根据 NSF (EPSCoR EPS-0814251) 和 NIH (1P20GM103653-01A1) 对 MKT 和 HSD 的支持, 特拉华州立大学对 MDM (标题 III HBGI) 和 KRS (NIH-INBRE) 的机构学生支持得到了感激的承认。多亏了线虫基因中心 (由 NIH 研究基础设施项目办公室 P40 OD010110) 为c. 线虫野生型 N2 菌株所支持。

下面给出的协议已经标准化, 以测试 BPA 暴露在早期胚胎发生中的影响, 并可能被修改, 用于与 BPS, BPF 或其他 EDCs 和毒物在C. 线虫胚胎。
1. 材料设置
<ol><li>500毫升的线虫生长培养基 (NGM)18培养基, 溶解1.5 克氯化钠, 1.25 克的蛋白胨, 和8.5 克的水中的琼脂到500毫升。热处理后 (121 摄氏度, 15 PSI, 20 分钟), 添加0.5 毫升的胆固醇 (5 毫克/毫升乙醇), 0.5 毫升1米 MgSO4, 0.5 毫升 1 m CaCl2和12.5 毫升磷酸钾的缓冲, pH 1 (7.4 g 的 108.3 PO 2, 35.6 克 K2HPO4, 水到1升)。</li>
	<li>混合25毫升1氮 KOH, 4 毫升漂白剂和71毫升水, 制备次氯酸盐溶液。使这个解决方案新鲜。</li>
	<li>通过混合3克2PO4, 6 g 的 Na2HPO4, 5 克氯化钠, 5 毫升1米的 MgSO4和水到1升, 准备 M9 缓冲区。</li>
	<li>通过混合 129 mL 0.02 米 K2HPO4, 871 毫升的 18 ml2PO4和5.85 克氯化钠, 准备 S 缓冲区。</li>
	<li>用热处理消毒 M9 和 S 缓冲器。</li>
	<li>在20毫升的 Luria 汤 (磅) 中生长一夜间大肠杆菌 (OP 50 菌株) 的培养。</li>
	<li>将 BPA 溶解在10% 乙醇中, 制成1毫米的库存溶液。在 S 缓冲区中进行后续稀释0.1 µM、0.5 µM、1µM、5µM 和10µM。 注: 稀释水中的缓冲液可大大降低乙醇的浓度;例如, 在本议定书所描述的最高 BPA 浓度 (10 µM) 中, 乙醇的浓度为0.1% 伏/v。</li>
</ol>2.线虫培养和同步
<ol><li>将 NGM 介质倒入100毫米板 (约25毫升/片), 并使其凝固。一旦固化, 种子的板块通过传播150µL 的大肠杆菌OP50 从液体培养一夜之间生长。孵化盘在37°c 过夜。</li>
	<li>第二天, 将 N2 蠕虫转移到新的板块上, 通过分块1厘米正方形的近似板。允许蠕虫生长3天, 在20摄氏度, 直到板块填充有良好喂养成熟的成年人, 含有可见的胚胎。</li>
	<li>要同步, 请通过吹打5毫升的 M9 缓冲板来清洗每个盘子。将液体转移到无菌的15毫升锥形离心管。 注意: 同步是为了杀死成年蠕虫并收集相对相同阶段的胚胎。</li>
	<li>离心机以 3000 x g 为4分钟室温, 以获得一个松散的成人蠕虫颗粒。</li>
	<li>使用10毫升吸管, 小心地取出上清, 以保持颗粒完好。加入5毫升的次氯酸盐溶液, 轻轻地混合。</li>
	<li>离心机在 3000 x g室温下4分钟。</li>
	<li>用10毫升吸管去除上清, 用7毫升的 M9 缓冲液冲洗。重复此步骤两次。</li>
</ol>3. 将蠕虫暴露于 BPA 中
<ol><li>在最后一次洗涤从上面步骤 2.7, 暂停大约100µL 蛋与 M9 缓冲。将大约50µL 的卵转移到2毫升离心管, 已经含有适当浓度的 BPA 稀释在 S 缓冲器中。调整 S 缓冲量以确保每管的 BPA (0.0 µM、0.1 µM、0.5 µM、1.0 µM、5.0 µM 和10µM) 的最终浓度正确。表1说明了使这些稀释的一种方法。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>最终的 BPA 浓度</td>
