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Environnement

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Published: July 29, 2019
doi:
10.3791/59367
, , , ,
1College of Environmental Sciences and Engineering,Tongji University, 2Shanghai Institute of Pollution Control and Ecological Security, 3Jiaxing Tongji Institute of Environment, 4College of Ecological Technique and Engineering,Shanghai Institute of Technology

Summary

持久性化学品的跨代和多代影响对于判断其对环境和人类健康的长期影响至关重要。我们提供了研究跨代和多代效应使用自由生活线虫卡埃诺哈布迪炎埃莱甘斯的新方法。

Abstract

有关化学品毒性的信息对于化学品的应用和废物管理至关重要。对于低浓度化学品,长期影响对于判断其对环境和人类健康的影响非常重要。在展示长期影响时,化学品对几代人的影响为最近的研究提供了新的见解。在这里,我们描述了使用自由生活线虫Caenorhabdiselegans研究多代化学物质影响的协议。提出了两个方面:(1)跨代(TG)和(2)多代效应研究,后者分为多代暴露(MGR)和多代残差(MGR)效应研究。TG效应研究是强大的,其目的很简单,即与父母接触化学品是否会对后代造成任何残留后果。在测量对父母的影响后,使用次氯酸钠溶液杀死父母并保留后代,以便促进对后代的影响测量。TG效应研究用于确定当后代的父母暴露于污染物时是否受到影响。MGE 和 MGR 效应研究用于系统化,用于确定连续的代接触是否会导致后代代代适应反应。小心的拾取和转移用于区分代,以方便对每一代人进行效果测量。我们还结合协议来测量运动行为、生殖、寿命、生化和基因表达变化。还给出了一些实例实验来说明跨代和多代效应研究。

Introduction

化学品的应用和废物管理高度依赖于其在特定浓度下的影响信息。值得注意的是,时间是影响和浓度之间的另一个基本要素。也就是说,化学品,特别是那些在实际环境中浓度低的化学品,需要时间来产生可衡量的效果。因此,研究人员在动物实验中安排了不同长度的暴露时间,甚至覆盖了整个生命周期。例如,老鼠接触尼古丁30天,90天或180天,以研究其毒性影响2。 然而,这种接触持续时间仍然不足以阐明污染物(例如持久性有机污染物[持久性有机污染物])的长期影响,这些污染物在环境中可以持续几代生物。因此,对几代人影响的研究越来越受到人们的重视。

代际效应研究主要有两个方面。第一个是跨代(TG)效应研究,它可以有力地测试与父母接触的化学物质是否会对后代造成任何后果3。第二个是多代效应研究,在接触和残余效应方面考虑更系统。一方面,多代暴露(MGE)效应用于说明动物对长期具有挑战性的环境的适应性反应。另一方面,多代残留效应用于证明接触后的长期残留后果,因为母体接触伴随着胚胎接触第一个后代和胚芽系接触第二个后代后代,使第三代作为第一代完全出暴露4。

虽然哺乳动物(例如,小鼠)是毒性研究中的模范生物体,特别是与人类有关的生物体,但在研究代际影响时,它们的应用相当耗时、昂贵和伦理上涉及5。 因此,包括甲壳类达芙尼亚麦格纳6号、昆虫果蝇黑色素7和斑马鱼达尼奥雷里奥8号在内的生物提供了替代选择。然而,这些生物要么缺乏与人类的相似性,要么在研究中需要特定的设备。

Caenorhabditis elegans是一种小型自由活线虫(长约 1 毫米),生命周期短(在 20 °C 时约为 84 小时)9。这种线虫与人类有许多保守的生物途径,因此被广泛用于说明各种应力或毒物的影响。值得注意的是,99.5%的线虫是甲虫,使这种生物极适合研究代际效应,例如重金属和磺胺的TG效应3,11,金纳米粒子和重重的MGE效应金属12和温度13,MGR效应的磺胺14,以及MGE和MGR效应的伽马辐照15和林丹4。此外,在化学品(如Zearalenone)对小鼠发育和繁殖的影响和16、17的C.elegans之间发现了类似的结果,这将为推断从这个小动物到人类的影响。

TG 和 MG 效应研究既耗时,也需要仔细的设计和性能。值得注意的是,上述研究中在生命阶段选择、暴露条件和代际分离方法方面存在差异。这种差异妨碍了结果之间的直接比较,也妨碍了对结果的进一步解释。因此,必须建立统一的协议,以指导TG和MG效应研究,并提供一个更大的图景,揭示各种有毒物质或污染物在长期后果中的相似模式。现行协议的首要目标将表明在研究C.elegans的跨代和多代效应方面的明确操作过程。这些协议将有利于那些有兴趣研究有毒物质或污染物的长期影响的研究人员。

