In general, 10 μg is the minimum amount of DNA required to measure a DNA adduct by LC/MS assay. The amount of DNA required depends on the adduct. The ionization efficiency of the adduct to electrospray ionization affects the sensitivity of the LC/MS assay. The ionization efficiencies of adducts can vary by 10-fold or more.1
The DNA processed from tissues, saliva, or blood should be performed by experienced technicians in the Hub. This helps maintain quality control and ensure reproducible results. Alternatively, the principal investigator’s laboratory must rigorously follow Lab Hub protocols when, for logistical reasons, the biofluids must be processed immediately before freezing.
DNA from biopsy specimens should be rinsed with chilled saline or Tris-EDTA buffer (pH 7.1 – 8.0) to remove blood, snap-frozen in liquid nitrogen, and stored at -80 °C.
To measure DNA adducts in white blood cells (WBCs), collect between 1 to 10 mL of blood, depending upon the DNA adducts being measured. Collect whole blood in tubes containing EDTA and centrifuge it at 2,500 rpm for 15 minutes. The thin layer of WBCs is between the upper plasma layer and the bottom red blood cell (RBC) layer. WBCs are a mixture of granulocytes, monocytes, and lymphocytes with variable lifespans. You may need to separate the different WBC subpopulations to get a reliable estimate of DNA adduct levels.2,3
The procedures used to isolate DNA depends on the types of adducts under investigation.
DNA from saliva is usually obtained by vigorously swishing the mouth with a saline solution and then collecting the oral rinse in a receptacle. Buccal cells are obtained using a soft-bristled cytobrush to brush the oral mucosa inside the cheeks from top to bottom approximately 30 times and then shake the brush in a saline solution (5 mL) in a 15 mL polypropylene tube.4
Typically, tissues are homogenized in a Tris-EDTA or HEPES-EDTA buffer (pH 7.0 or pH 8.0), followed by centrifugation at 3,000 g to retrieve nuclear pellets.
The cells in biofluids are also collected by centrifugation. The samples are digested with proteinase K and RNase. Then the DNA is isolated using phenol/chloroform extraction or silica-based ion exchange resins.
To measure lipid peroxidation DNA adducts you need scavengers, such as glutathione (GSH) or beta-mercaptoethanol (βME), in all buffers employed for DNA isolation. This will mitigate the artifactual formation of DNA adducts.4,5 Measurement of acetaldehyde adducts formed from exposure to tobacco or ingestion of ethanol requires NaCNBH3 in the buffer.6
Most classes of DNA adducts are measured in DNA that has undergone digestion with a cocktail of nucleases to yield the modified 2′-deoxynucleoside.1 Although, in some instances, thermal hydrolyses of DNA is preferred to recover some types of modified nucleobases.
DNA adducts formed from carcinogens in tobacco, cooked meat, environmental pollutants, and traditional herbal remedies can be measured in human biopsy specimens preserved with formalin.1
The formalin-fixed tissues must be processed in 10% neutral buffered formalin for 24 hours and then serial dehydrated with ethanol and paraffin-embedded in accredited histology/pathology laboratories. Prolonged fixation in formalin causes an inefficient reversal of DNA crosslinks and poor recovery of DNA.7
- The formalin-fixed paraffin-embedded (FFPE) tissue blocks can be stored at room temperature. The section blocks can be provided to the Hub for the processing of DNA. If the amount of FFPE provided are limiting, then prepare section cuts of 5 μm thickness and store them in Eppendorf tubes at -80 °C to minimize the oxidation of DNA.
- The recovery of DNA from human FFPE tissue section cuts of 1 cm2 x 10 μm thickness provides from 1 to 3 μg of DNA per section cut.7 Thus, a minimum of 10 section cuts are required for DNA adduct measurements in FFPE tissues.
Measuring and Reporting Quality
The quality of the isolated DNA is measured by UV spectroscopy.8 An absorbance ratio A260/A280 of 1.8 is considered high-quality DNA, with an A260 absorbance of 1.0 ODU estimated as 50 μg DNA/mL. The ratio A260/A230 is a secondary measure of nucleic acid purity. The A260/A230 values for “pure” nucleic acid are often higher than the A260/A280 values and commonly in the range of 2.0 – 2.2. A much lower ratio indicates that contaminants such as phenol, carbohydrates, or EDTA are in the DNA solution.
Following enzymatic digestion, the quality of DNA is further characterized by HPLC-UV at 260 nm to monitor the four 2′-deoxynucleosides.8,9 For DNA recovered from FFPE tissues, these analyses are particularly important for to ensure that the processing method fully reversed the DNA crosslinks.1,7 The presence of Guo or Urd reveals contamination with RNA.
The level of DNA adduct modification is usually reported as the adduct level per nucleotide, using the amount of DNA measured by UV spectroscopy, assuming 10 μg DNA is equal to 3.03 nmol nucleotide. (The average mw of nucleotide is 330 g). Alternatively, the relative amount of adduct can be reported to the level of non-modified dGuo or other targeted 2′-deoxynucleoside, as measured by HPLC-UV or LC/MS.
- Yun BH, Guo J, Bellamri M, et al. DNA adducts: Formation, biological effects, and new biospecimens for mass spectrometric measurements in humans. Mass Spectrometry Reviews. 2018;1–28.
- Godschalk RW, Van Schooten FJ, Bartsch H. A critical evaluation of DNA adducts as biological markers for human exposure to polycyclic aromatic compounds. Journal of Biochemical and Molecular Biology. 2003; 36:1-11.
- Wiencke JK, Kelsey KT, Varkonyi A, et al. Correlation of DNA adducts in blood mononuclear cells with tobacco carcinogen-induced damage in human lung. Cancer Research. 1995; 55:4910-4914.
- Paiano V, Maertens L, Guidolin V, et al. Quantitative Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry Analysis of Acrolein-DNA Adducts and Etheno-DNA Adducts in Oral Cells from Cigarette Smokers and Nonsmokers. Chemical Research in Toxicology. 2020; 33: 2197-2207.
- Chen H, Krishnamachari S, Guo J, et al. Quantitation of Lipid Peroxidation Product DNA Adducts in Human Prostate by Tandem Mass Spectrometry: A Method That Mitigates Artifacts. Chemical Research in Toxicology. 2019; 32:1850-1862.
- Balbo S, Meng L, Bliss RL, et al. Kinetics of DNA adduct formation in the oral cavity after drinking alcohol. Cancer Epidemiology, Biomarkers & Prevention. 2012; 21:601-608.
- Yun BH, Yao L, Jelakovic B, et al. Formalin-fixed paraffin-embedded tissue as a source for quantitation of carcinogen DNA adducts: aristolochic acid as a prototype carcinogen. Carcinogenesis. 2014; 35:2055-2061. (8) Laws GM, Adams SP. Measurement of 8-OHdG in DNA by HPLC/ECD: the importance of DNA purity. BioTechniques. 1996; 20:36-38.
- Gu D, Turesky RJ, Tao Y, et al. DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 4-aminobiphenyl are infrequently detected in human mammary tissue by liquid chromatography/tandem mass spectrometry. Carcinogenesis. 2012; 33:124-130.