition assay is derived from rat but shows 100% sequence homology to the hAR: The amino acid sequences of the LBDs of rat and human AR [GeneBank acces sion numbers J05454 and M20132 (National Center for Biotechnology Information 2009), respectively] are identical. The final concen tration of AR LBD was 50 nM. Binding affinity was determined using competition against the synthetic androgen Fluormone AL Green ligand (2 nM; Invitrogen). DHT was used as a positive control. We produced competition curves for DHT and TBECH diastereomers using concentrations ranging from 1 nM to 10 µM. All analyses were per formed in triplicate.
Before transfection, the cells were seeded onto 24well plates in an antibioticfree and phenolfree medium complemented with charcoalstripped FCS. The charcoalstripped serum was prepared by mixing serum with activated charcoal and Dextran T70 (Sigma Aldrich Sweden AB, Stockholm, Sweden). After 12 hr incubation at 4°C, the mixture was centrifuged to remove the charcoal/ dextran, and the supernatant was filtersterilized. At 90-95% confluence, the cells were transfected with 270 ng slpARELuc (sexlimiting pro tein-androgen response element-luciferase) reporter, 270 ng hAR expression vector (pCMVhAR), and 60 ng Renilla luciferase (pRL; Promega, Madison, WI, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. The slpARELuc vector contains four copies of an ARE that has been shown to be specific for AR activation while minimizing the influence of glucocorticoids (Verrijdt et al. 2002). At 24 hr post transfection, the medium was aspi rated and replaced with medium containing different concentrations DHT, testosterone, or different diastereomers of TBECH. After exposure (8 hr for testosterone and DHT, 40 hr for TBECH), the cells were lysed in situ using passive lysis buffer (Promega), and luciferase levels were meas ured using the Dual Luciferase Assay Kit (Promega) in a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). The luciferase values were normal ized to the corresponding Renilla values. All analyses were performed in triplicate.
Enzyme-linked immunosorbent assay (ELISA). For the ELISA, human LNCaP cells were cultured in culture flasks in EMEM (Invitrogen) supplemented with 10% FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1% antibiotic antimycotic mixture in an incubator at a stable environ ment of 95% humidity, 5% CO 2 , and 37°C. Before challenge, the cells were seeded onto 24well plates in cell culture media containing charcoalstripped serum.
LNCaP cells were challenged with 100 nM DHT or different combinations of the TBECH diastereomers at 1 µM. The cells were challenged for 5 days; the super natants were collected by gentle pipetting and stored at -80° C until use. PSA quantification was performed by coating each well, in 96well plates, with 100 µL of a 1:1 mix of sample and coating buffer (0.1 M sodium carbonate,

Results
Ligand docking. We determined ligand dock ing of TBECH in hAR and LNCaP AR LBD using molecular modeling techniques. The BFR TBECH can exist as four diastereomers (α, β, γ, and δ), which differ in the manner in which the bromine atoms are oriented in their molecular structures (Figure 1). These differ ent orientations of the bromine atoms in the molecule strongly affect their binding affinities in the AR LBD.
Docking simulations established that all four TBECH diastereomers occupied the same hAR LBP as did DHT (Figure 2A). In the LNCaP AR, the structure of the LBP volume 117 | number 12 | December 2009 • Environmental Health Perspectives differs from the hAR because of the T877A mutation. This mutation shortens the distance between ligands and Ala877 in the LBP with a concomitant reduction of ligand discrimina tion ( Figure 2B). The differences in distance between the key amino acids [Asn705 (N705), Gln711 (Q711), Arg752 (R752), Thr877 (T877), and Ala877 (A877)] and ligands are shown in Figure 2C and D.
