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，可能需要评估多达80,000种内分泌干扰物（EDCs），以确定其雌激素或抗雌激素活性。制定激素活性ED化合物的标准化测试方案是全球监管机构的主要目标。此外，在评估EDCs时，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需要认真考虑。我们的研究旨在确定包括植物雌激素含量（大豆黄素和染料木素）和总代谢能（ME）在内的饮食因素对未成熟小鼠和大鼠子宫重量和阴道开放（VO）终点的影响。我们还评估了VO作为选择最合适的饮食来比较EDCs的雌激素或抗雌激素活性的终点。在产后15天（PND）断奶的青春期前CD-1小鼠，在PND 15至PND 22期间饲喂含有预定水平雌激素的试验饲料，以确定膳食中植物雌激素对子宫重量的影响。小鼠从PND 15至PND 35或直到VO发生，以确定膳食中植物雌激素对VO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时出现VO的小鼠比例具有高度预测性（P < 0.0001）。总代谢能也与VO时间显著相关（P < 0.01），尽管该变量的可预测性不如植物雌激素含量强。饲料中植物雌激素含量高的小鼠比饲料中植物雌激素含量低的小鼠的VO时间显著加快（P < 0.05）。得出的结论是,1)膳食黄素和染料木素可以显著(P小于0.01)加快签证官在cd -老鼠的时候,2)进步的时候签证官是一个敏感的端点edc的雌激素活性,评价,应该评估edc的标准协议的一部分,3)的植物雌激素含量相同的饮食可以改变3到4折,4)不同的工厂日期相同的PMI 5002饮食产生显著(P小于。5)与CD-1小鼠和F344大鼠相比，CD Sprague Dawley （SD）大鼠对膳食植物雌激素的敏感性较低，这表明当以VO时间作为激素终点时，SD大鼠不是评价EDCs雌激素活性最敏感的模型。

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.