			<td>库存 BPA (100 微米)</td>
			<td>S 缓冲区</td>
			<td>同步蠕虫</td>
		</tr>
<tr>
<td>0.1 微米</td>
			<td>1 ul</td>
			<td>949 ul</td>
			<td>50 ul</td>
		</tr>
<tr>
<td>0.5 微米</td>
			<td>5 ul</td>
			<td>945 ul</td>
			<td>50 ul</td>
		</tr>
<tr>
<td>1微米</td>
			<td>10 ul</td>
			<td>940 ul</td>
			<td>50 ul</td>
		</tr>
<tr>
<td>5微米</td>
			<td>50 ul</td>
			<td>900 ul</td>
			<td>50 ul</td>
		</tr>
<tr>
<td>10微米</td>
			<td>100 ul</td>
			<td>850 ul</td>
			<td>50 ul</td>
		</tr>
</tbody></table>表 1: 库存 BPA, S 缓冲和同步蠕虫用于达到所需的浓度用于我们的研究。
<ol start="2"><li>把管子放在一个振动筛上, 在20°c 时轻轻摇动4小时, 在 25 rpm 的旋转振动筛或25倾斜/分钟的摇摆振动筛。</li>
	<li>4小时后, 将管子放在管架上, 允许蠕虫沉淀。</li>
	<li>丢弃上清并将蠕虫转移到种子板上 (NGM 板与 OP50 的大肠杆菌)。使液体 OP50大肠杆菌文化, 如1.5 所述, 并将2.1 中所述的板材播种。</li>
	<li>让蠕虫生长60小时, 在20摄氏度。每天检查盘子是否有污染。受污染的 NGM 板块通常是变色的, 并伴有个别的菌落。检查显微镜下的蠕虫是否人满为患。缺乏 OP50 的大肠杆菌草坪和集中的蠕虫在小斑点意味拥挤。</li>
</ol>4. 前接触适应性试验
<ol><li>将大约10个同步的年轻的成人蠕虫转移到新的非种子选手 NGM 板使用一个火消毒30克铂丝挑选。让蠕虫不受干扰的5分钟, 让他们的时间适应新的板块。</li>
	<li>对于适应反应, 用一根挂在木制串或牙签末端的眉毛, 用70% 乙醇浸泡消毒。用干净的无绒纸巾擦拭, 等待1分钟乙醇蒸发掉。用眉毛轻轻地触摸头上的蠕虫 (咽前的灯泡)。重复触摸, 允许十年代在触摸之间, 以允许蠕虫恢复。</li>
	<li>继续触摸 (允许十年代 interstimulus 间隔), 直到蠕虫不再向后移动。在这一点上, 蠕虫已经习惯了刺激。记录蠕虫对习惯所需的接触次数。</li>
	<li>用单向方差分析法和 Tukey 后的特殊试验, 对治疗和未治疗对照的平均适应率的差异进行研究。</li>
</ol>5. 蠕虫繁殖试验
<ol><li>使用铂金采摘将单个 L3 蠕虫转移到新鲜的和种子的盘子里。确保每个盘子只放置一个蠕虫。 注: L3 幼虫期后, 蠕虫发育为 L4 幼虫期和成年。及早采摘他们是方便的, 因为他们足够大, 以转移个别动物, 同时确保他们还没有产卵, 否则将错过在伯爵。</li>
	<li>计算每12–24个的卵数。计数后, 将父蠕虫转移到新的盘中。重复直到蠕虫停止产卵, 通常在5天后孵化20摄氏度。 注: 野生型蠕虫平均可产卵300只;因此, 每天将父母转移到新的盘子里可以防止过度拥挤。</li>
	<li>用单向方差分析法和 Tukey 后的临时试验, 对治疗后和未经治疗的对照组的平均卵数进行了比较。</li>
</ol>

