Why is exposure to DNA-damaging agents relevant to health?
Humans are frequently exposed to low levels of hazardous chemicals in the environment and diet, as well as some occupational exposures, which damage the genome. DNA adducts are dosimeters for chemical exposure and a measure of the biologically effective dose. DNA adducts serve as important biomarkers to formulate public health policy for exposure reduction and cancer prevention (Himmelstein et al., 2009; Jarabek et al., 2009).
DNA damage also occurs through biological processes, such as oxidative stress and inflammation, which produce reactive electrophiles. Thousands of lesions accumulate in the genome of the mammalian cell over 24 hours (Nelson, 2008).
The results of DNA damage include covalent modifications of nucleobases and phosphate groups, intra- and inter-strand cross-links, DNA-protein cross-links, single-strand breaks (SSBs), and double-strand breaks (DSBs) (Yun et al., 2018). Certain classes of DNA adducts induce depurination or depyrimidination, resulting in mutagenic abasic sites.
Fortunately, DNA repair enzymes efficiently remove much of the damage; however, some of the changes are persistent and can escape repair and induce mutations during cell division, leading to cancer. Epidemiological studies have linked lifestyle habits and chemical exposures with increased cancer risk. For example, tobacco smoking is a risk factor for lung and bladder cancer, and frequent consumption of processed or cooked red meats is linked to an increased risk of colorectal cancer.
What types of questions can be answered?
DNA adducts are measures of exposure to hazardous chemicals. Characterizing DNA adducts also serves to elucidate mechanisms of bioactivation of procarcinogens(Yun et al., 2018). Quantitative DNA adduct measurements are robust biomarkers of exposure to hazaradous chemicials, some of which can be linked to genetic polymorphisms in genes encoding metabolism and DNA repair enzymes that impact cancer risk susceptibilities.
The biomonitoring of DNA damage is most relevant when tumor formation commences, rather than many years later when cancer is diagnosed. However, environmental pollution, tobacco smoking, and diet generally represent long-term exposures, and current adduct levels of carcinogens from these exposures are likely to correlate with adduct levels that existed during the time of tumor initiation. Some DNA adducts can be linked to mutations in cancer-driver genes (Alexandrov et al., 2013). Linking chemical exposures, DNA adducts with susceptibility genes and mutations can strengthen the associations of chemicals in cancer etiology.
How are DNA adducts measured and caveats in study design?
Analytes: Many types of DNA adducts can be measured in various types of biospecimens. The types of DNA adducts analyses for a specific study requires discussion with the HHEAR Lab Hub.
Methods: Mass spectrometry is the only analytical tool able to provide the physicochemical data needed to identify the structures of the DNA damaging agents and the chemical agents responsible for DNA damage. Both gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) can quantify DNA adducts in human specimens. Contemporary LC/MS instruments, particularly high-resolution accurate mass spectrometry (HRAMS) instruments, can measure some DNA adducts at detection limits in the low attomole level, which is the level required for human biomonitoring.
Before GC/MS or LC/MS measurements, DNA needs to be digested to the modified nucleoside using a cocktail of nucleases or by acid or base hydrolysis to recover the modified nucleobases. The employment of stable isotopically labeled internal standards is required for quantitative measurements.
Types of biospecimens: DNA is typically isolated from tissues by chloroform phenol extraction or by silica-based ion exchange resins.
DNA adduct levels are assumed to be at a steady-state, where adduct levels reflect the balance between DNA adduct formation and repair. Though often unavailable, biopsy samples in the target tissue are preferred for studying the relationship between DNA damage and disease outcomes. Formalin-fixed paraffin-embedded tissues with a disease diagnosis are often available for retrospective screening of carcinogen DNA adducts (Hwa Yun et al., 2020). Also, alternative biospecimens, including buccal cells and white blood cells (WBCs) have served as surrogate specimens to screen for DNA damage. The DNA yields in the blood (~30 - 40 μg/mL) and saliva (~20 - 50 μg/mL saliva) (Godschalk et al., 2003; Garbieri et al., 2017) are greater than buccal cell scrapings (1 - 5 μg) (Hecht, 2017); however, the quantity of DNA from these biospecimens is sufficient to screen for many types of DNA adducts.
One limitation is that DNA adduct levels may not reflect adduct levels in the target organ. The majority of WBC turnover occurs within several days, the same panel of DNA adducts may not exist in the target organ, and the DNA repair rates differ among various cell types (Godschalk et al., 2003). WBC and saliva contain mixed cell types with different half-lives, and saliva contains bacteria. These caveats need to be considered in the study design for biomonitoring DNA adducts in surrogate biofluids. Nevertheless, oral cell DNA is a promising approach to measure DNA adducts as potential biomarkers for lung cancer susceptibility in cigarette smokers and the health risks associated with e-cigarettes (Hecht, 2017). Also, some types of DNA adducts, such as oxidized DNA bases can be measured in urine (Shih et al., 2019; Cooke et al., 2018).
How does HHEAR ensure the quality of its analyses?
All assays are well validated with respect to accuracy and precision. All assays have embedded positive and negative controls. The positive controls are used to check assay accuracy within each set of samples. The negative controls are generally synthetic oligonucleotide blanks or from non-exposed specimens. These can be used to eliminate the possibility of cross-contamination with compounds, which can be a problem, particularly for measurements of oxidative DNA adducts.
What sample quality and quantity are necessary?
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. Efficiencies can vary by 10-fold or more. (Yun et al., 2018).
More details on DNA isolation and quality assessment can be found here. It is preferable for the HHEAR Lab Hub to isolate the DNA to ensure quality control. However, there may be certain cases where this may not be possible, such as with biofluids, where cells need to be processed immediately.
Himmelstein MW, Boogaard PJ, Cadet J, et al. Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage. Critical Reviews in Toxicology. 2009; 39:679-694.
Jarabek AM, Pottenger LH, Andrews LS, et al. Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Critical Reviews in Toxicology. 2009; 39: 659-678.
Nelson DL, Cox MM. Lehninger Principles of Biochemistry. New York: W. H. Freeman, 2008. Alexandrov LB, Nik-Zainal S, Wedge DC, et al. (2013) Signatures of mutational processes in human cancer. Nature. 2013; 500:415-421.
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 Biochemistry and Molecular Biology. 2003; 36:1-11.
Garbieri TF, Brozoski DT, Dionisio TJ, et al. Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols. Journal of Applied Oral Science. 2017; 25:147-158.
Hecht SS. Oral Cell DNA Adducts as Potential Biomarkers for Lung Cancer Susceptibility in Cigarette Smokers. Chemical Research in Toxicology. 2017; 30:367-375.
Shih YM, Cooke MS, Pan CH, et al. Clinical relevance of guanine-derived urinary biomarkers of oxidative stress, determined by LC-MS/MS. Redox Biology. 2019; 20:556-565.
Cooke MS, Hu CW, Chang YJ, et al. Urinary DNA adductomics - A novel approach for exposomics. Environment International. 2018; 121:1033-1038.
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. 2020; 39:55-82