FAPESP (Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo, Proc. 2012/22190-3 and 2012/08616-8), CNPq (Proc. 454214/2014-6 and 429184/2016-6), CAPES, PRPUSP (Pr&#243;-Reitoria de Pesquisa da Universidade de S&#227;o 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 (Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior). M. H. G. Medeiros, P. Di Mascio, P. H. N. Saldiva, and A. P. M. Loureiro received fellowships from CNPq.
Some figures and tables present in this work were originally published in Oliveira A.A.F. et al. Genotoxic and epigenotoxic effects in mice exposed to concentrated ambient fine particulate matter (PM2.5) from S&#227;o Paulo city, Brazil. Particle and Fibre Toxicology.&#160;15, 40 (2018).

Four week old male A/J mice, specific pathogen free, were obtained from the Breeding Center of Laboratory Animals of Funda&#231;&#227;o Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil, and were treated accordingly to the Ethics Committee of the Faculty of Medicine, University of S&#227;o Paulo (protocol no 1310/09).
1. Collection of mice tissues
<ol>
	<li>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...

A major problem found&#160;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 ...

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,&#160;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 stress1,2. Although the modified DNA bases can be removed from DNA by base or nucleotide excision repair (BER or NER), the induction of an imbalance between the generation and removal of DNA lesions in favor of the former leads to a net increase of their levels in DNA overtime3. Outcomes are the increase of DNA mutation rates, reduced gene expression, and diminished protein activity2,4,5,6,7, effects that are closely related to the development of diseases. DNA mutations may affect diverse cellular functions, such as cell signaling, cell cycle, genome integrity, telomere stability, the epigenome, chromatin structure, RNA splicing, protein homeostasis, metabolism, apoptosis, and cell differentiation8,9. Strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases) pass through the knowledge of the mutation sources, among them, DNA lesions and their causes.
ROS generated endogenously in excess, due to pollutant exposure, persistent inflammation, disease pathophysiology (e.g., diabetes), etc., are important causes of biomolecule damage, including DNA and lipid damage1. As an example, the highly reactive hydroxyl radical (OH) formed from H2O2 reduction by transition metal ions (Fe2+, Cu+) oxidizes the DNA bases, DNA sugar moiety and polyunsaturated fatty acids at diffusion-controlled rates10. Among the 80 already characterized oxidized nucleobases3, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo, Figure 1), a lesion that is able to induce GT transversions in mammalian cells10,11. It is formed by the mono electronic oxidation of guanine, or by hydroxyl radical or singlet oxygen attack of guanine in DNA1. Polyunsaturated fatty acids are other important targets of highly reactive oxidants, such as &#8226;OH, which initiate the process of lipid peroxidation1,12. It gives rise to fatty acid hydroperoxides that may decompose to electrophilic aldehydes and epoxyaldehydes, such as malondialdehyde, 4-hydroxy-2-nonenal, 2,4-decadienal, 4,5-epoxy-(2E)-decenal, hexenal, acrolein, crotonaldehyde, which are able to form mutagenic exocyclic DNA adducts, such as malondialdehyde-, propano-, or etheno adducts1,12,13. The etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo,&#160;Figure 1) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo, Figure 1) have been suggested as potential biomarkers in the pathophysiology of inflammation14,15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59734/59734fig1.jpg" /> 
