Effects of In Utero and Transgenerational Exposure of Avy Mice to Bisphenol A

描述（由申请人提供）：该申请涉及广泛的挑战领域（08）基因组学和特定挑战主题，08- es -104，与环境暴露相关的表观遗传标记改变的鉴定。在豚鼠基因中含有内源性A粒子（IAP）的Avy小鼠中，暴露于富含染料木黄酮或甲基供体的母体饮食中会导致IAP位点的甲基化，并逐渐从黄色毛色转变为假豚鼠后代。然而，双酚A （BPA）暴露在怀孕的黑人女性身上会导致更多的黄色后代。具有去甲基化启动子的黄色毛色后代会患上糖尿病、肥胖症，在某些品系中还会患上癌症，但具有棕色毛色的深色兄弟姐妹则保持健康。这些老鼠是研究表观遗传学的经典动物模型，因为它们对环境变化非常敏感。虽然先前的一项研究已经记录了这些小鼠在子宫内暴露于BPA（一种在大多数塑料化合物中发现的环境雌激素污染物）后明显的皮毛颜色变化，但没有研究检查了细微的表型改变，包括行为、血清代谢物和激素浓度，以及可能发生在各种皮毛颜色的Avy小鼠身上的病理变化。此外，单代（F1）在子宫内暴露于双酚a可能会对其曾孙（F3）产生跨代影响，因为他们只通过他们的前辈接触到双酚a。虽然其他化合物，包括vinclozolin和己烯雌酚（DES）的跨代效应已经在啮齿动物和人类（以DES为例）中进行了研究，但迄今为止还没有一项研究检测了BPA的跨代效应。然而，这些研究与人类高度相关，我们无法轻易评估一代人暴露于内分泌干扰物（如双酚a）对后代的影响。然而，由于它们的妊娠期为18 - 21天，性成熟较早（6 - 8周龄），这种跨代研究很容易在小鼠身上完成。在此，我们将通过将黑色（a/a）雌性繁殖到可存活的黄色（Avy/a）雄性来确定BPA是否会产生跨代效应。在繁殖前两周和整个妊娠期间，这些雌性将通过饮食暴露于三种剂量（501/4g/kg/day， 5mg/kg/day或50mg/kg/day）中的一种。对照雌性将继续饲喂AIN-93G饲料。将对幼犬进行断奶前和断奶后的行为评估，以确定BPA是否会影响它们的焦虑水平、学习和记忆反应以及其他发育参数。每周抽血测量血清中代谢激素、性调节激素、游离脂肪酸和葡萄糖的浓度，并跟踪其体重和整体外观。在10周龄时，将繁殖得到的雄性和雌性分别控制一只雌性和一只雄性，以确定是否有任何跨代效应可以通过雄性和雌性生殖系传递。此外，它们的黑色毛色（a/a） （F1）兄弟姐妹将被繁殖，以确定BPA是否支持独立于Avy位点的跨代效应。同样的实验程序将在F2和最终的F3代上重复进行，F3代只会通过它们的前辈接触到BPA。这些研究将使我们能够检查行为、体重、血液代谢物和激素浓度的细微变化，以及可能在子宫内暴露于双酚a的存活黄鼠身上发生的病理变化。此外，本研究将检验BPA在a/a和Avy/a小鼠中可能产生的跨代效应，后者是典型的表观遗传动物模型。这些研究与人类有相当大的相关性，因为它们可能作为预警，提醒我们目前暴露于“内分泌干扰物”，如BPA，已经损害了我们后代的福祉。如果在这些跨代啮齿类动物研究中发现负面结果，它可能会支持公众和国会开始控制环境和消费品中的双酚a，以防止对我们的后代产生这种不利影响。此外，这些数据可能会让我们深入了解当前BPA暴露给我们的后代、孙辈甚至潜在的孙辈带来的即将到来的医疗状况，以便我们更好地准备对这些后代进行治疗干预。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08-ES-104, Identification of alterations in epigenetic marks related to environmental exposures. In Avy mice that have an intracisternal A particle (IAP) in their agouti gene, exposure to a maternal diet-enriched in genistein or methyl- donors results in methylation of the IAP site and a graded shift from yellow coat color to pseudoagouti offspring. However, bisphenol A (BPA) exposure of pregnant black females that are carrying Avy fetuses results in more yellow coat color offspring. Yellow coat color offspring with a de-methylated promoter develop diabetes, obesity, and, in certain strains, cancer, but their darker siblings with a brown coat color remain healthy. These mice are a classic animal model to study epigenetics, as they are exquisitely sensitive to environmental changes. While one previous study has documented the overt coat color changes that occur in these mice in response to in utero exposure to BPA, which is an environmental estrogenic contaminant found in most plastic compounds, no study has examined the subtle phenotypic alterations, including in behavior and serum metabolite and hormone concentrations, and pathological changes that might also occur in the various coat color Avy mice. Moreover, in utero exposure of a single generation (F1) to BPA could result transgenerational effects into their great grand-offspring (F3), whose only exposure is through their predecessors. While the transgenerational effects of other compounds, including vinclozolin and diethylstilbestrol (DES) have been examined in rodents and humans (in the case of DES), not a single study to date has examined the transgenerational effects of BPA. Yet, such studies are highly relevant to humans, where we cannot easily assess the effects that exposure of one generation to endocrine disruptors, such as BPA, has on proceeding generations. However, with their 18 to 21-day gestation period and early sexual maturity (6 to 8wks of age), such transgenerational studies can readily be accomplished in mice. Herein, we will determine whether BPA can exert transgenerational effects by breeding black (a/a) females to viable yellow (Avy/a) males. Two weeks prior to breeding and throughout gestation, these females will be exposed to one of three doses of BPA (501/4g/kg/day, 5mg/kg/day, or 50mg/kg/day) through their diet. Control a/a females will be maintained on the AIN-93G diet. Pre- and post-weaning behavioral assessments will be performed on the pups to determine whether BPA affects their anxiety levels, learning and memory responses, and other developmental parameters. Femoral blood will be drawn weekly to measure the serum concentrations of metabolic and sex-regulating hormones, free fatty acids, and glucose, and their weight and overall appearance tracked. At 10 wks of age, the resultingY0 Avy males and females will be bred to control a/a females and a/a males, respectively, to determine whether any transgenerational effects can be passed through the male and female germlines. Additionally, their black coat color (a/a) (F1) siblings will be bred to determine if BPA underpins transgenerational effects independent of the Avy locus. The same experimental procedures will be repeated on the F2 and eventually the F3 generation, whose only exposure to BPA will be through their predecessors. These studies will permit us to examine the subtle changes in behavior, body weight, blood metabolites and hormone concentrations, and pathological changes that might ensue in viable yellow mice exposed in utero to BPA. Additionally, this study will examine the putative transgenerational effects that BPA can exert in a/a and Avy/a mice, the latter being a classic epigenetic animal model These studies have considerable relevance to humans, as they might serve as forewarning that our current exposure to "endocrine disruptors", such as BPA, has already compromised the welfare of our descendents. Should negative outcomes be detected in these transgenerational rodent studies, it might buttress public and congressional support to begin to curb BPA in the environmental and consumer goods to prevent such adverse effects in our descendents. Additionally, these data might provide insight into the impending medical conditions that our current exposure to BPA has imposed on our offspring, grand-offspring and even potentially great grand- offspring so that we are better prepared to therapeutically intervene in these generations.