Protocol

1. 培养大肠杆菌OP50 通过在100 mL水中溶解4克氢氧化钠溶液制备1M氢氧化钠溶液。 在1L锥形烧瓶中溶解10克试金石、5克酵母提取物和10克氯化钠,制备溶质汤(LB)培养基。使用 1 M 氢氧化钠溶液将 pH 调整到 7.0。 将 LB 液体介质从步骤1.2压成 20 锥形烧瓶(最大允许体积:100 mL),每个介质为 50 mL 介质。用牛皮纸盖住圆锥形烧瓶。 在 121°C 和 0.105 MPa 下对 LB 液体介质进行消毒…

Representative Results

在这里,我们描述了在跨代(TG)、多代接触(MGE)和多代残留(MGR)效应研究中使用C.elegans研究化学物质对几代人的影响的协议。我们自己的研究结果作为例子提出。一项研究介绍了重金属对运动行为3的TG影响。其他两项研究显示硫磺胺和林丹对繁殖和生化及遗传指数测量4、14的MGE和MGR效应。 <p class="jove_conten…

Discussion

为了成功实施所述协议,应考虑以下建议。在无菌环境中执行总体实验操作。操作不当可能导致大肠杆菌菌株(如真菌和虫子)的污染,从而阻碍大肠杆菌的正常生长,从而影响实验结果。在描述培养C. elegan 的章节中,用肉眼或显微镜观察 NGM 琼脂上C. elegans的生长尺度。当琼脂的C.elegans在面积上超过75%,或养殖时间超过一周时,进行新一轮的接种,以避免C.elegans的过度…

Divulgations

The authors have nothing to disclose.

Materials

 agar powder OXOID, Thermo Fisher Scientific, UK 9002-18-0
79nnHT Fast Real-Time PCR System  Applied Biosystems 
96-well sterile microplate Costar，Corning，America
Autoclave sterilizer Tomy, Tomy Digital Biology, Japan
Biosafety cabinet LongYue, Shanghai longyue instrument equipment co. Ltd, China
calcium chloride Sinopharm chemical reagent company Ltd, China 10043-52-4
centrifuge  5417R Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany
Centrifuge tubes Axygen, Aixjin biotechnology (Hangzhou) co. Ltd, America
cholesterol Sinopharm chemical reagent company Ltd, China 57-88-5
Dimethyl sulfoxide VETEC, Sigmar aldrich (Shanghai) trading co. Ltd, America 67-68-5
disodium hydrogen phosphate Sinopharm chemical reagent company Ltd, China 7558-79-4
ethanol Sinopharm chemical reagent company Ltd, China 64-17-5
Filter Thermo, Thermo Fisher Scientific, America
incubator YiHeng17, Shanghai yiheng scientific instrument co. Ltd, China
inoculating loop
K2HPO4•3H2O Sinopharm chemical reagent company Ltd, China 16788-57-1
kraft paper
Mcroplate Reader Boitek, Boten apparatus co. Ltd, America
MgSO4•7H2O Sinopharm chemical reagent company Ltd, China 10034-99-8
Microscopes XTL-BM-9TD BM, Shanghai BM optical instruments manufacturing co. Ltd, China 
Petri dishes
Pipette Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany
Potassium chloride Sinopharm chemical reagent company Ltd, China 7447-40-7
potassium dihydrogen phosphate Sinopharm chemical reagent company Ltd, China 7778-77-0
Qiagen RNeasy kits Qiagen Inc., Valencia, CA, United States
QuantiTect SYBR Green RT-PCR kits Qiagen Inc., Valencia, CA, United States
RevertAid First Strand cDNA Synthesis Kit Thermo Scientific, Wilmington, DE, United States
sodium chloride Sinopharm chemical reagent company Ltd, China 7647-14-5
sodium hydroxide Sinopharm chemical reagent company Ltd, China 1310-73-2
sodium hypochlorite solution Aladdin, Shanghai Aladdin biochemical technology co. Ltd, China 7681-52-9
tryptone OXOID, Thermo Fisher Scientific, UK 73049-73-7
yeast extract OXOID, Thermo Fisher Scientific, UK 119-44-8
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Tags

Keywords: Caenorhabditis ElegansTrans-generational EffectsMulti-generational EffectsToxicantsNematodeHermaphroditesPharmaceutical SynthesisEnvironmental ManagementAquatic ToxicityDaphnia MagnaDrosphila MelanogasterE. ColiLB MediumNGM AgarsGravid NematodesAge-synchronizationSodium Hypochlorite Solution
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Li, Z., Ai, F., Zhang, J., Yu, Z., Yin, D. Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants. J. Vis. Exp. (149), e59367, doi:10.3791/59367 (2019).
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