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.
