The Impact of Estrogens in Rodent Diets, Bedding and Caging on EDC Studies

环境保护局(EPA)和经济合作与发展组织(OECD)估计，应对多达80000种内分泌干扰物(EDCs)进行评估，以确定其雌激素或抗雌激素活性。开发检测激素活性内分泌细胞的标准化方案是世界各地监管机构的主要目标。 我们的研究集中在用于EDC研究的研究动物的微环境中存在EDC的可能性(例如，饮食、笼养、卧铺和环境丰富)，以及这些化合物可能对研究结果，特别是生殖发育的影响。在过去，我们利用了啮齿动物子宫重量的变化或阴道开放(VO)的时间，这两种情况都是雌激素敏感的终点。目前，我们将重点放在VO的影响上，因为我们认为这是生殖发育的敏感指标。 在我们的研究中，青春期前的CD-1小鼠在出生后第15天(PND)15天断奶，并从PND 15到PND 35口服(通过饮食、水或灌胃)测试化合物或直到发生VO，以确定VO的影响。从断奶到VO，每天记录阴道开口度。我们已经证明，商业上可获得的啮齿动物饲料中存在的天然植物雌激素(如大豆苷元和染料木素)或添加到不含这些化合物的饲料中可以显著(P<0.01)加快CD-1小鼠的VO时间。我们还表明，饮食中的总代谢能(ME)可以显著影响这些激素敏感的终点，尽管这个变量的可预测性不如植物雌激素含量。 我们已经证明，雌激素真菌毒素玉米赤霉烯酮在商业上可获得的玉米芯垫料中普遍存在。189个样本中有154个(84%)受到玉米赤霉烯酮的天然污染，污染水平从100到7,000 ppb(平均500 ppb)不等。我们已经证明，5-10ppm的水平可以显著延长CD-1有毛和SKH-1无毛未成熟小鼠的VO时间。 双酚A(BPA)是一种广为人知的EDC化合物。我们一直担心，关于BPA或其他内分泌细胞的ED效应的动物研究没有充分或一致地控制可能影响研究结果的动物微环境的潜在EDC效应。使用高植物雌激素饮食或含有高或可变水平ME的饮食或使用玉米芯寝具可以显著改变EDC的研究。我们还担心，一些报告中的双酚A暴露途径可能没有充分评估人类暴露的风险。我们发现，关于低剂量BPA(50g/KgBWT/天)动物研究的报道不一致，高植物雌激素饮食的使用和皮下暴露是促成因素。我们的文献回顾进一步表明，BPA研究中使用的饮食、床上用品、笼子和水瓶的识别和报告并不一致。目前，我们正在测定BPA和其他雌激素单体从不同类型的动物笼子、环境富集物和水瓶中浸出的效果，以及它们对CD-1幼鼠VO时间的影响。

The Environmental Protection Agency (EPA) and Organization for Economic Cooperation and Development (OECD) estimates that as many as 80,000 endocrine disruptor compounds (EDCs) should be evaluated to determine their estrogenic or anti-estrogenic activity. The development of standardized protocols to test for hormonally active EDCs is a major goal of regulatory agencies worldwide. Our studies focus on the potential for EDCs being present in the micro-environment of the research animals used for EDC studies (e.g., diet, caging, bedding, and environmental enrichment) and the impact these compounds may have on study outcomes, especially reproductive development. In the past, we have utilized either changes in uterine weight or the timing of vaginal opening (VO) in rodents, both estrogen-sensitive endpoints. Currently, we have focused on the effects on VO, as we feel this is a sensitive indicator of reproductive development. In our studies, pre-pubertal CD-1 mice are weaned at post-natal day (PND) 15 and given test compounds orally (via diet, water or gavage) from PND 15 to PND 35 or until VO occur to determine the effects of on the timing of VO. Vaginal opening is recorded daily from weaning to time of VO. We have shown that natural phytoestrogens (e.g., daidzein and genistein) present in commercially available rodent diets or added to diets free of these compounds can significantly (P less than 0.01) accelerate the time of VO in CD-1 mice. We have also shown that the total metabolizable energy (ME) in the diet can significantly affect these hormone-sensitive endpoints, although the predictability of this variable was less powerful than the phytoestrogen content. We have shown that the estrogenic mycotoxin zearalenone is ubiquitious in commercially available corn-cob bedding. 154 of 189 (84%) of the samples were naturally contaminated with zearalenone at levels ranging from 100 to 7,000 ppb (mean 500 ppb). We have shown that levels of 5-10 ppm can significantly advance the time of VO in immature CD-1 haired and SKH-1 hairless mice. Bisphenol A (BPA) is a highly publicized EDC compound. We have been concerned that animal studies on the ED effects of BPA or other EDCs have not adequately or consistently controlled for the potential EDC effects of the animals micro-environment that could affect the study outcome. The use of high phytoestrogen diets or diets with high or variable levels of ME or the use of corncob bedding can dramatically alter EDC studies. We have also had concerns that the route of BPA exposure in some reports may not adequately assess risks to human exposures. We have found that reports on low dose BPA (50 g /kg bwt/day) animal studies are inconsistent, and the use of high phytoestrogen diets and the subcutaneous route of exposure are contributing factors. Our literature review further indicates that the identification and reporting of the diet, bedding, caging and water bottles used in BPA studies are not consistent. Currently, we are determining the effects of BPA and other estrogenic monomers leaching from the various types of animal cages, environmental enrichment and water bottles and their impact on the time of VO in immature CD-1 mice.