<ol>
	<li>Gore, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+Summary+to+EDC-2:+The+Endocrine+Society's+Second+Scientific+Statement+on+Endocrine-Disrupting+Chemicals.">Executive Summary to EDC-2: The Endocrine Society's Second Scientific Statement on Endocrine-Disrupting Chemicals.</a> Endocr. Rev. 36, 593-602 (2015).</li><li>Braun, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-life+exposure+to+EDCs:+role+in+childhood+obesity+and+neurodevelopment.">Early-life exposure to EDCs: role in childhood obesity and neurodevelopment.</a> Nat. Rev. Endocrinol. 13, 161-173 (2017).</li><li>Melnik, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summary+of+the+National+Toxicology+Program's+Report+of+the+Endocrine+Disruptors+Low-Dose+Peer+Review.">Summary of the National Toxicology Program's Report of the Endocrine Disruptors Low-Dose Peer Review.</a> Environ. Health Perspect. 110, 427-431 (2002).</li><li>Kortenkamp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+mixing+cocktails:+a+review+of+combination+effects+of+endocrine-disrupting+chemicals.">Ten years of mixing cocktails: a review of combination effects of endocrine-disrupting chemicals.</a> Environ. Health Perspect. 115 Suppl 1, 98-105 (2007).</li><li>Vandenberg, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormones+and+endocrine-disrupting+chemicals:+low-dose+effects+and+nonmonotonic+dose+responses.">Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses.</a> Endocr. Rev. 33, 378-455 (2012).</li><li>Eramo, S., Urbani, G., Sfasciotti, G. L., Brugnoletti, O., Bossu, M., Polimeni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenicity+of+bisphenol+A+released+from+sealants+and+composites:+a+review+of+the+literature.">Estrogenicity of bisphenol A released from sealants and composites: a review of the literature.</a> Ann Stomatol. (Roma). 1, 14-21 (2010).</li><li>Cantonwine, D. E., Ferguson, K. K., Mukherjee, B., McElrath, T. F., Meeker, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+Bisphenol+A+Levels+during+Pregnancy+and+Risk+of+Preterm+Birth.">Urinary Bisphenol A Levels during Pregnancy and Risk of Preterm Birth.</a> Environ. Health Perspect. 123, 895-901 (2015).</li><li>Vandenberg, L. N., Maffini, M. V., Sonnenschein, C., Rubin, B. S., Soto, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol-A+and+the+great+divide:+a+review+of+controversies+in+the+field+of+endocrine+disruption.">Bisphenol-A and the great divide: a review of controversies in the field of endocrine disruption.</a> Endocr. Rev. 30, 75-95 (2009).</li><li>Suzuki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+and+neonatal+exposure+to+bisphenol-a+enhances+the+central+dopamine+d1+receptor-mediated+action+in+mice:+enhancement+of+the+methamphetamine-induced+abuse+state.">Prenatal and neonatal exposure to bisphenol-a enhances the central dopamine d1 receptor-mediated action in mice: enhancement of the methamphetamine-induced abuse state.</a> Neuroscience. 117, 639-644 (2003).</li><li>Rosenfeld, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+and+phthalate+endocrine+disruption+of+parental+and+social+behaviors.">Bisphenol A and phthalate endocrine disruption of parental and social behaviors.</a> Front. Neurosci. 9, 1-15 (2015).</li><li>Patisaul, H. B., Adewale, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effects+of+environmental+endocrine+disruptors+on+reproductive+physiology+and+behavior.">Long-term effects of environmental endocrine disruptors on reproductive physiology and behavior.</a> Front. Behav. Neurosci. 3, 10 (2009).</li><li>Lee, D., Jacobs, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+issues+in+human+studies+of+endocrine+disrupting+chemicals.">Methodological issues in human studies of endocrine disrupting chemicals.</a> Rev. Endocr. Metab. Disord. 16, 289-297 (2015).</li><li>Mimoto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+estrogenic+hormone+receptor+in+Caenorhabditis+elegans.">Identification of an estrogenic hormone receptor in Caenorhabditis elegans.</a> Biochem. Biophys. Res. Commun. 364, 883-888 (2007).</li><li>Allard, P., Colaiacovo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+impairs+the+double-strand+break+repair+machinery+in+the+germline+and+causes+chromosome+abnormalities.">Bisphenol A impairs the double-strand break repair machinery in the germline and causes chromosome abnormalities.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 20405-20410 (2010).</li><li>Mersha, M. D., Patel, B. M., Patel, D., Richardson, B. N., Dhillon, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+BPA+and+BPS+exposure+limited+to+early+embryogenesis+persist+to+impair+non-associative+learning+in+adults.">Effects of BPA and BPS exposure limited to early embryogenesis persist to impair non-associative learning in adults.</a> Behav. Brain. Funct. 11, 27 (2015).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+the+BPA-Substitute+Bisphenol+S+Causes+Unique+Alterations+of+Germline+Function.">Exposure to the BPA-Substitute Bisphenol S Causes Unique Alterations of Germline Function.</a> PLoS Genet. 12, e1006223 (2016).</li><li>Rankin, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+revisited:+an+updated+and+revised+description+of+the+behavioral+characteristics+of+habituation.">Habituation revisited: an updated and revised description of the behavioral characteristics of habituation.</a> Neurobiol. Learn. Mem. 92, 135-138 (2009).</li><li>Hope, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C elegans: A Practical Approach. , (1999).</li><li>Sanchez, K., Mersha, M., Dhillon, H. S., Temburni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+Effects+of+Endocrine+Disrupting+Compounds+on+the+Development+of+Vertebrate+Neural+Network+Function+Using+Multi-electrode+Arrays.">Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays.</a> J. Vis. Exp. , (2017).</li><li>Hu, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WormBook The C. elegans Research Community. , (2007).</li></ol>

