In Vitro Effects of the Endocrine Disruptor p,p'-DDT on Human Follitropin Receptor

Abstract

Background: 1-chloro-4-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl]benzene (p,p'-DDT) is a persistent environmental endocrine disruptor (ED). Several studies have shown an association between p,p'-DDT exposure and reproductive abnormalities.

Objectives: To investigate the putative effects of p,p'-DDT on the human follitropin receptor (FSHR) function.

Methods and results: We used Chinese hamster ovary (CHO) cells stably expressing human FSHR to investigate the impact of p,p'-DDT on FSHR activity and its interaction with the receptor. At a concentration of 5 μM p,p'-DDT increased the maximum response of the FSHR to follitropin by 32 ± 7.45%. However, 5 μM p,p'-DDT decreased the basal activity and did not influence the maximal response of the closely related LH/hCG receptor to human chorionic gonadotropin (hCG). The potentiating effect of p,p'-DDT was specific for the FSHR. Moreover, in cells that did not express FSHR, p,p'-DDT had no effect on cAMP response. Thus, the potentiating effect of p,p'-DDT was dependent on the FSHR. In addition, p,p'-DDT increased the sensitivity of FSHR to hCG and to a low molecular weight agonist of the FSHR, 3-((5methyl)-2-(4-benzyloxy-phenyl)-5-{[2-[3-ethoxy-4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-benzamide (16a). Basal activity in response to p,p'-DDT and potentiation of the FSHR response to FSH by p,p'-DDT varied among FSHR mutants with altered transmembrane domains (TMDs), consistent with an effect of p,p'-DDT via TMD binding. This finding was corroborated by the results of simultaneously docking p,p'-DDT and 16a into the FSHR transmembrane bundle.

Conclusion: p,p'-DDT acted as a positive allosteric modulator of the FSHR in our experimental model. These findings suggest that G protein-coupled receptors are additional targets of endocrine disruptors.

Citation: Munier M, Grouleff J, Gourdin L, Fauchard M, Chantreau V, Henrion D, Coutant R, Schiøtt B, Chabbert M, Rodien P. 2016. In vitro effects of the endocrine disruptor p,p'-DDT on human follitropin receptor. Environ Health Perspect 124:991-999; http://dx.doi.org/10.1289/ehp.1510006.

Figure 1

Effects of p,p′‑DDT on follitropin (FSH)-stimulated cAMP production. (A) Chinese hamster ovary-FSH receptor (CHO-FSHR) cells were incubated with hFSH at 3 × 10^–2 IU/mL and 3 IU/mL and increasing concentrations of p,p′‑DDT were investigated (means ± SEM of four independent experiments performed in triplicate). The cAMP concentration measured in the presence of hFSH alone was arbitrarily set at 100%, and the differences were evaluated using the Mann–Whitney U test. (B) Dose–response curve of hFSH on CHO-FSHR cells with or without p,p′‑DDT (5 × 10^–6 M) (means ± SEM of eight independent experiments performed in triplicate). The maximum response to FSH was arbitrarily set at 100%, and the differences were evaluated using a two-way analysis of variance (ANOVA). (C) Basal cAMP production of CHO-FSHR and CHO treated with p,p′‑DDT (5 × 10^–6 M) (means ± SEM of four independent experiments performed in triplicate). The basal cAMP level in the absence of p,p′‑DDT was arbitrarily set at 1. (D) Cells were stimulated with 3 IU/mL hFSH in the presence of p,p′‑DDT (5 × 10^–6 M). The luminescence was recorded every minute (means ± SEM of five independent experiments performed in triplicate). The maximum response to FSH was arbitrarily set at 100%. For clarity, the curve depicting the early phase of the kinetics is enlarged on the right. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for the response in p,p′‑DDT–exposed compared with unexposed cells.

