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

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 (PM_2.5) 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 PM_2.5 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 biotransformation, or induce oxidative stress^1,2. 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 activity^2,4,5,6,7, 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 differentiation^8,9. 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'-deoxyguanosine (8-oxodGuo, Figure 1), a lesion that is able to induce GT transversions in mammalian cells^10,11. 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 ^•OH, which initiate the process of lipid peroxidation^1,12. 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 adducts^1,12,13. The etheno adducts 1,N²-etheno-2'-deoxyguanosine (1,N²-εdGuo, Figure 1) and 1,N⁶-etheno-2'-deoxyadenosine (1,N⁶-εdAdo, Figure 1) have been suggested as potential biomarkers in the pathophysiology of inflammation^14,15.

Figure 1. Chemical structures of the DNA lesions quantified in the present study. dR = 2´-deoxyribose. This figure has been modified from Oliveira et al.³⁴. Please click here to view a larger version of this figure.

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 DNA^1,16. 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 ("comet assay"). 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 used^1,16,18.

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 bases^1,12. 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 µg) 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 disadvantages^19,20.

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 saliva^1,19,20. 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 mode^1,19. 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 present^1,12,19,20.

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 samples^{12,15,20,21,22,23,24,25,26,27,28,29}. Fine particles (PM_2.5) 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 disease^30,31,32,33. 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 PM_2.5 exposure³⁴.

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 n^o 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) containing 2.63 mg of ketamine and 0.38 mg of xylazine, intraperitoneally.
2. Collect blood (0.5 - 1.5 mL) for complementary analyses (e.g., antioxidant enzyme activity, malondialdehyde levels).
3. Shave the abdominal hair from the pelvis to the xiphoid process. Make an incision in a vertical middle line in the hairless area. Make incisions in horizontal lateral lines in order to expose the abdominal organs.
4. Cut the abdominal aorta to promote exsanguination and to euthanize the animal.
5. Remove the tissues of interest (in this case, liver, kidneys and lungs).
   1. To remove the liver, cut the inferior cava vein and portal hepatic vein.
   2. To remove the kidneys, section the renal veins and arteries.
   3. To remove the lungs, make an incision in the diaphragm extremities and circumference close to the thoracic wall. Break the clavicles by opening a scissor in the interior of the thoracic cavity. Cut the extern bone from the xiphoid process toward the trachea, in order to expose the lungs and heart.
      1. Hold the lung with a forceps, section the trachea and the ligaments around the lungs. Remove carefully the block lungs plus heart. To remove the lungs out of the block, hold the heart with a forceps and cut all vessels in its base.
6. Wash the isolated tissues immediately in cold saline solution (0.9% NaCl), transfer to cryogenic tubes, and immediately dip the tubes into liquid nitrogen. After completing the work, store the tubes at -80 °C.
   ​CAUTION: Liquid nitrogen in direct contact with the skin, mucosa or eyes causes burns. Use proper individual protection to avoid contact. Work in a ventilated laboratory to avoid asphyxia due to liquid nitrogen vapor.

2. DNA extraction

Note: The DNA extraction method was modified from Loureiro et al. (2009)³⁵ to allow the analyses of the lesions studied here.