作者声明他们没有什么可透露的。

快速生长和发育的阶段, 如胚胎发生特别容易受到各种内分泌干扰化合物, 包括 BPA 的有害影响。我们提供详细的协议, 以研究暴露于 BPA 或其他毒物的影响, 在线虫无脊椎动物模型, 它允许方便的滴定范围的浓度, 以评估其对胚胎生存能力的影响 (图 2).对存活的成人进行后续行为实验, 从暴露于低剂量 BPA 的胚胎中发展出来, 可以对神经元功能的长期影响进行分析 (图 3)。本文的核心思想是在线虫模型中, 为在胚胎发生初期暴露出各种毒物提供详细的协议;其11.5 小时胚胎发育期的前4小时窗 (20 摄氏度) 对应于增殖期, 其次是器官形成。为此, 我们通过生育力和非联想学习分析提供了终点的验证。虽然 bpa 和其他 bisphenols 的作用的机械基础是这一领域研究的目标, 但我们并没有试图为 bpa 在这一着重于方法的文章中的作用提供一个机械性的解释。然而, 我们想指出的是,线虫模型提供了一个理想的系统来进一步剖析行动机制。例如, 在今后有关破译 bpa 作用机制的工作中, 可以生成和筛选线虫突变体, 以抵抗 bpa 的影响, 从而为破译通路铺平道路。此外, 我们的同伴论文讨论了 BPA 在脊椎动物神经网络同步水平上的作用, 为了解研究毒物效应潜在机制的基础提供了另一条途径19。
在本文所详述的方法中, 应仔细执行的一些关键步骤包括: 选择一个有良好喂养的C. 线虫的平板, 在解剖显微镜下可见成熟的卵子是至关重要的, 并且如果不遵循这一步骤, 将导致不充分的样本大小的胚胎, 为 EDC 暴露实验。同样重要的是要确保次氯酸盐溶液是新鲜的, 以避免获得一个不同步的人群, 这将干扰在早期胚胎发生中 BPA 暴露的能力。用 M9 (次氯酸盐曝光后) 清洗小球也是非常重要的一步。如果没有正确的做, 高浓度的漂白剂可能导致死亡的胚胎。此外, 胚胎应转移到种子 NGM 板后4小时。如果在 BPA 溶液中留出一段较长的时间, 胚胎很可能会发育成长期存活的 dauer 幼虫。这将极大地干扰生殖试验和行为化验。如果遗漏了这些步骤中的任何一个, 最好从一开始就开始该过程。如果出于任何原因, NGM 板的蠕虫被污染或没有足够的食物, 它会增加蠕虫的压力, 从而扭曲收集的数据。此类板材应丢弃, 不能在实验中使用。
以前, 一项卓越的研究成功地使用了线虫模型来证明由于生殖14中 DNA 修复机械的故障, BPA 导致的染色体异常。然而, 值得注意的是, 上述研究设计已使用连续 bpa 暴露的 C.线虫, 以高剂量 bpa 在整个生命周期。我们的方法旨在研究小剂量 BPA 暴露在早期胚胎发生, 以检测其对胚胎生存能力和微妙的长期影响对幸存者的影响。据我们所知, 目前尚无任何方法可将早期胚胎暴露于极低剂量的 BPA, 限制在特定时间内;因此, 这里提出了小说的方法。
此外, 这里提供的协议可以很容易地适应, 以研究其他 EDCs 在胚胎发生的扩散阶段的影响。然而, 为了研究晚期胚胎阶段, 该议定书将需要对重点的发展关键时间窗口进行关键的修改。此外, 我们的议定书将不适合的实验需要较长时间的毒物暴露, 因为它开辟了替代发展途径的可能性, 导致长期生存的 dauer 逮捕在第二个蜕皮周期, 这是耐到苛刻的条件20。进一步的修改和改进将需要更长的暴露时间通过补充的食物来源, 没有潜力代谢毒物或 EDC例如蒸压大肠杆菌。最后, 我们的方法可以用来评估不同的内分泌干扰化学物质对无脊椎动物模型系统中的生育力和神经元功能的影响, 如线虫.