Figure 2

Effects of p,p′‑DDT on calcitonin-, human chorionic gonadotropin (hCG)-, and forskolin (FSK)-stimulated cAMP production and on inhibition of phosphodiesterase (PDE) by IBMX. (A) Chinese hamster ovary–follitropin receptor (CHO-FSHR) cells were stimulated for 30 min with increasing concentrations of salmon calcitonin (sCT) with or without 5 × 10^–6 M p,p′‑DDT (means ± SEM of three independent experiments performed in triplicate). The maximum response to sCT alone was arbitrarily set at 100. (B) Basal and hCG-stimulated (hCG 10^–2 IU/mL and 100 IU/mL) cAMP production was measured in CHO-luteinizing hormone/hCGreceptor (CHO-LH/hCGR) cells with or without p,p′‑DDT (means ± SEM of three independent experiments performed in triplicate). The cAMP production in the absence of p,p′‑DDT was arbitrarily set at 100, and the differences were evaluated using the Mann–Whitney U test. (C) CHO-FSHR and CHO cells were stimulated with 10^–5 M forskolin [an adenylate cyclase (AC) agonist] and increasing doses of p,p′‑DDT (means ± SEM of three independent experiments performed in triplicate). The cAMP production in the presence of forskolin alone was arbitrarily set at 100, and the differences were evaluated using the Mann–Whitney U test. (D) CHO-FSHR cells were incubated with or without 1 mM IBMX for 2 hr and then stimulated or not with FSH 3 IU/mL with or without p,p′‑DDT 5 × 10^–6 M (means ± SEM of three independent experiments performed in triplicate). The cAMP production in the presence of FSH alone was arbitrarily set at 1, and the differences were evaluated using the Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001 for the response in p,p′–DDT-exposed compared with unexposed cells.

Figure 3

p,p′‑DDT targets the follicle stimulating hormone receptor (FSHR) transmembrane domain. (A) Chinese hamster ovary–follitropin receptor (CHO-FSHR) cells were stimulated for 30 min by increasing doses of 3-((5methyl)-2-(4-benzyloxy-phenyl)-5-{[2-[3-ethoxy-4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-benzamid (16a) in the presence of increasing concentrations of p,p′‑DDT (means ± SEM of six independent experiments performed in triplicate). The maximum response to 16a was arbitrarily set at 100, and the differences were evaluated using the Mann–Whitney U test. (B–F) Effects of p,p′‑DDT on mutant FSHR T3.32A (B), T3.32I (C), H7.42A (D), T3.32I-H7.42A (E) and rat FSHR (F) transiently expressed in CHO cells and stimulated for 30 min with increasing concentrations of FSH with or without p,p′‑DDT (means ± SEM of three independent experiments performed in triplicate). The maximum response to hFSH in the absence of p,p′‑DDT was arbitrarily set at 100. The basal activity measured in absence of FSH with (white columns) or without (black columns) p,p′‑DDT. The basal activity in absence of p,p′‑DDT was arbitrarily set at 1, and the differences were evaluated using the Mann–Whitney U test. (G) Side and top views of the putative binding mode of p,p′‑DDT and 16a in the transmembrane domain (TMD) of FSHR. p,p′‑DDT is shown as spheres [carbon (C), purple; chlorine (Cl), green; hydrogen (H), gray], and 16a is shown as sticks [C, white; nitrogen (N), blue; oxygen (O), red]. FSHR is shown as a ribbon representation. The helices are colored from blue for TM1 to red for TM7 and the intracellular TM8. Thr3.32 and His7.42, at the interface between the minor binding site (TM1-3,7) and the major binding site (TM3-7) are shown as spheres (black arrowhead: C, white; N, blue; O, red). p,p′‑DDT was docked to the minor binding pocket, and the best pose was used for subsequent docking of 16a in the major binding pocket as described in “Methods.” *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 for the response in p,p′‑DDT–exposed compared with unexposed cells.

Figure 4

Effects of p,p′‑DDT on human chorionic gonadotropin (hCG) and recombinant human thyroid stimulating hormone (rhTSH)–stimulated cAMP production in Chinese hamster ovary–follicle-stimulating hormone receptor (CHO-FSHR) cells. CHO-FSHR cells were stimulated for 30 min with increasing concentrations of hCG or rhTSH with or without 5 × 10^–6 M p,p′‑DDT (means ± SEM of three independent experiments performed in triplicate). The maximum response to hCG or rhTSH was arbitrarily set at 100. **p < 0.01, ****p < 0.0001 for the response in p,p′‑DDT–exposed compared with unexposed cells, Mann–Whitney U test.

Figure 5

Effects of p,p′‑DDE, o,p′‑DDT, and bisphenol A (BPA) on follicle stimulating hormone (FSH)-stimulated cAMP production. Chinese hamster ovary–follitropin receptor (CHO-FSHR) cells were stimulated with 3 × 10^–2 IU/mL human FSH (hFSH) (left) and 3 IU/mL hFSH (right) in the presence of increasing doses of p,p′‑DDE (A), o,p′‑DDT (B), or BPA (C) (means ± SEM of three independent experiments performed in triplicate). The response to hFSH alone was arbitrarily set at 1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for the response in p,p′‑DDE, o,p′‑DDT, or BPA-exposed compared with unexposed cells, Mann-Whitney U test.