Figure 1.&#160;Chemical structures of the DNA lesions quantified in the present study. dR = 2&#180;-deoxyribose. This figure has been modified from Oliveira et al.34. <a href="https://www.jove.com/files/ftp_upload/59734/59734fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Studies carried out in the early 1980s allowed the sensitive detection of 8-oxodGuo by high performance liquid chromatography coupled to electrochemical detection (HPLC-ECD). Quantification of 8-oxodGuo by HPLC-ECD in several biological systems subjected to oxidizing conditions led to the recognition of 8-oxodGuo as a biomarker of oxidatively induced base damage in DNA1,16. Although robust and allowing the quantification of 8-oxodGuo in the low fmol range17, HPLC-ECD measurements rely on the accuracy of the analyte retention time for analyte identification and on the chromatography resolution to avoid interferences of other sample constituents. As the electrochemical detection requires the use of salt (e.g., potassium phosphate, sodium acetate) in the mobile phase, the maintenance of adequate analytical conditions needs routine column and equipment cleaning time.
Alternatively, the use of the bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) and, afterwards, human 8-oxoguanine glycosylase 1 (hOGG1), for detection and removal of 8-oxoGua from DNA, emerged as a way for the induction of DNA alkali labile sites. The alkali labile sites are converted to DNA strand breaks and allow the very high sensitive indirect quantification of 8-oxoGua by alkaline single cell gel electrophoresis (&#34;comet assay&#34;). The high sensitivity and the accomplishment of the analyses without the need of cellular DNA extraction are the main advantages of this type of assay. It gives the lowest steady-state levels of 8-oxoGua in DNA, typically 7-10 times lower than the levels obtained by bioanalytical methods based on HPLC. However, it is an indirect measurement of 8-oxoGua and some drawbacks are the lack of specificity or the unknown efficiency of the repair enzymes used1,16,18.
Immunoassays are other set of methods used for the detection of 8-oxoGua1 and exocyclic DNA adducts, such as 1,N6-dAdo and 1,N2-dGuo12. Despite the sensitivity, a shortcoming of the use of antibodies for detection of DNA lesions is the lack of specificity due to cross-reactivity to other components of biological samples, including the normal DNA bases1,12. The exocyclic DNA adducts, including 1,N6-dAdo and 1,N2-dGuo, may also be detected and quantified by highly sensitive 32P-postlabeling assays12. The high sensitivity of 32P-postlabeling allows the use of very small amounts of DNA (e.g., 10 &#181;g) for detection of about 1 adduct per 1010 normal bases19. However, the use of radio-chemicals, lack of chemical specificity and low accuracy are some disadvantages19,20.
A shared limitation of the methods cited above is the low selectivity or specificity for the detection of the desired molecules. In this scenario, HPLC coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS and HPLC-MS3) evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva1,19,20. Advantages of HPLC-ESI-MS/MS methods are the sensitivity (typically in the low fmol range) and the high specificity provided by i) the chromatographic separation, ii) the characteristic and known pattern of molecule fragmentation inside the mass spectrometer collision chamber, and iii) the accurate measurement of the selected mass to charge ratio (m/z) in multiple reaction monitoring mode1,19. 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 present1,12,19,20.
Several methods based on HPLC-ESI-MS/MS have been used for quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in DNA extracted from different biological samples12,15,20,21,22,23,24,25,26,27,28,29. Fine particles (PM2.5) carry organic and inorganic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, aldehydes, ketones, carboxylic acids, quinolines, metals, and water-soluble ions, which may induce inflammation and oxidative stress, conditions that favor the occurrence of biomolecule damage and disease30,31,32,33. We present here validated 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 exposure34.

