Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

Exposure of human fetal kidneys to mild analgesics interferes with early nephrogenesis.

Acetaminophen, aspirin, and ibuprofen are mild analgesics commonly used by pregnant women, the sole current recommendation being to avoid ibuprofen from the fifth month of gestation. The nephrotoxicity of these three analgesics is well documented in adults, as is their interference with prostaglandins biosynthesis. Here we investigated the effect of these analgesics on human first trimester kidneys ex vivo. We first evaluated prostaglandins biosynthesis functionality by performing a wide screening of prostaglandin expression patterns in first trimester human kidneys. We demonstrated that prostaglandins biosynthesis machinery is functional during early nephrogenesis. Human fetal kidney explants aged 7-12 developmental weeks were exposed ex vivo to ibuprofen, aspirin or acetaminophen for 7 days, and analyzed by histology, immunohistochemistry, and flow cytometry. This study has revealed that these analgesics induced a spectrum of abnormalities within early developing structures, ranging from cell death to a decline in differentiating glomeruli density. These results warrant caution for the use of these medicines during the first trimester of pregnancy.

Characterization of Estrogenic Activity and Site-Specific Accumulation of Bisphenol-A in Epididymal Fat Pad: Interfering Effects on the Endocannabinoid System and Temporal Progression of Germ Cells.

The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3ß-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell-cell junction genes (i.e., zonula occcludens protein-1, vimentin and ß-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.

Ovarian dysfunction following prenatal exposure to an insecticide, chlordecone, associates with altered epigenetic features.

Chlordecone (CD) is an insecticide that was used in the French West Indies for several years to control the banana root borer pest. Given its nonsignificant degradation, it persists in the environment. CD is a carcinogenic compound with reproductive and developmental toxicity and is a recognized endocrine-disrupting chemical. In this study, we examined the effects of CD on female reproductive system of mice with the focus on epigenetic features in ovary. Our data show that gestational exposure to low dose of CD affects meiotic double-strand breaks repair in female embryos. In adult mice derived from CD-treated pregnant females, we observed delayed puberty, decreased number of primordial and increased number of atretic follicles. Gene expression analysis revealed that Rcbtb2 and Rbpms genes were not expressed in embryonic gonads. Estrogen signaling- and oocyte maturation-associated genes were downregulated in adult ovaries. The morphological changes were associated with altered epigenetic features: increased H2Aub and increased H3K27me3 and decreased H4ac and H3K4me3 in embryonic oocytes. The DNA damage-associated, γH2AX marks were detected in the follicles of treated but not control adult ovaries. We also found reduced H3K4me3 and H4ac in fully grown oocytes of the treated ovaries. The ChIP-seq analysis of H3K4me3 in adult ovaries showed that target genes of ZFP57 and TRIM28, which regulate pluripotency and imprinting, were significantly enriched in altered regions. Our study clearly demonstrates that gestational exposure to a low dose of CD impairs the function of female reproductive system and the changes are associated with altered epigenetic features.

Analysis of Endocannabinoid System in Rat Testis During the First Spermatogenetic Wave.

Endocannabinoids are lipid mediators, enzymatically synthesized and hydrolyzed, that bind cannabinoid receptors. Together with their receptors and metabolic enzymes, they form the "endocannabinoid system" (ECS). Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the main endocannabinoids studied in testis. In this study, using the first wave of spermatogenesis as an in vivo model to verify the progressive appearance of germ cells in seminiferous tubules [i.e., spermatogonia, spermatocytes, and spermatids], we analyzed the expression of the main enzymes and receptors of ECS in rat testis. In particular, the expression profile of the main enzymes metabolizing AEA and 2-AG as well as the expression of cannabinoid receptors, such as CB1 and CB2, and specific markers of mitotic, meiotic, and post-meiotic germ cell appearance or activities have been analyzed by RT-PCR and appropriately correlated. Our aim was to envisage a relationship between expression of ECS components and temporal profile of germ cell appearance or activity as well as among ECS components. Results show that expression of ECS components is related to germ cell progression. In particular, CB2 and 2-AG appear to be related to mitotic/meiotic stages, while CB1 and AEA appear to be related to spermatogonia stem cells activity and spermatids appearance, respectively. Our data also suggest that a functional interaction among ECS components occurs in the testis. Indeed, in vitro-incubated testis show that AEA-CB2 activity affects negatively monoacylglycerol-lipase levels via upregulation of CB1 suggesting a CB1/CB2-mediated relationship between AEA and 2-AG. Finally, we provide the first evidence that CB1 is present in fetal gonocytes, during mitotic arrest.

Bisphenol A induces hypothalamic down-regulation of the the cannabinoid receptor 1 and anorexigenic effects in male mice.

Bisphenol A is an environment-polluting industrial chemical able to interfere with the endocrine system. An obesogenic effect in perinatally exposed rodents has been described as estrogenic activity. We exposed male mice to Bisphenol A during fetal-perinatal period (from 10 days post coitum to 31 days post partum) and investigated the effects of this early-life exposure at 78 days of age. Body weight, food intake, fat mass, and hypothalamic signals related to anorexigenic control of food intake were analyzed. Results show that Bisphenol A exposure reduced body weight and food intake. In addition, the exposure decreased epididymal fat mass and adiposity, acting negatively on adipocyte volume. At hypothalamic level, Bisphenol A exposure reduced the expression of the cannabinoid receptor 1 and induced gene expression of cocaine and amphetamine-regulated transcript-1. This observation suggests that Bisphenol A induces activation of anorexigenic signals via down-regulation of the hypothalamic cannabinoid receptor 1 with negative impact on food intake.