Summary

该DNA氧化标志物的高效液相色谱法测量,8-氧代-7,8-二氢-2'-脱氧鸟苷,在培养细胞和动物组织

Published: August 01, 2015
doi:

Summary

该协议的目的是将DNA氧化标志物的检测,8-氧代-7,8-二氢-2'-脱氧鸟苷(8-氧dGuo)通过HPLC-ED,从培养的细胞或动物组织的DNA。

Abstract

氧化应激与许多生理和病理过程,以及生物外源性代谢相关,导致生物大分子的氧化,包括DNA。因此,高效的检测DNA氧化是多种学科的研究,包括药物和毒物的重要。氧化损伤的DNA的一个共同的生物标志物是-8-氧代-7,8-二氢-2'-脱氧鸟苷(8-氧代dGuo;经常误称为8-羟基-2'-脱氧鸟苷(8-OH-dGuo或8氧代dG的))。对于-8-氧代dGuo测量用高压液相色谱电化学检测器(HPLC-ED)几个协议已被描述。然而,这些主要适用于具有促氧化剂处理纯化DNA。此外,由于实验室之间方法上的差异,主要是由于在分析设备的差异,采用用于检测-8-氧代dGuo通过HPLC-ED公布的方法需要由每个实验室仔细优化。一个全面的协议,描述这样一个优化过程,是缺乏。这里,一个详细的协议是从培养的细胞或动物组织中所述的8-氧dGuo通过HPLC-ED的检测,在DNA中。它说明了如何DNA样品制备能够容易且快速地优化,以尽量减少可能发生的样品制备过程中不希望的DNA的氧化。这个协议显示了如何检测-8-氧代dGuo与氧化剂溴酸钾3处理培养的人肺泡腺癌细胞( ,A549细胞),以及从暴露在多环芳烃二苯并( 闪避,p)的屈小鼠的脾脏(DBC,前身为二苯(A,L)芘,DALP)。总体来说,这一工作说明了如何用HPLC-ED方法可以用于-8-氧代dGuo生物样品中的检测可以容易地优化。

Introduction

活性氧(ROS),其稳态水平期间许多病理学病症和xenotoxic代谢能增加,有助于氧化性DNA损伤的频率增加。在几个可能的核碱基的氧化产物,氧化性DNA损伤可容易地使用稳定标记测量-8-氧代-7,8-二氢-2'-脱氧鸟苷(8-氧dGuo),它是2的氧化形式之一' -deoxyguanosine(dGuo)1。 -8-氧代dGuo是最丰富的DNA损伤2,因此,已研究以更详细的DNA氧化标志物尽管多种DNA氧化产物3的存在。在人类中,这种损伤可以通过碱基切除修复了8羟基鸟嘌呤糖苷酶1(hOGG1基因)4修复。如果左未修理,8-氧dGuo可以向形成的碱基对替代突变( ,G到T颠换)4。重要的是,8-氧dGuo是一个既定的标记FO- [R DNA损伤有关的启动和促进癌变2。因此,8-氧代- dGuo准确定量是氧化性DNA损伤5的有用和理想的生物标志物。

人们普遍混乱关于对氧化损伤的形式的2-脱氧鸟苷,此外,化合物(S)的正确名称常规测量作为氧化性DNA损伤6的生物标志物的正确名称的文献。的8-氧dGuo( 图1中示出)的6,8-二酮和6-烯醇,8-酮互变异构形式都在文献5,7中讨论的两个最突出的互变异构体。的6,8-二酮的形式是在7.4的生理pH值的最突出的形式,并且是最突出的DNA氧化产物7。因此,8-氧dGuo,而不是8羟基dGuo是用于该氧化产物6的最适当的名称。同样重要的是要注意,2-脱氧鸟苷(dGuo),而不是nucleob酶鸟嘌呤(卦)或核糖鸟苷(国),分别是由大多数方法6检测。