<table border="0" cellpadding="0" cellspacing="0" style="width:1260px;" width="1262">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">[15N5]-2&#8217;-deoxyadenosine</td>
			<td style="width:369px;">Cambridge Isotope Laboratories</td>
			<td style="width:472px;">NLM-3895-25</td>
			<td style="width:65px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">[15N5]-2&#8217;-deoxyguanosine</td>
			<td style="width:369px;">Cambridge Isotope Laboratories</td>
			<td style="width:472px;">NLM-3899-CA-10</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">acetonitrile</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">412413000</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">alkaline phosphatase from bovine intestinal mucosa</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">P5521</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">ammonium acetate</td>
			<td style="width:369px;">Merck</td>
			<td style="width:472px;">101116</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">calf thymus DNA</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">D1501</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">cell lysis solution</td>
			<td style="width:369px;">QIAGEN</td>
			<td style="width:472px;">158908</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">chloroform</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">412653</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">deferoxamine</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">D9533</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">deoxyribonuclease I (DNase I)</td>
			<td style="width:369px;">Bio Basic Inc</td>
			<td style="width:472px;">DD0649</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">ethanol</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">414542</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">formic acid</td>
			<td style="width:369px;">Sigma-Aldrich</td>
			<td style="width:472px;">F0507</td>
			<td></td>
		</tr>
		<tr height="242">
			<td height="242" style="height:242px;width:353px;">HPLC-ESI-MS/MS system</td>
			<td style="width:369px;">HPLC: Agilent 1200 series&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ESI-MS/MS: Applied Biosystems/MDS Sciex Instruments</td>
			<td style="width:472px;">HPLC: binary pump (G1312B), isocratic pump (G1310A), column oven with a column switching valve (G1316B), diode array detector (G1315C), auto sampler (G1367C).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ESI-MS/MS: Linear Quadrupole Ion Trap mass spectrometer, Model 4000 QTRAP.</td>
			<td></td>
		</tr>
		<tr height="138">
			<td height="138" style="height:138px;width:353px;">HPLC/DAD system</td>
			<td style="width:369px;">Shimadzu</td>
			<td style="width:472px;">Two pumps (LC-20AT), photo diode array detector (DAD-20AV), auto-injector (Proeminence SIL-20AC), column oven (CTO-10AS/VP)</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">HPLC column (50 x 2.0 mm i.d., 2.5 &#181;m, C18)</td>
			<td style="width:369px;">Phenomenex</td>
			<td>00B-4446-B0</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">HPLC column (150 x 2.0 mm i.d., 3.0 &#181;m, C18)</td>
			<td style="width:369px;">Phenomenex</td>
			<td style="width:472px;">00F-4251-B0</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">HPLC column (250 x 4.6 mm i.d., 5.0 &#181;m, C18)</td>
			<td style="width:369px;">Phenomenex</td>
			<td style="width:472px;">00G-4252-E0</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">HPLC C18 security guard cartridge (4.0 x 3.0 mm i.d.)</td>
			<td style="width:369px;">Phenomenex</td>
			<td style="width:472px;">AJO-4287</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">isoamyl alcohol</td>
			<td style="width:369px;">Sigma-Aldrich</td>
			<td style="width:472px;">M32658</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">isopropyl alcohol (isopropanol)</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">A412790010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">ketamine</td>
			<td style="width:369px;">Ceva</td>
			<td style="width:472px;">Commercial name: Dopalen</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">magnesium chloride</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">349377</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">magnesium chloride</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">M2393</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">methanol</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">L022909K7</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">phosphodiesterase I from Crotalus atrox</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">P4506</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">protein precipitation solution</td>
			<td style="width:369px;">QIAGEN</td>
			<td style="width:472px;">158912</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">proteinase K</td>
			<td style="width:369px;">Sigma-Aldrich</td>
			<td style="width:472px;">P2308</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">ribonuclease A</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">R5000</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">sodium chloride</td>
			<td style="width:369px;">Sigma-Aldrich</td>
			<td style="width:472px;">S9625</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">SPE-C18 (Strata-X)</td>
			<td style="width:369px;">Phenomenex</td>
			<td style="width:472px;">8B-S100-TAK</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">tris(hydroxymethyl)-aminomethane</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">489983</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">xylazine</td>
			<td style="width:369px;">Syntec do Brasil</td>
			<td style="width:472px;">Commercial name: Xilazin</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of three dna lesions by mass spectrometry and assessment of their levels in tissues of mice exposed to ambient fine particulate matter

