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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2'-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2'-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

Abstract

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo) and 1,N6-etheno-2'-deoxyadenosine (1,N6-εdAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.

We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

Introduction

Some reactive oxygen species (ROS) are able to oxidize carbon double bonds of DNA bases and some carbons in the deoxyribose moiety, generating oxidized bases and DNA strand breaks1. As a negatively charged molecule rich in nitrogen and oxygen atoms, DNA is also a target for electrophilic groups that covalently react with the nucleophilic sites (nitrogen and oxygen), giving products that are called DNA adducts2. So, DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransfor....

Protocol

Four week old male A/J mice, specific pathogen free, were obtained from the Breeding Center of Laboratory Animals of Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil, and were treated accordingly to the Ethics Committee of the Faculty of Medicine, University of São Paulo (protocol no 1310/09).

1. Collection of mice tissues

  1. Anesthetize the animal with xylazine and ketamine. For a mouse with 30 g of body weight, inject a solution (no more than 2 mL) c.......

Representative Results

The average DNA concentrations (± SD) obtained from mice liver (~ 1 g tissue), lung (~ 0.2 g tissue) and kidney (~ 0.4 g tissue) were, respectively, 5,068 ± 2,615, 4,369 ± 1,021, and 3,223 ± 723 µg/mL in the final volume of 200 µL. A representative chromatogram obtained by HPLC-DAD of the purified DNA is shown in Figure 3. The presence of the four 2'-deoxynucleosides, free from the RNA ribonucleosides, which elute immediately.......

Discussion

A major problem found in the 8-oxodGuo analyses by HPLC methods is the possible induction of its formation during the workup procedures of DNA extraction, DNA hydrolysis, and concentration of DNA hydrolysates22,38. In order to minimize the problem of 8-oxodGuo artifactual formation, it is recommended the addition of deferoxamine to all DNA extraction, storage and hydrolysis solutions, the use of the sodium iodide chaotropic method and avoidance of phenol in .......

Acknowledgements

FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Proc. 2012/22190-3 and 2012/08616-8), CNPq (Proc. 454214/2014-6 and 429184/2016-6), CAPES, PRPUSP (Pró-Reitoria de Pesquisa da Universidade de São Paulo), INCT INAIRA (MCT/CNPq/FNDCT/CAPES/FAPEMIG/FAPERJ/FAPESP; Proc. 573813/2008-6), INCT Redoxoma (FAPESP/CNPq/CAPES; Proc. 573530/2008-4), NAP Redoxoma (PRPUSP; Proc. 2011.1.9352.1.8) and CEPID Redoxoma (FAPESP; Proc. 2013/07937-8). T. F. Oliveira and A. A. F. Oliveira received scholarships from FAPESP (Proc. 2012/21636-8, 2011/09891-0, 2012/08617-4) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Níve....

Materials

NameCompanyCatalog NumberComments
[15N5]-2’-deoxyadenosineCambridge Isotope LaboratoriesNLM-3895-25
[15N5]-2’-deoxyguanosineCambridge Isotope LaboratoriesNLM-3899-CA-10
acetonitrileCarlo Erba Reagents412413000
alkaline phosphatase from bovine intestinal mucosaSigmaP5521
ammonium acetateMerck101116
calf thymus DNASigmaD1501
cell lysis solutionQIAGEN158908
chloroformCarlo Erba Reagents412653
deferoxamineSigmaD9533
deoxyribonuclease I (DNase I)Bio Basic IncDD0649
ethanolCarlo Erba Reagents414542
formic acidSigma-AldrichF0507
HPLC-ESI-MS/MS systemHPLC: Agilent 1200 series            ESI-MS/MS: Applied Biosystems/MDS Sciex InstrumentsHPLC: binary pump (G1312B), isocratic pump (G1310A), column oven with a column switching valve (G1316B), diode array detector (G1315C), auto sampler (G1367C).                                                            ESI-MS/MS: Linear Quadrupole Ion Trap mass spectrometer, Model 4000 QTRAP.
HPLC/DAD systemShimadzuTwo pumps (LC-20AT), photo diode array detector (DAD-20AV), auto-injector (Proeminence SIL-20AC), column oven (CTO-10AS/VP)
HPLC column (50 x 2.0 mm i.d., 2.5 µm, C18)Phenomenex00B-4446-B0
HPLC column (150 x 2.0 mm i.d., 3.0 µm, C18)Phenomenex00F-4251-B0
HPLC column (250 x 4.6 mm i.d., 5.0 µm, C18)Phenomenex00G-4252-E0
HPLC C18 security guard cartridge (4.0 x 3.0 mm i.d.)PhenomenexAJO-4287
isoamyl alcoholSigma-AldrichM32658
isopropyl alcohol (isopropanol)Carlo Erba ReagentsA412790010
ketamineCevaCommercial name: Dopalen
magnesium chlorideCarlo Erba Reagents349377
magnesium chlorideSigmaM2393
methanolCarlo Erba ReagentsL022909K7
phosphodiesterase I from Crotalus atroxSigmaP4506
protein precipitation solutionQIAGEN158912
proteinase KSigma-AldrichP2308
ribonuclease ASigmaR5000
sodium chlorideSigma-AldrichS9625
SPE-C18 (Strata-X)Phenomenex8B-S100-TAK
tris(hydroxymethyl)-aminomethaneCarlo Erba Reagents489983
xylazineSyntec do BrasilCommercial name: Xilazin

References

  1. Cadet, J., Davies, K. J. A., Medeiros, M. H. G., Di Mascio, P., Wagner, J. R. Formation and repair of oxidatively generated damage in cellular DNA. Free Radical Biology and Medicine. 107, 13-34 (2017).
  2. Barnes, J. L., Zubair, M., John, K., Poirier, M. C., Martin, F. L.

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