Endocrine Disruption in Human Fetal Testis Explants by Individual and Combined Exposures to Selected Pharmaceuticals, Pesticides, and Environmental Pollutants

Abstract

Background: Numerous chemicals are capable of disrupting androgen production, but the possibility that they might act together to produce effects greater than those of the most effective component in the mixture has not been studied directly in human tissues. Suppression of androgen synthesis in fetal life has been associated with testis maldescent, malformations of the genitalia at birth, and poor semen quality later in life.

Objectives: Our aim was to investigate whether chemicals can act together to disrupt androgen production in human fetal testis explants and to evaluate the importance of mixture effects when characterizing the hazard of individual chemicals.

Methods: We used an organotypic culture system of human fetal testes explants called FEtal Gonad Assay (FEGA) with tissue obtained at 10 and 12 gestational wk (GW 10-12), to screen 27 chemicals individually for their possible anti-androgenic effect. Based on the results of the screen, we selected 11 compounds and tested them as mixtures.

Results: We evaluated mixtures composed of four and eight antiandrogens that contained the pharmaceuticals ketoconazole and theophylline and several previously untested chemicals, such as the pesticides imazalil and propiconazole. Mixtures of antiandrogens can suppress testosterone synthesis in human fetal testicular explants to an extent greater than that seen with individual chemicals. This revealed itself as a shift towards lower doses in the dose-response curves of individual antiandrogens that became more pronounced as the number of components increased from four to eight.

Conclusions: Our results with the FEGA provide the foundations of a predictive human mixture risk assessment approach for anti-androgenic exposures in fetal life. https://doi.org/10.1289/EHP1014.

Figure 1.

Concentration–response data for individual chemicals from the organotypic culture system in human fetal testes. (A,B) The graphs show the experimental data as $\text{mean}\pm \text{SEM}$ of at least three independent studies together with the regression curves (solid lines) and their respective 95% confidence intervals (CI) (dashed lines). Testosterone production is represented as relative to the first day of culture (D0) production and the control level, see text for more details. Gray areas indicate the cytotoxic concentration ranges, the vertical dashed line the concentrations expected to inhibit testosterone secretion in human fetal testis by 50%.

Figure 1.

Concentration–response data for individual chemicals from the organotypic culture system in human fetal testes. (A,B) The graphs show the experimental data as $\text{mean}\pm \text{SEM}$ of at least three independent studies together with the regression curves (solid lines) and their respective 95% confidence intervals (CI) (dashed lines). Testosterone production is represented as relative to the first day of culture (D0) production and the control level, see text for more details. Gray areas indicate the cytotoxic concentration ranges, the vertical dashed line the concentrations expected to inhibit testosterone secretion in human fetal testis by 50%.

Figure 2.

Single compound histopathology of treated human fetal testes explants at the highest nontoxic concentrations after a culture of 96 h. (A,B) Steroidogenic Leydig cells were labeled by immunostaining of CYP11A1 in cultured explants of gestational week (GW) 10–12. The 3,3′-diaminobenzidine tetrahydrochloride staining appears brown in all photos, and sections were counterstained with hematoxylin. Testis cords and interstitial tissue could be easily identified in all the sections (dashed lines represent testis cords). Bar$=20\mathrm{\mu }\mathrm{m}$.

Figure 3.

Predicted and observed testosterone secretion in human fetal testis by four chemical mixtures. Experimental data are shown as $\text{mean}\pm \text{SEM}$ (blue) of at least four independent experiments. Testosterone production is represented as relative to the first day of culture (D0) production and the control level, see text for more details. The mixture effects were predicted according to dose addition (DA) (thick red curve), with dashed curves the respective 95% confidence intervals (CIs) (dotted orange lines).

Figure 4.

Histopathology of explants at high-effect concentrations of the mixtures. Steroidogenic Leydig cells and apoptotic cells were labeled in cultured explants of GW 10–12 human fetal testis with an immunostaining of CYP11A1 (left panel) and an anticleaved caspase-3 antibody (right panel), respectively. The 3,3′-diaminobenzidine tetrahydrochloride staining appears brown, and sections were counterstained with hematoxylin. Testis cords are highlighted by dashed lines, and the Leydig cells within the interstitial tissue can be identified by their brown labeling. Bar$=20\mathrm{\mu }\mathrm{m}$.

Figure 5.

The impact of co-exposures on the dose–response curves of individual mixture components. A) The concentration–response curves on the right in each panel show the responses for each compound tested on its own, and the curves on the left, the response if tested in combination with seven other compounds in mixture III (red solid lines). Testosterone production is represented as relative to the D0 production and the control level, see text for more details. B) The shift in the concentration–response curve of BPA in the presence of mixtures I–IV. C) Comparison of the concentrations of BPA associated with 50% testosterone synthesis suppression in the presence of mixtures I–IV.