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

环境保护署（EPA）和经济合作与发展组织（OECD）估计，多达80，000种内分泌干扰物化合物（EDCs）可以进行评估，以确定其雌激素或抗雌激素活性。开发标准化方案来测试药物活性艾德化合物是全球监管机构的主要目标。此外，在评估内分泌干扰物时，需要认真考虑市售啮齿动物饮食中存在的植物雌激素对生殖和致癌性终点的作用。我们的研究旨在确定膳食因素，包括植物雌激素含量（大豆黄酮和染料木素）和总代谢能（ME）对子宫重量和阴道开口（VO）的未成年小鼠和大鼠的终点的影响。我们还评估了VO作为选择最合适饮食的终点，以比较EDCs的雌激素或抗雌激素活性。青春期前CD-1小鼠在出生后第15天（PND）断奶，并从PND 15至PND 22喂食含有预定水平膳食雌激素的试验饮食，以确定膳食植物雌激素对子宫重量的影响。从PND 15至PND 35或直到VO发生，给小鼠喂食测试饮食，以确定饮食植物雌激素对VO发生时间的影响。从断奶到VO时每天记录阴道开口。饮食中的植物雌激素含量对PND 24时表现出VO的小鼠比例具有高度预测性（P <0.0001）。总ME也显着（P <0.01）与VO的时间相关，虽然这个变量的可预测性不如植物雌激素含量。与低植物雌激素含量的饮食相比，高植物雌激素含量的饮食显著（P <0.05）加速了小鼠VO的时间。结果表明：1）大豆黄酮和染料木黄酮可显著提高（P小于0.01）加速CD-1小鼠的VO时间，2）VO时间的提前是评价EDCs雌激素活性的敏感终点，并且应该是评价EDCs的标准方案的一部分，3）相同饮食的植物雌激素含量可以变化3至4倍，4）相同PMI 5002日粮的不同加工日期产生显著的CD-1小鼠和F344大鼠的VO时间（p <0.05），但CD Sprague道利（SD）大鼠的VO时间未发生变化，和5）CD Sprague道利大鼠对膳食植物雌激素的敏感性低于CD-1小鼠或F344大鼠，提示SD大鼠不是评价EDCs雌激素活性的最敏感模型，当使用VO时间作为激素终点时。 我们最近报道了开放式和封闭式配方实验室动物饲料的优点/缺点及其对研究的重要性。 此外，我们还报告了各种啮齿动物垫料中的内毒素、大肠菌群和粉尘水平。 我们得出结论，纸垫料是进行LPS吸入研究的最佳垫料类型，含有高水平内毒素的啮齿动物垫料可能会改变啮齿动物呼吸和免疫学研究的结果。 我们已经表明，雌激素真菌毒素玉米赤霉烯酮是无处不在的市售玉米芯垫料。 189个样品中有154个（84%）天然受到玉米赤霉烯酮污染，污染水平为100至7，000 ppb（平均500 ppb）。 我们已经表明，5-10 ppm的水平可以显着提前未成熟的CD-1毛和SKH-1无毛小鼠VO的时间。 最近的研究集中在： 1)回顾有关双酚A的文献，测定啮齿动物饲料、垫料、笼子和水瓶中的雌激素含量及其对双酚A研究的影响。 我们的文献综述表明，低剂量BPA（50 g /kg bwt/天）动物研究的结果不一致，使用高植物雌激素饮食和皮下暴露途径是影响因素。 我们的文献综述进一步表明，BPA研究中使用的饲料、垫料、笼舍和水瓶的识别和报告并不一致。 我们的结论是，含有降低水平的植物雌激素（<20 g/g饲料）和低水平的代谢能（3.1 k cal/饲料）的饲料，无雌激素的垫料，笼子和水瓶可用于评价内分泌干扰化合物（如BPA）的雌激素活性的研究。 2)当要推断结果以确定对人类的潜在不良影响时，应使用BPA暴露的口服途径。 此外，我们正在确定BPA和其他雌激素单体从各种类型的动物笼和水瓶中浸出的影响，以及它们对未成熟CD-1小鼠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) may be evaluated to determine their estrogenic or anti-estrogenic activity. The development of standardized protocols to test for hormonally active ED compounds is a major goal of regulatory agencies worldwide. Also, the role of phytoestrogens present in commercially available rodent diets on reproduction and carcinogenicity endpoints when EDCs are being evaluated requires serious consideration. Our studies were aimed at determining the effects of dietary factors including the phytoestrogen content (daidzein and genistein) and total metabolizable energy (ME) on uterine weights and vaginal opening (VO) endpoints of immature mice and rats. We also assessed VO as an endpoint for selecting the most appropriate diets(s) for comparing the estrogenic or anti-estrogenic activity of EDCs. Pre-pubertal CD-1 mice were weaned at post-natal day (PND) 15 and fed test diets containing predetermined levels of dietary estrogens from PND 15 to PND 22 to determine the effects of dietary phytoestrogens on uterine weights. Mice were fed the test diets from PND 15 to PND 35 or until VO occurs to determine the effects of dietary phytoestrogens on the time of VO. Vaginal opening was recorded daily from weaning to time of VO. The phytoestrogen content of the diet was highly predictive (P less than 0.0001) of the proportion of mice exhibiting VO at PND 24. Total ME was also significantly (P less than 0.01) correlated with the time of VO, although the predictability of this variable was less powerful than the phytoestrogen content. The time of VO in mice was significantly (P less than 0.05) accelerated in mice fed diets high in phytoestrogens as compared to low phytoestrogen content diets. It was concluded that 1) dietary daidzein and genistein can significantly (P less than 0.01) accelerate the time of VO in CD-1 mice, 2) the advancement in the time of VO is a sensitive endpoint for evaluating the estrogenic activity of EDCs, and should be part of the standard protocol for evaluating EDCs, 3) the phytoestrogen content of the same diet can vary 3 to 4 fold, 4) different mill dates of the same PMI 5002 diet produced significant (p less than .05) in the time of VO in CD-1 mice and F344 rats but not in CD Sprague Dawley (SD) rats, and 5) the CD Sprague Dawley rat is less sensitive to dietary phytoestrogen when compared with the CD-1 mouse or the F344 rat suggesting that the SD rat is not the most sensitive model for evaluating the estrogenic activity of EDCs when using the time of VO as the hormonal endpoint. We have recently reported the advantages/disadvantages of open and closed formula laboratory animal diets and their importance to research. In addition, we have reported the endotoxin, coliform and dust levels in the various types of rodent bedding. We concluded that paper bedding is the optimal bedding type for conducting LPS inhalation studies and that rodent bedding containing high levels of endotoxin may alter the results of respiratory and immunologic studies in rodents. 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 form 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. Recent studies have focused on: 1) reviewing the literature on BPA and determining the estrogenic content of rodent diets, bedding, cages and water bottles and its impact on Bisphenol A studies. Our literature review indicated that the results of 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. We concluded that a diet containing reduced levels of phytoestrogens (<20 g/g diet) and low levels of metabolizable energy (3.1 k cal/ diet), estrogen-free bedding, cages and water bottles be used for studies evaluating the estrogenic activity of endocrine disrupting compounds such as BPAs. 2) That the oral route of BPA exposure should be used when results are to be extrapolated to determine potential adverse effects in humans. Also, we are determining the effects of BPA and other estrogenic monomers leaching from the various types of animal cages and water bottles and their impact on the time of VO in immature CD-1 mice.