1. Transfer the tubes containing the tissues to dry ice.
2. Use a culture plate placed on ice as a base to cut a piece of tissue with a scalpel. Weight 1 g for immediate use. The remaining tissue should be kept on dry ice until it returns to storage at -80 °C.
   NOTE: It is important to avoid thawing of the remaining tissue to prevent the formation of artifacts if repetitions of the analyses are needed.
3. To each 1 g of tissue in 50 mL capped tubes, add 10 mL of the commercial cell lysis solution containing 0.5 mM deferoxamine and keep on ice.
   NOTE: Add deferoxamine to the volume of solution for immediate use. For each 100 mL of solution, add 0.0328 g of the deferoxamine mesylate salt.
4. Homogenize the tissues using a tissue homogenizer until a homogeneous solution without tissue fragments is obtained. Keep the tube cold (on ice) during homogenization. Use a low speed to avoid heating.
5. Add 150 µL of proteinase K solution (20 mg/mL) to each homogenized sample. Shake the tubes by inversion and keep them at room temperature overnight.
6. Add 40 µL of ribonuclease A solution (15 mg/mL), shake by inversion, and keep the tubes at room temperature for 2 h.
   NOTE: Prepare ribonuclease A solution in sodium acetate buffer 10 mM, pH 5.2 to avoid precipitation. Heat the solution at 100 °C for 15 min before use to obtain a solution free from deoxyribonuclease.
7. Add 5 mL of the commercial protein precipitation solution, vortex vigorously, and centrifuge at 2,000 x g, 4 °C, for 10 minutes.
8. Transfer the supernatants to 50 mL capped tubes containing 10 mL of cold isopropanol. Invert the tubes gently several times until the observation of the precipitated DNA.
   NOTE: The protocol can be paused here, keeping the tubes at -20 °C.
9. Collect the precipitated DNA using a Pasteur pipette closed at the end. Transfer it to tubes containing 4 mL of 10 mM Tris buffer, 1 mM deferoxamine, pH 7.0.
10. After the DNA is completely dissolved in the above solution (do not vortex), add 4 mL of a chloroform solution containing 4% of isoamyl alcohol.
11. Invert the tubes 10 times for homogenization, centrifuge at 2,000 x g, 4 °C, for 10 minutes to separate the two phases, and transfer the upper phase to a new tube.
12. Repeat the steps 2.10 and 2.11 two more times.
13. Add 8 mL of absolute ethanol and 0.4 mL of a 5 M NaCl solution to precipitate the DNA.
14. Collect again the precipitated DNA and transfer it to 3 mL of 70% ethanol. Repeat this step one more time.
15. Discard the ethanol solution with caution and invert the tubes containing the precipitated DNA on absorbent paper to remove the excess of the solution.
16. Add 200 µL of 0.1 mM deferoxamine solution to dissolve the DNA. Maintain the tubes at 4 °C until the DNA is completely rehydrated (overnight).
17. Determine the DNA concentration by measuring the absorbance at 260 nm and its purity by the 260/280 nm absorbance ratio.
    NOTE: To determine the DNA concentration, transfer an aliquot of 10 µL of the DNA solution to 990 µL of ultrapure water (100x dilution). Multiply the absorbance at 260 nm (it should be below 1) by 50 (50 µg/mL is the concentration of double stranded DNA when the absorbance of a 1 cm path length solution at 260 nm is 1) and by the dilution used (100x) to obtain the DNA concentration in µg/mL. If the absorbance at 260 nm is above 1, additional dilutions are necessary. The 260/280 nm absorbance ratio should be equal or above 1.8 for the desired DNA purity, but ratios around 1.6 are acceptable.

3. DNA enzymatic hydrolysis

1. Analysis recipe
   1. 1,N⁶-εdAdo and 1,N²-εdGuo analyses: To an aliquot containing 150 µg of DNA, add 7.5 µL of 200 mM Tris/MgCl₂ buffer (pH 7.4), 1.4 µL of the internal standard solution containing 250 fmol/µL of [¹⁵N₅]1,N⁶-εdAdo and [¹⁵N₅]1,N²-εdGuo, and 15 units of deoxyribonuclease I. Adjust the final volume to 200 µL with ultrapure water, subtracting the volumes of enzymes to be used on step 3.2.1.
   2. 8-oxodGuo analyses: To an aliquot containing 80 µg of DNA, add 3.8 µL of 200 mM Tris/MgCl₂ buffer (pH 7.4), 2 µL of the internal standard solution containing 1,000 fmol/µL of [¹⁵N₅]8-oxodGuo, and 8 units of deoxyribonuclease I. Adjust the final volume to 100 µL with ultrapure water, subtracting the volumes of enzymes to be used on step 3.2.2.
      ​NOTE: The internal standards [¹⁵N₅]1,N⁶-εdAdo, [¹⁵N₅]1,N²-εdGuo and [¹⁵N₅]8-oxodGuo can be synthetized and characterized as described^35,36,37. The quantities of the internal standards in the injected sample volumes should be the same as those in the injected calibration curve volumes.
2. Incubate the samples at 37 °C for 1 hour.
   1. Samples from step 3.1: Add 0.006 units of phosphodiesterase I from Crotalus atrox and 15 units of alkaline phosphatase from bovine intestinal mucosa.
   2. Samples from step 3.2: Add 0.0032 units of phosphodiesterase I from Crotalus atrox and 8 units of alkaline phosphatase from bovine intestinal mucosa.
3. Incubate the samples at 37 °C for 1 hour.
4. Centrifuge the samples at 14,000 x g for 10 minutes.
5. Samples from step 3.2.1: Separate 10 µL of each sample for quantification of the deoxynucleosides (dAdo, dGuo) by HPLC/DAD (step 9). Subject the residual volume to solid phase extraction (step 4).
6. Samples from step 3.2.2: Transfer 80 µL of the supernatant to vials for injections of 50 µL (1,000 fmol of [¹⁵N₅]8-oxodGuo) in the HPLC-ESI-MS/MS system. Reserve the remaining 20 µL for quantification of dGuo by HPLC/DAD (step 9).

