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. 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 17α-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.

内分泌干扰型药物、非甾体抗炎药（non-steroidal anti-inflammatory drugs, NSAIDs）与17α-炔雌醇（17α-ethinyl-estradiol, EE2）是水生生态系统、地表水及饮用水中检出的核心污染物之一，因无法被污水处理厂完全去除而广泛存在。治疗剂量的暴露会对啮齿类动物模型的性腺发育与生殖能力产生负面影响，但低剂量长期暴露的影响尚未明确。本研究中，我们针对从妊娠第8.5天（8.5 dpc）的胚胎期直至性成熟阶段，通过饮用水暴露于两种环境相关浓度的布洛芬、2-羟基布洛芬、双氯芬酸与EE2混合污染物的F1代暴露小鼠及其F2代后代的生殖系统，探究了长期暴露的影响。

在成年F1与F2代动物中，低环境浓度的布洛芬、2-羟基布洛芬、双氯芬酸与EE2混合污染物长期暴露，会影响生殖器官成熟、雌性的动情周期、雄性的精子发生过程，以及F2代雄性的部分精子参数。转录组学（Transcriptomics）进一步揭示了基因表达模式与相关通路的显著变化，这些变化是睾丸、卵巢生理功能以及配子前体生殖细胞的调控机制发生改变的基础。因此，暴露于高浓度药物混合物的F1代雌雄个体及其F2代后代的生殖能力，通过幼崽总数量与产仔间隔时间进行评估后发现，在5月龄后其生殖能力受到了影响。这表明暴露于这类药物混合污染物会产生跨代影响。

整体实验设计：将两种含环境相关浓度布洛芬/2-羟基布洛芬+双氯芬酸+EE2的混合物（分别记为D1与D2）以及空白对照（C，无添加）加入孕鼠及其后代的饮用水中，暴露周期从妊娠第8.5天（8.5 dpc）持续至动物安乐死时（雌性为8周龄，雄性为10周龄）。为最小化近交繁殖，使用不同窝别F0代雌雄个体进行交配，随后将受孕雌鼠分为三组：对照组（无暴露）、低剂量组（IBU/2hIBU+DCF+EE2 D1）、高剂量组（IBU/2hIBU+DCF+EE2 D2），每组各6-7只受孕雌鼠。

布洛芬、2-羟基布洛芬、双氯芬酸与EE2的暴露剂量，基于环境饮用水样本中检出的平均浓度（分别为5、40、10与1-2 ng/L）与最高浓度（分别为50-100、85-100、50与20-50 ng/L）进行换算。因此，动物饮用水中的实际换算剂量为：① D1组：布洛芬11.3 ng/L、2-羟基布洛芬90 ng/L + 双氯芬酸22.5 ng/L + EE2 2.25 ng/L；② D2组：布洛芬113 ng/L、2-羟基布洛芬225 ng/L + 双氯芬酸112 ng/L + EE2 45 ng/L。对照组则给予添加了0.001%稀释乙醇的饮用水。

实验动物饲养于聚苯砜（polyphenylsulfone, PPSU）笼盒中，饲养环境为可控条件：光暗周期12h/12h，温度23℃；饮用水置于包裹铝箔的PPSU饮水瓶中，饲料选用不含鱼蛋白、大豆与苜蓿的SAFE D131饲料，市政自来水可自由饮用。