In Silico, In Vitro, and In Vivo Studies Indicate the Endocrine-Disrupting Effects of Cyproconazole Stereoisomers

The widespread use of chiral triazole fungicide cyproconazole (CPZ) in agricultural fields has led to frequent detection of CPZ in the environment. The restriction of CPZ in the EU raised wide concerns regarding its potential endocrine-disrupting effects (EDEs). The present study was conducted to evaluate EDEs of CPZ stereoisomers <i>in vitro</i>, <i>in silico</i>, and <i>in vivo</i>. The reporter gene assay indicated that all CPZ stereoisomers were agonists to the human estrogenic receptor α. (2<i>S</i>,3<i>S</i>)-(+)- and (2<i>R</i>,3<i>S</i>)-(-)-CPZ exhibited stronger binding capacities to ERα compared with (2<i>R</i>,3<i>R</i>)-(-)- and (2<i>S</i>,3<i>R</i>)-(+)-CPZ. Our computational studies showed consistent results with reporter gene assay, elucidating the stereoselective binding mode of CPZ to estrogen receptor. In zebrafish embryos, the 96h-lethality of CPZ stereoisomers ordered (2<i>R</i>,3<i>R</i>)-(-)- > (2<i>R</i>,3<i>S</i>)-(-)- > (2<i>S</i>,3<i>S</i>)-(+)- > Rac- > (2<i>S</i>,3<i>R</i>)-(+)-CPZ. Stereoselective developmental toxicity of CPZ was observed while (2<i>R</i>,3<i>S</i>)-(-)-CPZ is the most toxic isomer. The estrogenic hormones were significantly decreased in (2<i>S</i>,3<i>R</i>)-(+)- and (2<i>R</i>,3<i>S</i>)-(-)-CPZ groups and enhanced in (2<i>S</i>,3<i>S</i>)-(+)- and (2<i>R</i>,3<i>R</i>)-(-)-CPZ, along with the gene expression in hypothalamic-pituitary-gonad axis altered. CPZ shows no thyroid hormone activity. These data clarified that CPZ is a new-found endocrine disruptor threatening human health and each stereoisomer of CPZ showed stereoselective EDEs by regulating the nuclear receptor-mediated gene expression.