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…

Disclosures

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

References

  1. Helbock, H. J., Beckman, K. B., Shigenaga, M. K., Walter, P. B., Woodall, A. A., Yeo, H. C., Ames, B. N. DNA oxidation matters: The HPLC-electrochemical assay of 8-oxo-deoxyguianosine and 8-oxo-guanine. Proc. Natl. Acad. Sci. 95 (1), 288-293 (1998).
  2. Valavanidis, A., Vlachogianni, T., Fiotakis, C. 8-hydroxy-2′ -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis. J. Environ. Sci Health C Environ. Carcinog. Ecotoxicol. Rev. 27 (2), 120-139 (2009).
  3. Cadet, J., Bellon, S., Douki, T., Frelon, S., Gasparutto, D., Muller, E., Pouget, J. P., Ravanat, J. L., Romieu, A. Radiation-induced DNA damage: formation, measurement, and biochemical features. J Environ Pathol Toxicol Oncol. 23 (1), 23-23 (2004).
  4. Weiss, J. M., Goode, E. L., Ladiges, W. C., Ulrich, C. M. Polymorphic variation in hOGG1 and risk of cancer: a review of the functional and epidemiologic literature. Mol. Carcinog. 42 (3), 127-141 (2005).
  5. Culp, S. J., Cho, B. P., Kadlubar, F. F., Evans, F. E. Structural and Conformational Analyses of 8-hydroxy-2′-deoxyguanosine. Chem. Res. Toxicol. 2 (6), 416-422 (1989).
  6. Cooke, M. S., Loft, S., Olinski, R., Evans, M. D., Bialkowski, K., Wagner, J. R., Dedon, P. C., Møller, P., Greenberg, M. M., Cadet, J. Recommendations for standardized description of and nomenclature concerning oxidatively damaged nucleobases in DNA. Chem. Res. Toxicol. 23 (4), 705-707 (2010).
  7. Jang, Y. H., Goddard, W. A. 3. r. d., Noyes, K. T., Sowers, L. C., Hwang, S., Chung, D. S. First principles calculations of the tautomers and pKa values of 8-oxoguanine: implications for mutagenicity and repair. Chem. Res. Toxicol. 15 (8), 1023-1035 (2002).
  8. Park, J. -. H., Gopishetty, S., Szewczuk, L. M., Troxel, A. B., Harvey, R. G., Penning, T. M. Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) by PAH o-quinones: involvement of reactive oxygen species and copper(ii)/copper(i) redox cycling. Chem. Res. Toxicol. 18 (6), 1026-1037 (2005).
  9. Mangal, D., Vudathala, D., Park, J. H., Lee, S. H., Penning, T. M., Blair, I. A. Analysis of 7,8-dihydro-8-oxo-2′-deoxyguanosine in cellular DNA during oxidative stress. Chem. Res. Toxicol. 22 (5), 788-797 (2009).
  10. Ravanat, J. L., Douki, T., Duez, P., Gremaud, E., Herbert, K., Hofer, T., Lasserre, L., Saint-Pierre, C., Favier, A. Cellular background level of 8-oxo-7,8-dihydro-2′-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up. Carcinogenesis. 23 (11), 1911-1918 (2002).
  11. Gossen, J. A., De Leeuw, W. J. F., Tan, C. H. T., Zwarthoff, E. C., Berends, F., Lohman, P. H. M., Knook, D. L., Vijg, J. Efficient rescue of integrated shuttle vectors from transgenic mice: A model for studying mutations in vivo. Proc. Natl. Acad. Sci. U.S.A. 86 (20), 7971-7975 (1989).
  12. Van Campen, L. E., Murphy, W. J., Franks, J. R., Mathias, P. I., Toraason, M. A. Oxidative DNA damage is associated with intense noise exposure in the rat. Hear Res. 164 (1-2), 164-161 (2002).
  13. European Standards Committee on Oxidative DNA Damage (ESCODD). Measurement of DNA oxidation in human cells by chromatographic and enzymic methods. Free Radic. Biol. Med. 34 (8), 1089-1099 (2003).
  14. Rebelo, I. A., Piedade, J. A., Oliveira-Brett, A. M. Development of an HPLC method with electrochemical detection of femtomoles of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine in the presence of uric acid. Talanta. 63 (2), 323-331 (2004).
  15. Ravanat, J. -. L., Turesky, R. J., Gremaud, E., Trudel, L. J., Stadler, R. H. Determination of 8-oxoguanine in DNA by gas chromatography-mass spectrometry and HPLC-electrochemical detection: overestimation of the background level of the oxidized base by the gas chromatography-mass spectrometry assay. Chem. Res. Toxicol. 8 (8), 1039-1045 (1995).
  16. Kawanishi, S., Murata, M. Mechanism of DNA damage induced by bromate differs from general types of oxidative stress. Toxicology. 221 (2-3), 172-178 (2006).
  17. Tahara, S., Kaneko, T. Susceptibility of mouse splenic cells to oxidative DNA damage by x-ray irradiation. Biol. Pharm. Bull. 27 (1), 105-108 (2004).
  18. Garratt, L. W., Mistry, V., Singh, R., Sandhu, J. K., Sheil, B., Cooke, M. S., Sly, P. D. Interpretation of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine is adversely affected by methodological inaccuracies when using a commercial ELISA. Free Radic. Biol. Med. 48 (11), 1460-1464 (2012).
  19. Cooke, M. S., Collins, A., Olinski, R., Rozalski, R., Loft, S. Harmonising measurements of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and urine. Free Radic. Res. 46 (4), 541-553 (2012).
  20. Cadet, J., Douki, T., Ravanat, J. L. Measurement of oxidatively generated base damage in cellular DNA. Mutat Res. 711 (1-2), 3-12 (2011).
  21. Chomczynski, P., Mackey, K., Drews, R. DNAzol: a reagent for the rapid isolation of genomic DNA. Biotechniques. 22 (3), 550-553 (1997).
  22. Collins, A. R., Cadet, J., Möller, L., Poulsen, H. E., Viña, J. Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells. Arch Biochem Biophys. 423 (1), 57-65 (2004).
  23. Badouard, C., Ménézo, Y., Panteix, G., Ravanat, J. L., Douki, T., Cadet, J. Determination of new types of DNA lesions in human sperm. Zygote. 16 (1), 9-13 (2008).
  24. Cadet, J., Douki, T., Gasparutto, D., Ravanat, J. L. Oxidative damage to DNA: formation, measurement and biochemical features. Mutat Res. 531 (1-2), 1-2 (2003).
  25. . . Validation of analytical procedures: text and methodology Q2(R1). , (2015).

Play Video

Cite This Article
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).

View Video