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&#39;-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&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), 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.

The average DNA concentrations (&#177; SD) obtained from mice liver (~ 1 g tissue), lung (~ 0.2 g tissue) and kidney (~ 0.4 g tissue) were, respectively, 5,068 &#177; 2,615, 4,369 &#177; 1,021, and 3,223 &#177; 723 &#181;g/mL in the final volume of 200 &#181;L. A representative chromatogram obtained by HPLC-DAD of the purified DNA is shown in Figure 3. The presence of the four 2&#39;-deoxynucleosides, free from the RNA ribonucleosides, which elute immediately...

Watch this Scientific Journal Video about Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter at JoVE.

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-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.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

The methods presented here allow the quantification of DNA lesions induced by oxidative stress, contributing to the understanding of pathophysiological mechanisms that may be target for prevention and treatment of diseases. Selectivity and sensitivity are the main advantages. Selectivity is an essential advantage when working with complex biological samples.HPLC coupled to tandem mass spectrometry evolved as the gold standard for this type of quantification with biological matrices. Oxidative stress is a common event to different pathophysiological processes. Increased levels of etheno adducts have been detected in tissues or urine of patients with inflammatory diseases.The DNA lesions also provide information on endogenous events triggered by exposure to pollutants. If the DNA lesions are increased, the exposure may be at the level that increases the risk of cancer development, for example. The lesions can be quantified in DNA from any cellular system and alternatively in urine, plasma, saliva culture medium with adaptations in sample preparation.To begin, use a culture plate placed on ice as a base to cut a piece of tissue with a scalpel. Weigh one gram of the tissue in each 50-milliliter capped tube for immediate use. Keep the remaining tissue on dry ice before storing at minus 80 degrees Celsius.To each tube, add 10 milliliters of the commercial cell lysis solution containing 0.5-millimolar deferoxamine, and keep on ice. Set the tissue homogenizer to a low speed, around 60 to 90 rpm. Homogenize the tissues on ice for a few minutes until the solution is without tissue fragments.Then, add 150 microliters of proteinase K solution to each homogenized sample. Shake the tubes by inversion, and keep them at room temperature overnight. In the morning, add 40 microliters of ribonuclease A solution, shake by inversion, and keep the tubes at room temperature for two hours.Add five milliliters of commercial protein precipitation solution, vortex vigorously, and centrifuge at 2, 000 times g, four degrees Celsius, for 10 minutes. Transfer the supernatants to 50-milliliter capped tubes containing 10 milliliters of cold isopropanol. Invert the tubes gently several times until observation of precipitated DNA.Use a Pasteur pipette closed at the end to collect the precipitated DNA. Transfer the collected DNA to tubes containing four milliliters of 10-millimolar Tris buffer and one-millimolar deferoxamine at pH seven to dissolve. After the DNA is completely dissolved in the tubes, add four milliliters of a chloroform solution containing 4%isoamyl alcohol to each tube.Invert the tubes 10 times for homogenization, centrifuge at 2, 000 times g, four degrees Celsius, for 10 minutes, and transfer the upper phases to new tubes. Repeat the wash of the upper phase with the chloroform solution two more times. Then, add eight milliliters of absolute ethanol and 0.4 milliliters of a five-molar sodium chloride solution to precipitate the DNA.Again, collect the precipitated DNA, and transfer it to three milliliters of 70%ethanol. Repeat the wash with 70%ethanol one more time. Discard the ethanol solution with caution, and invert the tubes containing the precipitated DNA on absorbent paper to remove excess solution.After preparing DNA hydrolysis samples with 15N5 isotopic standard of 1, N6-etheno-deoxyadenosine and 15N5 isotopic standard of 1, N2-etheno-deoxyguanosine according to the manuscript, transfer 10 microliters of each sample into tubes for quantification of normal deoxynucleosides by HPLC-UV. Subject the residual volume to solid-phase extraction. To perform HPLC, first elute a C18 column attached to a C18 SecurityGuard cartridge with a gradient of 0.1%formic acid and methanol.Set up the gradient program to run from zero to 25 minutes with zero to 18%methanol, from 25 to 27 minutes with 18 to 0%methanol, then from 27 to 37 minutes with 0%methanol at a flow rate of one milliliter per minute and 30 degrees Celsius. After that, inject a volume between two and six microliters for each sample reserved for normal deoxynucleosides quantification. Set the DAD detector at 260 nanometers for integration of the deoxyguanosine and deoxyadenosine peaks.To perform solid-phase extraction for analyzes of 1, N6-etheno-deoxyadenosine