Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

The effect of perinatal exposure to ethinyl oestradiol or a mixture of endocrine disrupting pesticides on kisspeptin neurons in the rat hypothalamus.

Early life exposure to endocrine disruptors is considered to disturb normal development of hormone sensitive parameters and contribute to advanced puberty and reduced fecundity in humans. Kisspeptin is a positive regulator of the hypothalamic-pituitary-gonadal axis, and plays a key role in the initiation of puberty. In the adult, Kiss1 gene expression occurs in two hypothalamic nuclei, namely the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), which are differentially regulated by peripheral sex steroid hormones. In this study we determined the effects on puberty onset and Kiss1 mRNA levels in each of the two nuclei after long-term perinatal exposure of rats to ethinyl oestradiol (EE2) or to five different pesticides, individually and in a mixture. Rat dams were per orally administered with three doses of EE2 (5, 15 or 50 µg/kg/day) or with the pesticides epoxiconazole, mancozeb, prochloraz, tebuconazole, and procymidone, alone or in a mixture of the five pesticides at three different doses. Kiss1 mRNA expression was determined in the AVPV and in the ARC of the adult male and female pups in the EE2 experiment, and in the adult female pups in the pesticide experiment. We find that perinatal EE2 exposure did not affect Kiss1 mRNA expression in this study designed to model human exposure to estrogenic compounds, and we find only minor effects on puberty onset. Further, the Kiss1 system does not exhibit persistent changes and puberty onset is not affected after perinatal exposure to a pesticide mixture in this experimental setting. However, we find that the pesticide mancozeb tends to increase Kiss1 expression in the ARC, presumably through neurotoxic mechanisms rather than via classical endocrine disruption, calling for increased awareness that Kiss1 expression can be affected by environmental pollutants through multiple mechanisms.