近年来, 对环境毒物的接触, 特别是那些倾向于干扰发展的化合物受到了严格的科学审查。每天使用的1000多种化学物质被归类为内分泌干扰化合物 (EDC)1。已经指出, 包括胚胎、婴儿和儿童阶段在内的快速增长和发展时期特别容易受到甚至是低剂量的药物的有害影响。它们的作用已被证明导致生殖和神经发育紊乱2。根据美国环境保护局和美国国家毒理学计划小组的指导方针, 低剂量可以定义为任何剂量低于已报告的水平, 导致一个可观察到的生物变化或损害3。除了个别 EDCs 的低剂量效应外, 在环境中低浓度发现的各种 EDCs 的混合物有可能导致实质性累积效应4。
双酚 a (BPA 或 44 '-(丙烷-22-己)) 是一种聚合剂, 在通常使用的物品中发现, 如水瓶, 商店收据, 牙科密封胶, 饮料和食品罐的衬里5。由于其与 17-β雌二醇 (E2) 的结构相似性及其与 ERα和 ERβ雌激素受体的亲和力, BPA 被归类为内分泌干扰化学 (EDC)5,6。虽然更弱, BPA 与雌激素受体的亲和力已经被证明影响了两性的生殖系统和扰乱神经功能的剂量, 被认为是安全的7,8。通过 epigenetically 调节机制对 DNA 甲基化的变化进行了观察, 以引起暴露于 BPA9的小鼠的长期神经缺损。具体地说, BPA 也被牵连作为一个可能的罪魁祸首, 增加多动症率, 注意力不足和增加对药物的敏感性, 由于在小鼠边缘前脑慢性暴露后 D1 巴胺受体增加看到9,10. EDCs 对人类健康的有害影响的重要证据是基于相关研究, 其主要重点是在低剂量的5下, 将人口长期暴露于环境毒物;然而, 在对未经证实的炒作11、12的批评中, 人们接受了对人类研究和操纵实验控制的推论的限制。
由于对哺乳动物的秀丽线虫 '基因的保护, 包括它的类固醇激素受体基因, 研究人员利用这个基因可驯服的实验室模型来解开 EDCs 的功能和机械效应。13. 使用C. 线虫进行的实验表明, BPA 和 BPS 等化合物可能导致细胞凋亡、胚胎致死、双链 DNA 断裂修复机制和神经功能14、15 ,16。
我们的实验室先前已经表明, 即使是低剂量暴露限于早期胚胎发生, 也导致了存活的成年人的行为缺陷的生育力降低了15。对反复刺激的适应是模型系统中的非联想学习的一种形式, 包括线虫, 我们的方法论将这种非联想学习的形式作为行为输出来检测胚胎暴露对毒物17具体地说, 我们为研究在C. 线虫上的 BPA 暴露提供了详细的协议, 包括它对胚胎杀伤力的直接影响和对成人行为的长期影响, 并在中描述了总体示意图. 图 1。提供生殖和非联想学习化验的代表性结果, 以突出我们的方法的有效性。