4. Solid phase extraction for analyses of 1, N⁶-εdAdo and 1, N²-εdGuo

1. Load the cartridges (SPE-C18, 30 mg/mL, 33 µm, 1 mL) with 1 mL of the following sequence of solutions: 100% methanol, deionized water, hydrolyzed DNA sample, deionized water, 10% methanol, 15% methanol, and 100% methanol (to be collected).
   NOTE: Do not leave the cartridges dry between the applications of the different solutions. Add the next solution immediately after the previous solution enters the cartridge completely.
2. Vacuum dry the last elution fraction (100% methanol) containing the adducts.
3. Resuspend the dried samples in 83.1 μL of ultrapure water immediately prior to the HPLC-ESI-MS/MS analysis, to obtain 200 fmol of each internal standard in 50 µL of each sample.

5. Preparation of calibration curves

1. Prepare at least five points in the interval of 300 to 6,000 fmol of 8-oxodGuo standard, with the fixed amount of 1,000 fmol of [¹⁵N₅]8-oxodGuo in each point. Consider these amounts in the volume injected.
2. Prepare at least five points in the interval of 1 to 40 fmol of 1,N⁶-εdAdo and 1,N²-εdGuo, with fixed amounts of 200 fmol of [¹⁵N₅]1,N⁶-εdAdo and [¹⁵N₅]1,N²-εdGuo in each point. Consider these amounts in the volume injected.
3. Prepare at least five points in the interval of 0.05 - 1 nmol of dGuo and dAdo. Consider these amounts in the volume injected.

6. Preparation of DNA samples for method validation

1. 1,N⁶-εdAdo and 1,N²-εdGuo analyses: Add varying amounts of 1,N⁶-εdAdo and 1,N²-εdGuo (e.g., 1.75, 8.75, 17.5, and 35 fmol) and fixed amounts of [¹⁵N₅]1,N⁶-εdAdo and [¹⁵N₅]1,N²-εdGuo (350 fmol) to 100 µg of calf thymus DNA and carry out the enzymatic hydrolysis as described in step 3. Process the samples in quadruplicate in two different days. Use the samples for method accuracy and precision assessment.
   NOTE: The final volume of the DNA hydrolysates will be 200 µL (step 3), from which 10 µL will be separated for quantification of deoxynucleosides by HPLC/DAD (step 9). The remaining solution (190 µL) will be subjected to solid phase extraction (step 4), the dried fraction will be resuspended in 83.1 µL (step 4.3), from which 50 µL will be injected in the HPLC-ESI-MS/MS system. The amounts of 1,N⁶-εdAdo and 1,N²-εdGuo injected will be 1, 5, 10, and 20 fmol, with 200 fmol of [¹⁵N₅]1,N⁶-εdAdo and [¹⁵N₅]1,N²-εdGuo in each sample.
2. 8-oxodGuo analyses: Add varying amounts of 8-oxodGuo (e.g., 734, 1,468, 2,938, and 4,408 fmol) and a fixed amount of [¹⁵N₅]8-oxodGuo (2,000 fmol) to 100 µg of calf thymus DNA and carry out the enzymatic hydrolysis as described in step 3. Process the samples in quadruplicate in two different days. Use the samples for method accuracy and precision assessment.
   NOTE: The final volume of the DNA hydrolysates will be 100 µL (step 3), from which 50 µL will be injected in the HPLC-ESI-MS/MS system. The amounts of 8-oxodGuo injected will be 367, 734, 1469, and 2204 fmol, with 1,000 fmol of [¹⁵N₅]8-oxodGuo in each sample.
3. Add 13.125 fmol of 1,N⁶-εdAdo (to obtain 7.5 fmol in the injection volume) and 35 fmol of 1,N²-εdGuo (to obtain 20 fmol in the injection volume) to eight samples of 100 µg of calf thymus DNA.
   1. Add the internal standards [¹⁵N₅]1,N⁶-εdAdo and [¹⁵N₅]1,N²-εdGuo (200 fmol) to four of the samples. Proceed with the DNA hydrolysis and solid phase extraction of all samples.
   2. Add the internal standards [¹⁵N₅]1,N⁶-εdAdo and [¹⁵N₅]1,N²-εdGuo (200 fmol) to the other four samples.
   3. Use the samples to calculate the recovery of the adducts from solid phase extraction.