精确检测和8-氧代dGuo量化是具有挑战性的,原因是:在该DNA样品的消化ⅰ)变异,ⅱ)dGuo的不定氧化成8-氧dGuo可能发生样品制备过程中,和iii)需要在分析HPLC-ED法8验证有效。在这个协议中,我们通过提供条件,有利的完整的DNA消化和ii)通过包含金属螯合剂和螯合剂处理的溶液和一种特殊的DNA分离试剂,而ⅲ)仅部分通过包含寻址旨在实现ⅰ)阳性对照,从而提供了该方法能够检测-8-氧代dGuo生物样品中。进一步验证超出了本文的范围。但是,我们相信,这一协议将有助于准用户确定在何种程度上他们需要正式验证协议,这取决于它们的目的。所需的方法的正式验证步骤的清单还设置。在8氧dGuo检测方法的开发和部署,公布的方法进行了回顾和巩固。因此,该方法消除了需要从几个公布来源往往缺乏重要的实验细节,同时还提供测试快速和简单的方式,如果该方法用于-8-氧代dGuo的检测和定量已成功地采用了收集信息。这种适应方法采用成功地分析DNA样本培养细胞和小鼠组织。这个视频文章将有助于其他群体建立一种有效的方法可靠的检测和8-氧dGuo定量用HPLC-ED。

Protocol

确保所有牧,住房,处理和实验遵守当地的法律和法规,而且实验的协议之前,开始任何研究批准。对于所描述的实验中,动物护理,处理和治疗批准了加拿大卫生署动物管理委员会。见“试剂表”,供应商的信息。 1.采集生物样品细胞或动物组织生长在含有10%胎牛血清,青霉素100单位/ ml和链霉素100μg/ ml的F12-K媒体人肺泡腺癌A549细胞。 种子细胞在每一…

Representative Results

dGuo观察到有4.7分钟而-8-氧代dGuo具有约6.4分钟( 图2A和B)的保留时间,保留时间。有一个在两种分析物之间的峰高约1000倍的差异,如在图2C中看到的。伏安-8-氧代dGuo和dGuo通过运行标准在+0.2的范围内的工作电位,获得至1.1 V的最佳工作电位为-8-氧代dGuo被确定为+ 0.5V,和0.9 v对于dGuo( 图3)。这些电位是与文献14,15中所述的其它玻璃碳电极?…

Discussion

虽然-8-氧代dGuo已被报告为脱氧核糖核酸氧化的有用的生物标志物,其可靠的量化可以构成挑战。虽然有几种公开的方法存在,有必要对协议的全面的,描述概述,以允许研究人员部署在他们的实验室的方法。这里,我们提出的HPLC为基础的协议,将允许新用户建立一种有效的方法-8-氧代dGuo检测和定量的详细概述。

已经描述了8个氧代dGuo量化三种主要的方法。这些包括酶联免疫…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

该研究是由加拿大卫生部基因组学研究和发展倡议(GRDI)和加拿大监管策略生物技术(华润雪花)。作者有没有利益冲突。

Materials

8-oxo-dGuo standard Cayman Chemical Company 89320 Inappropriately referred to as "8-hydroxy-2'-deoxy Guanosine" – see Fig. 1 and text for details
Alkaline phosphatase  Sigma-Aldrich P5931 From E.coli
Chelex 100 Sigma-Aldrich C7901 Chelates heavy metals
Desferoxamine mesylate Sigma-Aldrich D9533
dGuo standard Sigma-Aldrich D7145
Dibasic sodium phosphate Sigma-Aldrich S9390
DNA from salmon sperm Sigma-Aldrich D1626 Sodium salt
DNase I Sigma-Aldrich D4527 TypeII, from bovine pancreas
DNAzol Invitrogen 10503-27
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) Sigma-Aldrich E4884 The compound would not completely dissolve until solution pH is adjusted to 8.0 with e.g. NaOH
F12-K media ATCC 30-2004
Foetal bovine serum ATCC 30-2020
Guard column Chromatographic Specialties YBA 99S03 0204GC Protects colum from contamination; may also lead to pressure build-up
Magnesium chloride Sigma-Aldrich M8266
Monobasic sodium phosphate Sigma-Aldrich S9638
Penicillin-Streptomycin Invitrogen 15140-122
Phosphate buffered saline Invitrogen 15190-250
Phosphodiesterase I enzyme  Sigma-Aldrich P3243 Type II from Crotalus adamaneus venom
Teflon homogenizer Thomas Scientific 7724T-1 or 7724T-5 for 1 or 5 mL, respectively Volume (holding capacity) depends on the amount of sample to be processed.
Trypsin Invitrogen 15050-065
YMC-BASIC column with bonded spherical silica Chromatographic Specialties YBA 99S03 1546WT

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Citazione di questo articolo
Chepelev, N. L., Kennedy, D. A., Gagné, R., White, T., Long, A. S., Yauk, C. L., White, P. A. HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in Cultured Cells and Animal Tissues. J. Vis. Exp. (102), e52697, doi:10.3791/52697 (2015).

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