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

Abstract

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 (alpha, beta, gamma, and delta) 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 gamma and delta diastereomers to be more potent activators of hAR than the alpha and beta 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.

Keywords: androgen; brominated flame retardant; endocrine disruptor.

Figure 1

The molecular structures of the BFRs examined, along with structures of DHT and testosterone for comparison. (A) TBECH-α{ rac-(1R,2R)-1,2-dibromo-(4S)-4-[(1R)1,2-dibromoethyl]cyclohexane}. (B) TBECH-β {rac-(1R,2R)-1,2-dibromo-(4S)-4-[(1S)1,2-dibromoethyl]cyclohexane}. (C) TBECH- γ {rac-(1R,2R)-1,2-dibromo-(4R)-4-[(1R)1,2-dibromoethyl]cyclohexane}. (D) TBECH-δ {rac-(1R,2R)-1,2-dibromo-(4R)-4-[(1S)1,2-dibromoethyl]cyclohexane}. (E) DHT. (F) Testosterone.

Figure 2

The four key amino acids (N705, Q711, R752, and T877/A877) in the active site of the hAR (A) and LNCaP AR (B) shown with the natural ligands DHT, testosterone (T), and the four TBECH diastereomers (C and D). The closest distances between the amino acids and the ligands for hAR (C) and for LNCaP AR (D). See “Materials and Methods” for details of simulations.

Figure 3

Competition curves for binding of DHT 50:50 TBECH-γδ, 50:50 TBECH-αβ, and purified TBECH-β to the rat AR. The amino acid sequences of the LBDs of rat and human AR are identical, so the results can be extrapolated to hAR. Each value represents the mean of three assays.

Figure 4

In vitro analysis of AR activation using HepG2 cells were transfected with both the slp-ARE-Luc reporter vector and AR expression vector pCMVhAR. (A) Time-dependent activation of AR after exposure to 100 nM T and 10 nM DHT. (B) Time-dependent activation of AR after exposure to 50:50 TBECH-γδ or 25:75 TBECH-γδ at a final concentration of 1 μM. All values were normalized against the controls; the control levels were arbitrarily set to 1, and maximal induction was set to 100%. n= 4 per exposure group. *p < 0.05, **p < 0.01, and ^#p < 0.001 by Student’s t-test compared with controls.

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.

Figure 6

Determination of PSA expression in LNCaP cells treated with DHT or one of the four mixtures of TBECH diastereomers [TBECH-β, 50:50 TBECH-αβ, 50:50 TBECH-γδ, or 25:75 TBECH-γδ) at a final concentration of 100 nM and 1 μM for 5 days. C, control. n= 4 per exposure group. *p < 0.05 and ^#p < 0.001 (Student’s t-test).