7. HPLC-ESI-MS/MS analysis of 8-oxodGuo

1. Infusing the 8-oxodGuo standard into the equipment, set the ESI-MS/MS parameters for the best detection of its fragmentation pattern by multiple reaction monitoring (MRM): m/z 284 [M+H]+ → m/z 168 [M - 2'-deoxyribose + H]⁺.
   1. Use the same parameters for detection of [¹⁵N₅]8-oxodGuo: m/z 289 [M+H]⁺m/z → 173 [M - 2'-deoxyribose + H]⁺.
      NOTE: Use an equipment equivalent or better than the equipment used in this work (see the Table of Materials). The ESI-MS/MS parameters were set as described in Table 1.
2. Filter (using 0.22 µm porous membranes) and degasify (using a sonicator) all the water based HPLC solvents.
3. Use the following chromatography conditions for the analyses, mounting the system as shown in Figure 2.
   NOTE: Column A is connected to the binary pump. Its eluent is directed to UV detection and waste in the first 16 min and from 32 to 46 min of the chromatography, as shown in Figure 2A. This is the column through which the sample is eluted immediately after injection. Column B is connected to the isocratic pump and the mass spectrometer. It receives the eluent of column A only in the 16 - 32 min interval, when the valve is switched to the position shown in Figure 2B. The valve switching allows the connection between the two columns, which are eluted by the binary pump gradient. The configuration shown in Figure 2B permits further peak separation and narrowing. Additionally, only the chromatographic fraction of interest reaches the mass spectrometer, improving sensitivity and selectivity.
   1. Elute a 50 x 2.0 mm i.d., 2.5 µm, C18 column (column A of Figure 2) coupled to a C18 security guard cartridge (4.0 x 3.0 mm i.d.) with a gradient of 0.1% formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B) at a flow rate of 150 µL/min and 25 °C.
      1. Use the following gradient program for the binary pump: from 0 to 25 min, 0 - 15% of solvent B; 25 to 28 min, 15 - 80% of solvent B; 28 to 31 min, 80% of solvent B; 31 to 33 min, 80 - 0 % of solvent B; 33 to 46 min, 0% of solvent B.
      2. Use the switching valve to direct the first 16 min of eluent to waste and the 16 - 32 min fraction to a second column (150 x 2.0 mm i.d., 3.0 µm, C18, column B of Figure 2) connected to the ESI source and conditioned by the isocratic pump with a solution of 15% methanol in water containing 0.1% formic acid (150 µL/min).
         NOTE: Before using the switching valve program of step 7.3.1.2, check if the 8-oxodGuo standard elutes from the first column after 16 min. It is important to close the valve at 32 min to use the gradient of the binary pump to elute 8-oxodGuo from the second column and get a sharp chromatographic peak. The lesion 8-oxodGuo elutes from the second column at approximately 36 min. Variations of the retention time of the analyte may occur depending on the column and equipment used. Adaptations of the HPLC solvent gradient program may be necessary.

Figure 2. System of two columns used for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) analyses. A) Configuration used in the first 16 min and from 32 to 46 min of the chromatography; B) Configuration used in the interval 16 - 32 min, allowing further separation and peak narrowing in column B prior to elution to the ESI source of the mass spectrometer. This figure has been republished from Oliveira et al.³⁴. Please click here to view a larger version of this figure.

