Chemical preparation

Sulforaphane was removed from −20 °C and either 0mg, 1.5mg, 3mg, 6mg, 9mg, 12mg, 15mg, or 24mg was combined with 0.05mL of corn oil to generate sulforaphane treatments that would deliver 0, 7.5, 15, 30, 45, 60, 75, and 120 mg/kg . The sulforaphane and corn oil was combined with vinclozolin dissolved in 0.45mL of corn oil (see below). The vinclozolin + sulforaphane + corn oil mixture was vortexed for one minute and was visually inspected to ensure that the vinclozolin did not fall out of solution and the sulforaphane was completely in solution. In all cases sulforaphane went into solution within 1 minute of mixing.

Vinclozolin (Sigma Aldrich, St. Louis, MO, United States,45705), that weighed either 10mg, 15mg, 20mg, or 25mg was dissolved in 0.45mL tocopherol stripped corn oil (Millipore, Santa Ana, CA, United States, 02901415) to generate vinclozolin treatments that delivered 50, 75, 100, and 125 mg/kg respectively. The mixture was heated at 60 °C and mixed with Genemate Rotator (H-6800) for 3 hours. Upon removal from the oven vinclozolin never fell out of solution.

Once sulforaphane, vinclozolin, or their combination was dissolved in corn oil, Mice were gavaged with the appropriate mixture for the specific experiment (see below). Gavage volume (μL) received by the pregnant dam was always 2.5 multiplied by the weight of the pregnant dam. In this research we use high concentrations of vinclozolin that induce urogenital defects in100% of the male fetuses. Although these vinclozolin concentrations are not relevant to human health, they provide a highly reproducible (penetrant) phenotype that allows us to make strong conclusions about the rescue effects that we induce with sulforaphane. The dose of sulforaphane that we use in this study is difficult to compare to concentrations in supplements currently used in humans as they typically contain precursors to sulforaphane rather than the parent compound (e.g., Avmacol).