<table><tbody><tr><td>NaCl</td><td>Fisher</td><td>7647-14-5</td><td></td></tr><tr><td>Peptone</td><td>Fisher</td><td>S25761B</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>A11470</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>7487-88-9</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P286-1</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>7758-11--4</td><td></td></tr><tr><td>KOH</td><td>Fisher</td><td>1310-58-3</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>n/a</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>BP332-500</td><td></td></tr><tr><td>LB</td><td>Fisher</td><td>BP1426-500</td><td></td></tr><tr><td>BPA</td><td>Sigma-Aldrich</td><td>239658-250g</td><td></td></tr><tr><td>BPS</td><td>Sigma-Aldrich</td><td>103039-100g</td><td></td></tr><tr><td>EtOH</td><td>Sigma-Aldrich</td><td>64-17-5</td><td></td></tr><tr><td>E.coli OP50</td><td>CGC</td><td></td><td>Repository at U of Minnesota</td></tr><tr><td>N2 (Wild-type C. elegans) worms</td><td>CGC</td><td></td><td>Repository at U of Minnesota</td></tr><tr><td>Platinum wire pick</td><td>Genesee Scientific</td><td>59-AWP</td><td></td></tr><tr><td>Petri plates</td><td>Fisher</td><td>07-202-011</td><td></td></tr><tr><td>Dissection Microscope</td><td>AmScope</td><td>SM-2TYY</td><td></td></tr></tbody></table>

long-term behavioral and reproductive consequences of embryonic exposure to low-dose toxicants

Bisphenols, 如双酚 a (BPA) 和双酚 S (BPS) 是聚合剂广泛用于生产塑料和许多日常使用的产品。根据其化学结构和雌二醇的生物学特性, 将其归类为内分泌干扰物 (EDC)。长期暴露于 EDCs, 即使在低剂量, 已与各种健康缺陷, 包括癌症, 行为紊乱和不孕, 在早期发育期显示更大的脆弱性。基因处理线虫模型的细胞和分子研究秀丽线虫表明, 接触 BPA 会导致细胞凋亡、胚胎致死和 DNA 修复机制的破坏。我们以前报告过, 暴露的C. 线虫胚胎对低剂量的不同 bisphenols 减少生育力。此外, 我们已经表明, 暴露在发育的早期阶段的影响持续到成年, 通过量化的习惯行为, 一种非联想学习的形式来衡量。在这里, 我们提供了胚胎暴露于低剂量 EDCs 的详细协议, 以及相关的繁殖力和前接触习惯的测定, 以及具有代表性的结果。