and 1, N2-etheno-deoxyguanosine, first load the cartridges with a series of solutions at a volume of one milliliter. Add 100%methanol, deionized water, hydrolyzed DNA sample, deionized water, 10%methanol, 15%methanol, and lastly 100%methanol for collection. Then, proceed according to the manuscript.After preparing DNA hydrolysis samples 15N5 isotopic standard of 8-oxo-deoxyguanosine, transfer 80 microliters of each sample into vials for analysis in the HPLC-ESI-MS/MS system. Reserve the remaining 20 microliters for quantification of deoxyguanosine on HPLC-UV, as done previously. For analysis of 8-oxo-deoxyguanosine on HPLC-ESI-MS/MS, elute the C18 column A coupled to a C18 SecurityGuard cartridge with a gradient of solvent A and B at a flow rate of 150 microliters per minute and 25 degrees Celsius.Run the binary pump with zero to 15%of solvent B during the first 25 minutes, 15 to 80%of solvent B from 25 to 28 minutes, 80%of solvent B from 28 to 31 minutes, 80 to 0%of solvent B from 31 to 33 minutes, and 0%of solvent B from 33 to 46 minutes. Direct the first 16 minutes of column A eluent to waste. Condition column B by the isocratic pump with a solution of 15%methanol in water containing 0.1%formic acid at a flow rate of 150 microliters per minute.After six minutes, check the chromatogram to ensure the 8-oxo-deoxyguanosine standard elutes from column A.During the 16 to 32 minutes interval, switch the valve to the position allowing connection between column A and column B.Close the valve at 32 minutes to elute 8-oxo-deoxyguanosine from the second column and get a sharp chromatographic peak. For analysis of 1, N6-etheno-deoxyadenosine and 1, N2-etheno-deoxyguanosine, elute a C18 column coupled to a C18 SecurityGuard cartridge with a gradient of solvent A and B at a flow rate of 130 microliters per minute and 25 degrees Celsius. Run the binary pump with 0%of solvent B for the first 10 minutes, zero to 20%of solvent B for 10 to 39 minutes, 20 to 75%of solvent B from 39 to 41 minutes, 75%of solvent B from 41 to 46 minutes, 75 to 0%of solvent B from 46 to 47 minutes, and 0%of solvent B from 47 to 60 minutes.Use the switching valve to direct the first 35 minutes of eluent to waste and the 35 to 42 minutes fraction to the ESI source. Be sure that the adduct standards elute from the column in the set interval. Integrate the peaks of 8-oxo-deoxyguanosine 15N5 isotopic standard of 8-oxo-deoxyguanosine, 1, N6-etheno-deoxyadenosine, 15N5 isotopic standard of 1, N6-etheno-deoxyadenosine, 1, N2-etheno-deoxyguanosine, and 15N5 isotopic standard of 1, N2-etheno-deoxyguanosine from the HPLC-ESI-MS/MS analyses.Calculate the area ratios for the calibration curves and the samples. Establish the calibration curves, and calculate the amounts of lesions in each injected sample. Integrate the peaks of deoxyguanosine and deoxyadenosine from the HPLC-UV analyses.Use the areas to establish the calibration curves, and calculate the amounts of deoxyguanosine and deoxyadenosine in animals in each injected sample. Calculate the molar fractions for 8-oxo-deoxyguanosine over deoxyguanosine, 1, N6-etheno-deoxyadenosine over deoxyadenosine, and 1, N2-etheno-deoxyguanosine over deoxyguanosine, which give the number of lesions per one million normal deoxyguanosine or deoxyadenosine. A representative chromatogram of the purified DNA obtained by HPLC-UV shows the presence of the four normal deoxynucleosides, free from the RNA ribonucleosides, demonstrating the DNA purity.Representative chromatograms from HPLC-ESI-MS/MS of 8-oxo-deoxyguanosine, 1, N6-etheno-deoxyadenosine, and 1, N2-etheno-deoxyguanosine in mice tissue DNA samples are shown. The chromatogram obtained with UV detection shows the four normal deoxynucleosides eluting from the first column until about 10 minutes, with a good separation from 8-oxo-deoxyguanosine, eliminating undesired interferences. An important thing to remember for exact quantifications is to have always the same quantities of internal standards in the injection volume in the calibration curves and samples.The methods presented here may be adapted for the quantification of other modified deoxynucleosides. Expanding the band of modified deoxynucleosides allows a better understanding of the underlying pathophysiological mechanisms. We can investigate the role of oxidative damage in different situations such as exposure to xenobiotics, diabetes, and cell malignant transformation, therefore aiding the development of preventive and treatment strategies.

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Research

JoVE Journal

Immunology and Infection

8.5K vistas.  Universidade de São Paulo. Los métodos presentados aquí permiten la cuantificación de las lesiones del ADN inducidas por el estrés oxidativo, contribuyendo a la comprensión de los mecanismos fisiopatológicos que pueden ser objetivo para la prevención y el tratamiento de enfermedades. La selectividad y la sensibilidad son las principales ventajas. La selectividad es una ventaja esencial cuando se trabaja con muestras biológicas complejas.HPLC acoplado a espectrometría de masas en tándem evolucionó como...

Video: Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter