An experimental evaluation of the efficacy of perinatal sulforaphane supplementation to decrease the incidence and severity of vinclozolin-induced hypospadias in the mouse model

Abstract

Determining the mechanisms of toxicity induced by pollutants has long been a research priority in lieu of considering the mechanisms of resilience that prevent deleterious impacts. Protective mechanisms in many taxa can be therapeutically targeted to enhance resilience to synthetic toxicants. For example, the environmental sensor, Nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2 or Nrf2), a transcription factor, facilitates transcription of many protective genes. Hypospadias is a common malformation of the penis. The risk of being born with hypospadias increases with pollutant exposure. We use vinclozolin-induced hypospadias in the mouse as a model to test the hypothesis that pollutant-induced birth defects can be prevented and reduced in severity by augmenting natural mechanisms of resilience. Pregnant mice were exposed to the demasculinizing toxicant, vinclozolin, in combination with increasing doses of the NRF2 activator, sulforaphane. The sulforaphane dose that most effectively increased masculinization (anogenital distance) was identified and used to test the hypothesis that sulforaphane reduces the hypospadias-inducing potency of vinclozolin. Finally, a Nrf2 knockout study was conducted to test whether NRF2 was required for the sulforaphane-induced rescue effects. Sulforaphane supplementation to vinclozolin exposed embryos increased anogenital distance in a nonlinear fashion typical of Nrf2 activators. The most effective dose of sulforaphane (45 mg/kg) reduced the occurrence and severity of vinclozolin-induced hypospadias and corrected penis morphogenesis. The sulforaphane-induced rescue effect was dependent on the presence of Nrf2. Nrf2 plays a critical role in protecting the fetus from vinclozolin and reduces the incidence and severity of hypospadias, the most common birth defect in boys in many countries. This work lays a foundation for developing prenatal supplements that will protect the fetus from pollutant-induced hypospadias. Studying the protective mechanisms that drive resilience to toxicants will facilitate innovation of protective therapies.

Keywords: Birth defects prevention; Hypospadias; Mechanisms of resilience; Preventive toxicology; Sulforaphane; Vinclozolin.

Figure 1.

Sulforaphane dose response crossed with a quantity of vinclozolin that induces 100% urogenital abnormalities. A) Experiment dosing regimen for the sulforaphane dose response. All pregnant CD1 mice were exposed to 125 mg/kg of vinclozolin and one of eight quantities of sulforaphane from embryonic day (E) 13.5-18.5. Embryos collected at E18.5 were evaluated for anogenital distance. B) Anogenital distance increased with increasing concentrations of sulforaphane up to 45 mg/kg. Error bars = 95% confidence intervals.

Figure 2.

Vinclozolin dose response crossed with the most effective quantity of sulforaphane A) Experimental dosing regimen for sulforaphane (SFN) and vinclozolin (VCZ) exposures (mg/kg). Pregnant CD1 mice were exposed to one of two doses of sulforaphane and one of five doses of vinclozolin from E13.5-18.5. Sulforaphane reduces the potency of vinclozolin for B) hypospadias incidence, C) hypospadias severity, D) PUO incidence, and E) septation height. Gray shaded areas represent 95% confidence intervals, circles are vinclozolin only exposed mice and triangles are vinclozolin + sulforaphane exposed mice. Best fit lines were generated from generalized linear mixed effects models using the appropriate distribution for the error variance for each data set (B=binomial, C=linear, D=binomial, and E=Poisson).

Figure 3.

Test of the role of Nrf2 in the sulforaphane-mediated rescue of hypospadias and anogenital distance. A) Experimental dosing regimen for the Nrf2 knockout study. Pregnant dams heterozygous for Nrf2 were dosed with one of two quantities of sulforaphane (SFN) and one of two doses of vinclozolin (VCZ). Fetuses were collected at E18.5 and wildtype (+/+) and knock out (−/−) individuals were scored for B) hypospadias incidence, C) hypospadias severity, and D) anogenital distance. Error bars indicate 95% confidence intervals, circles indicate KO mice and triangles indicate WT mice.