8. HPLC-ESI-MS/MS analysis of 1, N ⁶ -εdAdo and 1, N ² -εdGuo

1. Infusing the 1,N⁶-εdAdo and 1,N²-εdGuo standards into the equipment, set the ESI-MS/MS parameters for the best detection of their fragmentation patterns by multiple reaction monitoring (MRM): m/z 276 [M+H]⁺ → m/z 160 [M 2'-deoxyribose + H]⁺ for detection of 1,N⁶-εdAdo and m/z 292 [M+H]⁺ → m/z 176 [M - 2'-deoxyribose + H]⁺ for detection of 1,N²-εdGuo.
   1. Use the same parameters for detection of [¹⁵N₅]1,N⁶-εdAdo (m/z 281 [M+H]⁺ m/z → 165 [M - 2'-deoxyribose + H]⁺) and [¹⁵N₅]1,N²-εdGuo (m/z 297 [M+H]⁺ → m/z 181 [M - 2'-deoxyribose + H]⁺). Set the ESI-MS/MS parameters as described in Table 1.

ESI-MS/MS parameters	8-oxodGuo	Etheno adducts
Curtain Gas	20 psi	20 psi
Nebulizing Gas	55	50
Ion Source Gas	50 psi	40 psi
Collision-induced Dissociation Gas	Medium	Medium
Ion Spray Voltage	5000	4500
ESI Probe Temperature	450	450
Declustering Potential	31 V, 8-oxodGuo	41 V, 1,N6-εdAdo
	31 V, [15N5]8-oxodGuo	41 V, [15N5]1,N6-εdAdo
		45 V, 1,N2-εdGuo
		45V, [15N5]1,N2-εdGuo
Collision Energy	23 eV, 8-oxodGuo	25 eV, 1,N6-εdAdo
	23 eV, [15N5]8-oxodGuo	25 eV, [15N5]1,N6-εdAdo
		27 eV, 1,N2-εdGuo
		27 eV, [15N5]1,N2-εdGuo
Collision Cell Exit Potential	16 V, 8-oxodGuo,	8 V, 1,N6-εdAdo
	16 V, [15N5]8-oxodGuo	8 V, [15N5]1,N6-εdAdo
		16 V, 1,N2-εdGuo
		16 V, [15N5]1,N2-εdGuo
Entrance Potential	10 V	10 V

Table 1. Parameters used in the ESI-MS/MS equipment for detection of the DNA lesions. This table has been modified from Oliveira et al.³⁴.

2. Filter (using 0.22 µm porous membranes) and degasify (using a sonicator) all the water based HPLC solvents.
3. Use the following chromatography conditions for the analyses.
   1. Elute a 150 x 2.0 mm i.d., 3.0 µm, C18 column coupled to a C18 security guard cartridge (4.0 x 3.0 mm i.d.) with a gradient of 5 mM ammonium acetate, pH 6.6 (solvent A) and acetonitrile (solvent B) at a flow rate of 130 µL/min and 25 °C.
      1. Use the following gradient program for the binary pump: from 0 to 10 min, 0% of solvent B; 10 to 39 min, 0 - 20% of solvent B; 39 to 41 min, 20 - 75% of solvent B; 41 to 46 min, 75% of solvent B; 46 to 47 min, 75 - 0% of solvent B; 47 to 60 min, 0% of solvent B.
      2. Use the switching valve to direct the first 35 min of eluent to waste and the 35 - 42 min fraction to the ESI source. Be sure that the adduct standards elute from the column in the set interval (35 - 42 min). Make adjustments if necessary.

9. Quantification of normal 2'-deoxyribonucleosides by HPLC-UV

1. Use an equipment similar to the equipment used in this work (see the Table of Materials).
2. Elute a 250 mm x 4.6 mm i.d., 5 µm, C18 column attached to a C18 security guard cartridge (4.0 x 3.0 mm i.d.) with a gradient of 0.1% formic acid and methanol.
   1. Use the following gradient program: from 0 to 25 min, 0 to 18% methanol; from 25 to 27 min, 18 to 0% methanol; from 27 to 37 min, 0% methanol) at a flow rate of 1 mL/min and 30 °C.
   2. Inject 5 µL of each sample reserved for 2'-deoxynucleosides quantification.
   3. Set the DAD detector at 260 nm for integration of the dGuo and dAdo peaks.