早先与线虫有关的研究报告说, 在胚胎发育和成年期间持续暴露于高 BPA 浓度 (≥1毫米) 会降低生育力14。我们实验室报告的后续研究表明, 在其发育的开始阶段, 在4小时内暴露于 BPA 的胚胎显示, 作为成人的可存活卵子数量减少了15 (图 2)。该方法的两个主要特点是, (i) 所使用的 BPA 浓度明显低于 (0.1 µM 到10µM 范围), (ii) 暴露量仅限于早期胚胎期。除了减少的繁殖力, 即使在较低的剂量, 我们的适应化验显示, 蠕虫暴露在 BPA 作为胚胎需要更多的刺激变得习惯时, 与单独的车辆暴露在车上15 (图 3)。这些影响是值得注意的, 因为它们是基于低剂量的 BPA 暴露, 遵循的理由是, 对神经元功能的微妙影响可能不是形态学上明显的, 可能会通过行为改变来辨别。简而言之, 这里提出的代表性结果强调了这样一个事实, 即 BPA 对胚胎的有害影响影响其发育, 包括可通过成人行为分析在线虫中评估的神经元功能。此处提供的协议可用于测试低剂量以及其他潜在毒物 (包括内分泌破坏性化合物) 的长期影响。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56771/56771fig1.jpg"> 图 1: 实验设计的示意图表示形式.成熟的蠕虫收集和暴露在次氯酸盐溶液, 以收集同步胚胎。然后胚胎被暴露在不同浓度的 BPA 中4小时。暴露的胚胎被转移到种子 NGM 板上, 在那里他们被允许生长60小时, 在20摄氏度。为了在发育的早期阶段检测暴露的有效性, L4 蠕虫被转移到单独的平板上, 并进行了生殖试验, 并对年轻成年人进行了前接触适应试验。<a href="//cloudflare.jove.com/files/ftp_upload/56771/56771fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56771/56771fig2.jpg"> 图 2: BPA 降低了生育力.暴露于1µM 和更高浓度的 BPA 显著地降低了与对照组相比的卵数量 (由水平线表示);n = 10, *p < 0.05)。误差线表示扫描电镜. 用方差分析法进行 Tukey 的后验。此数字已从 Mersha et, 201515中进行了修改。<a href="//cloudflare.jove.com/files/ftp_upload/56771/56771fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56771/56771fig3.jpg"> 图 3: 胚胎 BPA 暴露的长期影响反映在成人行为中.暴露于 BPA 浓度低至0.1 µM 的胚胎影响成人的非联想前接触习惯 (n = 60, *p < 0.05)。误差条表示 SEM;方差分析, Tukey 的后临时测试。此数字已从 Mersha et, 201515中进行了修改。<a href="//cloudflare.jove.com/files/ftp_upload/56771/56771fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants at JoVE.com

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

接触环境毒物会对发展产生严重影响, 并具有长期影响。提供详细的协议, 以说明一个战略使用一个有效的实验室模型, 以研究早期胚胎接触双酚 a 的影响。我们提供生育力和行为分析, 以监测我们的毒物暴露的生物化验的有效性。

胚胎暴露于低剂量毒物的长期行为和生殖后果

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

long-term-behavioral-reproductive-consequences-embryonic-exposure-to

Research

JoVE Journal

Developmental Biology

6.0K 次观看.  Johns Hopkins School of Medicine. 接触环境毒物会对发展产生严重影响, 并具有长期影响。提供详细的协议, 以说明一个战略使用一个有效的实验室模型, 以研究早期胚胎接触双酚 a 的影响。我们提供生育力和行为分析, 以监测我们的毒物暴露的生物化验的有效性。

视频: Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

适度的产前酒精暴露在成年大鼠社会行为的量化

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

科研

行为学

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

环境科学

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

应用多电极阵列评价内分泌干扰物对脊椎动物神经网络功能发育的影响

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

发育生物学

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

环境污染物对斑马鱼幼虫的神经行为影响研究

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues and organs. Ecotoxicological effects of microplastics on the development of bird embryos are not known. The bird egg is a complete development and nutrition system, and the entire embryo development occurs in the eggshell. Therefore, a direct record of bird embryo development under the stress of pollutants such as microplastics is highly limited by the opaque eggshell in traditional hatching. In this study, the effects of microplastics on quail embryo development were visually monitored by hatching without an eggshell. The main steps include the cleaning and disinfection of fertilized eggs, the incubation before exposure, the short-term incubation after exposure, and the sample extraction. The results show that compared with the control group, the wet weight and body length of the microplastics-exposed group displayed a statistical difference and the liver proportion of the whole exposed group significantly increased. Additionally, we evaluated external factors that affect the incubation: temperature, humidity, egg rotation angle, and other conditions. This experimental method provides valuable information on the ecotoxicology of microplastics and a novel way to study the adverse effects of pollutants on the development of embryos.

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

微塑料对鸟胚胎发育的生态毒性影响，无需蛋壳孵化

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

蒸汽腔体建设用于公开小鼠酒精在所有三个学期人类发展的等效

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

医学

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

使用鸡胚胎作为评估发育性心脏病的有力工具

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

生物学

用于斑马鱼胚胎毒性评估的 Startle 试验

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

作者聚焦：使用 Zebrafish 胚胎惊吓反应测定进行高通量毒性筛选

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

使用斑马鱼振动惊吓反应筛查系统评估化学品的毒性

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...