Transcriptomic analysis of adult ovaries and 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 sexual maturity) on the reproductive tract in F1 exposed mice and their F2 offspring. In adult F1 and F2 animals, chronic exposure to low environmental doses of IBU, 2hydroxy-IBU, DCF, and EE2 mixtures, affected reproductive organ maturation, estrous cyclicity (in females), spermiogenesis (in males), and some sperm parameters in F2 males. Transcriptomics further revealed significant changes in gene expression patterns and associated pathways, underlying the modified mechanisms on testis and ovarian physiology and on germ cells, the precursors of gametes. Consequently, fertility of F1 male and female animals exposed to the higher dose of pharmaceuticals and that of their F2 offsprings, measured through the total number of pups and the time between litters, was affected after 5 months of age. This suggested that exposure to these drug cocktails has an inter-generational impact. 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 (8 week-old for females and 10 week-old 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.