Synergistic disruption of external male sex organ development by a mixture of four antiandrogens

Abstract

Background: By disrupting the action of androgens during gestation, certain chemicals present in food, consumer products, and the environment can induce irreversible demasculinization and malformations of sex organs among male offspring. However, the consequences of simultaneous exposure to such chemicals are not well described, especially when they exert their actions by differing molecular mechanisms.

Objectives: To fill this gap, we investigated the effects of mixtures of a widely used plasticizer, di(2-ethylhexyl) phthalate (DEHP); two fungicides present in food, vinclozolin and prochloraz; and a pharmaceutical, finasteride, on landmarks of male sexual development in the rat, including changes in anogenital distance (AGD), retained nipples, sex organ weights, and malformations of genitalia. These chemicals were chosen because they disrupt androgen action with differing mechanisms of action.

Results: Strikingly, the effect of combined exposure to the selected chemicals on malformations of external sex organs was synergistic, and the observed responses were greater than would be predicted from the toxicities of the individual chemicals. In relation to other hallmarks of disrupted male sexual development, including changes in AGD, retained nipples, and sex organ weights, the combined effects were dose additive. When the four chemicals were combined at doses equal to no observed adverse effect levels estimated for nipple retention, significant reductions in AGD were observed in male offspring.

Conclusions: Because unhindered androgen action is essential for human male development in fetal life, these findings are highly relevant to human risk assessment. Evaluations that ignore the possibility of combination effects may lead to considerable underestimations of risks associated with exposures to chemicals that disrupt male sexual differentiation.

Keywords: DEHP; antiandrogens; azole fungicides; combination effects; cumulative effects; dose addition; finasteride; independent action; male sexual differentiation; mixtures; phthalates; prochloraz; vinclozolin.

Figure 1

Outline of the study design adopted for dose–response analyses of antiandrogens and their mixtures. Mated dams were dosed from GD7 to PND16. Male offspring were examined for multiple signs of disrupted sexual development, including AGD on PND0, retained nipples (PND13), weights of prostate and LABC (PND16), and malformations of the genital organs (PND16, PND47).

Figure 2

Prediction and assessment of combination effects of antiandrogens with mixed modes of action on hallmarks of male sexual development in rats exposed gestationally to a combination of DEHP, vinclozolin, prochloraz, and finasteride with a mixture ratio of 3:5:5:0.01. Doses shown are total mixture doses. (A) Changes in anogenital index at PND1. (B) Weight changes of the LABC at PND16 normalized in relation to body weight and untreated controls. (C) Number of retained nipples at PND13. (D) Weight changes of ventral prostates at PND16 normalized to body weight and untreated controls. The predicted mixture effects were derived by using dose addition. Error bars for group means indicate SEM.

Figure 3

Synergistic effects on induction of genital malformations on PND16 in male rats exposed gestationally to combinations of DEHP, vinclozolin, prochloraz, and finasteride with a mixture ratio of 3:5:5:0.01. Doses shown are total mixture doses. Genital malformations include enlarged preputial clefts and urethral openings located toward the base of the genital tubercle, similar to hypospadias in humans. Black circles indicate the mean likelihood of malformation, based on 10–15 litters per group, with 95% confidence intervals; the solid blue line is the best-fit regression model (Weibull) with the 95% confidence belt (dashed blue lines); the green curve and green shaded area indicate the lower and upper estimates of combination effects according to dose addition; and the gray curve represents combined effects predicted by using independent action.

Figure 4

Combination effects in male rats after gestational exposure to low doses of vinclozolin (VZ; 5 mg/kg/day), finasteride (FIN; 0.01 mg/kg/day), prochloraz (PZ; 5 mg/kg/day), and DEHP (3 mg/kg/day). Doses NR NOAELs (left) and 10× NR NOAELs (right). See “Materials and Methods” for details on predicted values for dose addition (DA) and independent action (IA). Male offspring were evaluated for changes in AGD (A) and genital malformations at PND16 (cleft phallus; B) and at PND47 (hypospadias; C). Error bars are 95% confidence intervals. The light green part of the bar in B indicates the lower and upper estimates of combination effects according to dose addition. *Statistically significant compared with controls.