Ligand-receptor interaction energies. The substantial differences in distances between the amino acids in the LBP and ligand observed for the four TBECH diastereomers mani fested as significant differences in interaction energies. The natural ligands DHT and tes tosterone bound to the hAR with interaction energies of -53.8 and -48.9 kcal/mol, respec tively (Table 1). Among the four TBECH diastereomers, TBECHδ displayed the closest similarities to DHT (Table 2, Figure 2C). The LBP inter action energy with TBECHδ was -40.1 kcal/mol, whereas those for TBECHα, β, and γ were -34.8, -26.8, and -35.3 kcal/ mol, respectively. Interaction of DHT and TBECH diastereomers with the key amino acids in the hAR LBD are shown in Figure 2A and C. In the LNCaP AR, the mutation T877A resulted in increased interaction ener gies, which suggest stronger inter actions with the key amino acids in the mutated receptor (Tables 1 and 2, Figure 2B,D).
Competition assays. We tested the different diastereomers of TBECH for receptor binding affinity using the PolarScreen AR competi tive assay. In contrast to TBECHβ, TBECH diastereomers α, γ, and δ cannot be isolated; this prompted analysis of combinations of these in the receptor binding assay. We determined the binding affinity using competition against dexa methasone, (Figure 3). In this assay, DHT had a halfmaximal inhibi tory concentration (IC 50 ) of 21.5 nM with a goodness of fit (R 2 ) of 0.91. The relative affinity of the TBECH diastereomers was 655 nM (R 2 = 0.96) for TBECHβ, 191 nM (R 2 = 0.97) for a 50:50 mixture of TBECHα and β (TBECHαβ), 47.4 nM (R 2 = 0.98) for a 50:50 mixture of TBECHγ and δ (50:50 TBECHγδ), and 35.9 nM (R 2 = 0.99) for a 25:75 mixture of TBECHγδ. This indicates that TBECHγδ binds AR with an affinity very similar to that of DHT, whereas TBECHβ has the lowest affin ity of the TBECH diastereomers (Figure 3). AR activation assays. We determined the activation capacity of the different TBECH diastereomers using trans activa tion studies in HepG2 and LNCaP cells. We used HepG2 cells to allow comparison with an earlier study (Larsson et al. 2006), and we used LNCaP cells because they contain an AR mutation that is frequently seen in prostate cancer. Before exposure, the cells were transfected with the slpARELuc reporter vector and the hAR expression vector pCMVhAR together with an internal control (pRL). We used the slpARELuc vector because it contains four copies of an ARE that is highly specific for AR inter actions. The optimal time and concentra tion of exposure was determined for DHT, testosterone, and different combination of the TBECH diastereomers β, 50:50 αβ, 50:50 γδ, and 25:75 γδ.
Stimulation of HepG2 cells with testoster one and DHT resulted in maximal induction after 8-12 hr of exposure ( Figure 4A), whereas the TBECHγδ diastereomers (both 50:50 and 25:75) showed a slower response with maximal induction after 24 hr ( Figure 4B). DHT was also a stronger inducer than was testosterone, which required 10fold higher concentrations (100 nM) to induce hAR acti vation to the same level as DHT.
AR activity decreased after further stimu lation at 24 hr and 48 hr, suggesting early activation and fast cellular metabolism of DHT. We also assessed the timedependent AR activation in response to different com binations of TBECHγδ (50:50 and 25:75) using a final concentration of 1 µM. The two TBECHγδ combinations activated the AR     equally. Activation of AR reached statistical significance after 2 hr and maximal induction after 24 hr ( Figure 4B).
To determine the optimal concentrations of DHT and TBECH isoforms for AR activa tion, we stimulated HepG2 cells with DHT in a dosedependent manner for 8 hr and with TBECH for 40 hr. Exposure to 50:50 TBECHγδ resulted in a halfmaximal effective concentration (EC 50 ) of 14.9 nM (R 2 = 0.96), whereas exposure to 25:75 TBECHγδ resulted in an EC 50 of 22.7 nM (R 2 = 0.94; Figure 5). This suggests that TBECHγ may be a better inducer of AR than TBECHδ. Both of these diastereomers induce AR at con centrations that are comparable to those of DHT (10.5 nM; R 2 = 0.92) and indicate that these TBECH diastereomers are highly potent androgen agonists. Determination of AR acti vation by 50:50 TBECHαβ demon strated that these diastereomers are less potent, with an EC 50 of 174 nM (R 2 = 0.94), one order of magnitude higher than DHT or TBECHγδ. The weakest inducer of AR was TBECHβ, with an EC 50 of 294 nM (R 2 = 0.84). We also observed that TBECHγ and δ are complete agonists to DHT, whereas TBECHα and β are partial agonists because they conferred only partial induction. Determination of relative induction by the different compounds showed a 23.7 ± 5.0fold induction after exposure to DHT, a 24.4 ± 5.1fold induction with 50:50 TBECHγδ, a 25.5 ± 1.3fold induction with 25:75 TBECHγδ, an 8.4 ± 1.2fold induc tion with TBECHαβ, and a 2.4 ± 0.3fold induction with TBECHβ.
