Transcriptomic analysis of 30dpp ovaries and 35dpp testes exposed to the cocktails of pharmaceuticals ibuprofen (IBU), 2hydroxyIBU (2hIBU), diclofenac (DCF) and 17a ethinyl-estradiol (EE2) in mice

The endocrine disrupting pharmaceuticals, non-steroidal anti-inflammatory drugs (NSAIDs) and 17a-ethinyl-estradiol (EE2), are among the most relevant molecules found in aquatic ecosystems, surface and drinking water due to their incomplete removal by wastewater treatment plants. Exposure to therapeutic doses has a negative impact on gonadal development and fertility in rodent models; however, the effects of their chronic exposure at lower doses are not known. 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, 8.5 dpc, 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 inter-generational impact (Int. J. Mol. Sci. 2023, 24, 5890. /doi: 10.3390/ijms24065890) Overall design: Two mixtures containing environmentally relevant doses of IBU/2hIBU + DCF + EE2 (D1 and D2) were added or not (control; C) to the drinking water of pregnant mice and their progeny from 8.5 days post-coitum (dpc) until animal sacrifice (30 days post-partum, dpp, for females and 35 dpp for males): F0 males and females from different litters were mated to minimize inbreeding and mated females were divided in three groups: control (no exposure), IBU/2hIBU+DCF+EE2 dose 1 (D1), and IBU/2hIBU+DCF+EE2 dose 2 (D2) (n= 6-7 females per exposure group). The IBU, 2hIBU, DCF and EE2 doses were calculated on the basis of their mean concentrations (5, 40, 10 and 1-2 ng/L, respectively) and maximum concentrations (50-100, 85-100, 50 and 20-50 ng/L, respectively) found in environmental drinking water samples. Thus, the calculated doses in the animal drinking water were: i) D1: IBU 11.3 ng/L/2hIBU 90 ng/L + DCF 22.5 ng/L + EE2 2.25 ng/L; and ii) D2: IBU 113 ng/L/2hIBU 225 ng/L + DCF 112 ng/L + EE2 45 ng/L. The control group was exposed to diluted ethanol (0.001%). Animals were reared in polyphenylsulfone (PPSU) cages in controlled environmental conditions (light/darkness: 12h/12h, 23°C) and drinking water was put in PPSU bottles wrapped in aluminum foil, and were fed with SAFE D131 that does not contain fish proteins, soy and alfalfa. Municipal tap water was provided ad libitum.