Cocktails of NSAIDs and 17α Ethinylestradiol at Environmentally Relevant Doses in Drinking Water Alter Puberty Onset in Mice Intergenerationally

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) and 17α-ethinyl-estradiol (EE2) are among the most relevant endocrine-disrupting pharmaceuticals found in the environment, particularly in surface and drinking water due to their incomplete removal via wastewater treatment plants. Exposure of pregnant mice to NSAID therapeutic doses during the sex determination period has a negative impact on gonadal development and fertility in adults; however, the effects of their chronic exposure at lower doses are unknown. In this study, we investigated the impact of chronic exposure to a mixture containing ibuprofen, 2hydroxy-ibuprofen, diclofenac, and EE2 at two environmentally relevant doses (added to the drinking water from fetal life until puberty) on the reproductive tract in F1 exposed mice and their F2 offspring. In F1 animals, exposure delayed male puberty and accelerated female puberty. In post-pubertal F1 testes and ovaries, differentiation/maturation of the different gonad cell types was altered, and some of these modifications were observed also in the non-exposed F2 generation. Transcriptomic analysis of post-pubertal testes and ovaries of F1 (exposed) and F2 animals revealed significant changes in gene expression profiles and enriched pathways, particularly the inflammasome, metabolism and extracellular matrix pathways, compared with controls (non-exposed). This suggested that exposure to these drug cocktails has an intergenerational impact. The identified Adverse Outcome Pathway (AOP) networks for NSAIDs and EE2, at doses that are relevant to everyday human exposure, will improve the AOP network of the human reproductive system development concerning endocrine disruptor chemicals. It may serve to identify other putative endocrine disruptors for mammalian species based on the expression of biomarkers.

Adenomyotic Lesions Are Induced in the Mouse Uterus after Exposure to NSAID and EE2 Mixtures at Environmental Doses

“…This exposure time covered the main steps of migration, proliferation, and differentiation/maturation of germ cells and somatic cell lineages from the early embryonic stages until sexual maturity. The two doses in the drinking water were calculated based on their mean concentrations (D1: 5, 40, 10, and 1 ng/L, respectively) and maximum concentrations (D2: 50, 100, 50, and 20 ng/L, respectively) found in environmental drinking water samples (ANSES referral 2013-SA-0081) [78], as previously reported [52]. The control group was exposed to 0.001% ethanol (vehicle).…”

“…These mating combinations were simplified in the manuscript and named CxC, CxD1, CxD2, D1xC, D2xC, and D2xD2, respectively. For this study, animals were reared according to the protocols described previously [52,53]. All animal experiments were carried out in accordance with procedures approved by the Réseau des Animaleries de Montpellier (approval number 34-366 for B.B.-B.…”

“…For immunofluorescence (IF) analysis, tissue sections were processed as previously described [52,80] and incubated with the following primary antibodies: polyclonal rabbit anti-FOXL2 (homemade [81], 1/300), anti-α-SMA (Abcam ab124964, 1/500, Cambridge, UK), anti-Cx43 (Sigma Aldrich C6219, 1/400, St. Louis, MO, USA), and monoclonal mouse anti-PCNA (Sigma Aldrich P8825, 1/500), anti-pan-cytokeratin (Thermo Fisher MA5-13156, 1/400, Waltham, MA, USA). Sections were then incubated with donkey or goat secondary antibodies conjugated with Alexa Fluor 555, 488, or 649 (Thermo Fisher, 1/1000), followed by Hoechst staining.…”

“…Recently, we showed that chronic exposure to a mixture containing IBU, 2h-IBU, DCF, and EE2 at two environmentally relevant doses, intergenerationally impairs ovary development and physiology in F1 mice and their offspring. This leads to early puberty onset in F1-exposed postnatal females and their F2 offspring [52] and altered reproductive organ maturation in adult F1 and F2 females [53]. F1 and F2 females also showed irregular estrous cyclicity, although 17βestradiol (E2) concentration in serum was not significantly affected [53].…”