In vitro ELISA assays. We used LNCaP cells to determine the ability of TBECH to induce endogenous gene expression of PSA, a downstream target of the AR. The results revealed a 2.61 ± 0.15fold induction of PSA after treatment with 100 µM DHT ( Figure 6). PSA expression increased in response to all four TBECH diastereomer combinations in a dosedependent manner. Exposure to 1µM of the TBECH diastereomers resulted in a 3.11 ± 0.31fold induction by TBECHβ, a 3.22 ± 0.26fold induction by 50:50 TBECHαβ, 3.47 ± 0.12fold induction by 50:50 TBECHγδ, and a 3.74 ± 0.17fold induction by 25:75 TBECHγδ. The equiva lent inducibility by the different compounds is in agreement with the modeling data that show similar distances to the key amino acids in the AR LBP of the mutated LNCaP AR.

Discussion
TBECH belongs to a group of BFRs that are found in a variety of products, for example, oriented strand boards, particle boards, and rigid foam and soft foam used in insulation and as stuffing in furnishings, respectively, or as an additive in polystyrene and polyurethane. The discovery of TBECH's presence in both sediments and organisms (Gauthier et al. 2009;Nyholm et al. 2008;Santillo et al. 1997;Tomy et al. 2008), along with its potent activation of AR at nanomolar concentrations, suggests that these compounds constitute a serious threat to both humans and wildlife. Using the Waste Minimization Prioritization Tool, the U.S. EPA (2000) ranked TBECH as one of the 10% most hazardous compounds to ecosys tems. TBECH is an additive flame retardant, blended into material during manufacturing. In some processes this may involve thermal procedures and certainly, in the event of fire, elevated temperatures are likely to cause an interconversion among the different TBECH diastereomers. Thus, although TBECHβ is the most abundant isoform, it is reasonable to believe that all four diastereomers are present and cause a threat to the environment.
A wellknown characteristic of steroid receptors is that they bind their natural ligand with high specificity. Reported IC 50 and EC 50 values for sex steroid receptors by their natu ral ligands and the aryl hydrocarbon receptor (AhR) by TCDD (2,3,7,8tetrachloro dibenzo pdioxin) are listed in Table 3. Interestingly, when investigating substances with reported high endocrinedisrupting effects such as nonyl phenol or bisphenol A (both are estro genic) and vinclozolin (an androgen antag onist), none of the substances, with the exception of TCDD, binds to or activates the steroid receptors by > 1% of the endogenous ligand (Table 4). In our study, we found DHT to have an IC 50 of 21.5 nM, which correlates well with IC 50 values reported in other studies (Table 3). When we examined the TBECH diastereomer binding activities, we found that 50:50 TBECHγδ binds to the AR with 22% Figure 5. Determination of AR activation in response to TBECH diastereomers. HepG2 cells were stimulated with DHT for 8 hr or with TBECH diastereomers for 40 hr at concentrations ranging from 1 nM to 10 µM. Both combinations of TBECH-γδ were more potent AR activators than TBECH-β or TBECH-αβ. All values were normalized against the controls that were arbitrarily set to 1. n = 4 per exposure group.  ER binds 17β-estradiol, AR binds DHT, progesterone receptor (PR) binds progesterone, and AhR binds TCDD. a Data from Blair et al. (2000). b Data from Pillon et al. (2005). c Data from Cabeza et al. (2004). d Data from Xu et al. (2005). e Data from Viswanath et al. (2008). f Data from Lenasi and Breskvar (2004). g Data from Bonefeld-Jørgensen et al. (2007), h Data from Westerink et al. (2008).  Fang et al. (2003), with the values obtained with natural ligands of each receptor set to 100%. a Data from Blair et al. (2000). b Data from Pillon et al. (2005). c Data from Scippo et al. (2004). d Data from Bonefeld-Jørgensen et al. (2007). e Data from Legler et al. (1999). f Data from Fang et al. (2003). g Data from Viswanath et al. (2008) and 6). Furthermore, comparison of activation potential demonstrated that both TBECHγδ mixtures were was as potent as DHT at activat ing the hAR ( Figure 5). The only other envi ronmental contaminant inferred to be able to maximally activate a ligated receptor is TCDD (Table 4). This indicates that the TBECH diastereomers are extremely potent AR agonists compared with other known pollutants with proven endocrinedisrupting effects.