10. Quantification of the DNA lesions

1. Integrate the peaks of 8-oxodGuo, [¹⁵N₅]8-oxodGuo, 1,N⁶-εdAdo, [¹⁵N₅]1,N⁶-εdAdo, 1,N²-εdGuo, and [¹⁵N₅]1,N²-εdGuo from the HPLC-ESI-MS/MS analyses.
   1. Calculate the area ratios of 8-oxodGuo/[¹⁵N₅]8-oxodGuo, 1,N⁶-εdAdo/[¹⁵N₅]1,N⁶-εdAdo, and 1,N²-εdGuo/[¹⁵N₅]1,N²-εdGuo for the calibration curves and the samples.
   2. Plot the calibration curves using the area ratios obtained in step 10.1.1 in the y axis and the amounts of analytes present in each point in the x axis.
   3. Calculate the amounts (fmol) of lesions in each injected sample using the ratios calculated in step 10.1.1 and the calibration curves of step 10.1.2.
2. Integrate the peaks of dGuo and dAdo from the HPLC-UV analyses.
   1. Plot the calibration curves using the areas obtained in step 10.2 in the y axis and the amounts of analytes present in each point in the x axis.
   2. Calculate the amounts (nmol) of dGuo and dAdo in each injected sample using the areas obtained in step 10.2 and the calibration curves of step 10.2.1.
3. Calculate the amounts (nmol) of dGuo and dAdo present in each sample injected in the HPLC-ESI-MS/MS system, considering that the amounts calculated in step 10.2.2 are present in the sample volume of 5 µL, while 50 µL were injected in the HPLC-ESI-MS/MS system.
   NOTE: To calculate the amount of dGuo in the samples used for 8-oxodGuo analysis, just multiply the amount (nmol/µL) obtained in step 10.2.2 by 50. To calculate the amounts of dAdo and dGuo in the samples used for analyses of 1,N⁶-εdAdo and 1,N²-εdGuo, consider the concentration step after solid phase extraction. The volume of 50 µL injected in the HPLC-ESI-MS/MS system corresponds to 114.32 µL of the original sample. The amounts (nmol/µL) obtained in step 10.2.2 should be multiplied by 114.32 to obtain the correct values.
4. Calculate the molar fractions 8-oxodGuo/dGuo, 1,N⁶-εdAdo/dAdo, 1,N²-εdGuo/dGuo. The ratios (fmol lesion/nmol normal deoxynucleoside) give the number of lesions per 10⁶ normal dGuo or dAdo.

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 before the corresponding 2'-deoxy...

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 hydrolysates^22,38. 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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
[15N5]-2’-deoxyadenosine	Cambridge Isotope Laboratories	NLM-3895-25
[15N5]-2’-deoxyguanosine	Cambridge Isotope Laboratories	NLM-3899-CA-10
acetonitrile	Carlo Erba Reagents	412413000
alkaline phosphatase from bovine intestinal mucosa	Sigma	P5521
ammonium acetate	Merck	101116
calf thymus DNA	Sigma	D1501
cell lysis solution	QIAGEN	158908
chloroform	Carlo Erba Reagents	412653
deferoxamine	Sigma	D9533
deoxyribonuclease I (DNase I)	Bio Basic Inc	DD0649
ethanol	Carlo Erba Reagents	414542
formic acid	Sigma-Aldrich	F0507
HPLC-ESI-MS/MS system	HPLC: Agilent 1200 series            ESI-MS/MS: Applied Biosystems/MDS Sciex Instruments	HPLC: 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 system	Shimadzu	Two 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)	Phenomenex	00B-4446-B0
HPLC column (150 x 2.0 mm i.d., 3.0 µm, C18)	Phenomenex	00F-4251-B0
HPLC column (250 x 4.6 mm i.d., 5.0 µm, C18)	Phenomenex	00G-4252-E0
HPLC C18 security guard cartridge (4.0 x 3.0 mm i.d.)	Phenomenex	AJO-4287
isoamyl alcohol	Sigma-Aldrich	M32658
isopropyl alcohol (isopropanol)	Carlo Erba Reagents	A412790010
ketamine	Ceva	Commercial name: Dopalen
magnesium chloride	Carlo Erba Reagents	349377
magnesium chloride	Sigma	M2393
methanol	Carlo Erba Reagents	L022909K7
phosphodiesterase I from Crotalus atrox	Sigma	P4506
protein precipitation solution	QIAGEN	158912
proteinase K	Sigma-Aldrich	P2308
ribonuclease A	Sigma	R5000
sodium chloride	Sigma-Aldrich	S9625
SPE-C18 (Strata-X)	Phenomenex	8B-S100-TAK
tris(hydroxymethyl)-aminomethane	Carlo Erba Reagents	489983
xylazine	Syntec do Brasil	Commercial name: Xilazin