Recently, TBECH was shown to be pres ent in beluga whales and herring gull eggs at nanomolar concentrations (Gauthier et al. 2009;Tomy et al. 2008). Lowlevel expo sure to endocrinedisrupting compounds can induce functional, developmental, behavioral, and trans generational disturbances, as shown after lowlevel exposure to the fungicide vin clozolin, which acts as an androgen antagonist in rats and mice (Anway et al. 2006Elzeinova et al. 2008;Skinner et al. 2008). Therefore, although lowlevel exposure to the most common TBECH, TBECHβ, may not induce high AR activation, these compounds may nonetheless induce trans generational effects at the observed levels.
The natural ligands testosterone and DHT showed maximal hAR activation already after 8 hr (Figure 4). A recent study showed that HepG2 cells rapidly metabolized testosterone and DHT, resulting in 82% and 46% reduc tion, respectively, 21 hr after addition of the hormone (Simon and Mueller 2006). In con trast, determination of timedependent hAR activation by TBECH indicates slower receptor activation and prolonged induction times, sug gesting that these compounds are more stable in this cell line. The stability of TBECH is further supported by the discovery of its pres ence in the environment (Gauthier et al. 2009;Tomy et al. 2008) as well as its ability to be maternally transferred in zebrafish (Nyholm et al. 2008).
Although LNCaP is an androgen dependent cell line with a mutated AR (T877A), it retains the androgen binding and ligand specificity in the LBP of the AR (Gaddipati et al. 1994;Wang et al. 1997). This mutation is frequently detected and has been reported to be present in 30% of hormone refractory prostate cancer patients (Taplin et al. 1999). PSA is a welldefined androgenregu lated glycoprotein present in LNCaP cells and is widely used as a marker for prostate cancer diagnosis (Wang et al. 1997). In the present study, we found that both DHT and the four TBECH diastereomers induce comparable PSA expression in LNCaP cells when exposed to 100 nM of the compounds. TBECHβ was located farthest away from R752 in the hAR. This could explain its poor ability to activate the hAR. However, the LNCaP AR mutation (T877A) enables the different diastereom ers to bind with a closer distance to the key amino acids in the LBP, which could explain TBECHβ's ability to induce PSA expres sion in LNCaP cells. The higher affinity of TBECHβ to LNCaP AR and the frequency of the T877A mutation suggest that these compounds are active in a large proportion of prostate tumors and that they may contribute to the etiology of prostate cancer.
The present study provides important data on the ability of TBECH to bind and acti vate the hAR with high affinity. Combining the results from the molecular modeling, the competition assay, and the activation assay, the TBECHδ diastereomer appears to be the most potent, followed by TBECHγ and TBECHα, with TBECHβ being the least potent activator of AR. Furthermore, as the modeling experiments are in agreement with results obtained from the in vitro studies, this demonstrates that modeling is a powerful tool when identifying potential endocrine dis ruptors. However, it remains unknown how TBECH interacts with AR in other species, such as threespined stickleback and zebrafish, that both have 11ketotestoterone as their most potent activator of the AR (Hossain et al. 2008;Olsson et al. 2005). Therefore, future studies are needed to determine the interaction of TBECH with AR from other species in order to determine its effects on species present in the environment.