Development of Methodology for the Analysis of Oxidatively Damaged DNA in Archived Human Blood Samples

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The different methods used to detect DNA damage for human blood samples and the best one to date: Radiation, chemical mutagens (carcinogens), and oxidative radicals cause DNA breakdown, which in normal course is repaired by the action of enzymes and other antioxidants. However, if the damage-to-repair homeostasis is collapsed, cells eventually proceed for mutation, apoptosis, and death. For assessing to what extent these factors damage DNA until 70’s the labeled (35P) single-stranded (ss)DNA had to be separated from dsDNA and radioactivity was to be measured. The first non-radioactive fluorimetric method was devised for human leukocytes by Birnboim & Jevcak (1981, p. 1889). DNA strand breaks in cell lysates could be detected by exposing them to an alkaline solution for DNA unwinding, followed by ethidium bromide fluorescent staining. Unwinding proportionately reduced the fluorescence of dsDNA as against sonicated lysates in which DNA was already denatured and unwound. In this experiment, 60Co γ-ray exposure to leukocytes progressively decreased the fluorescence due to DNA breaks. Rydberg & Johanson (1978, pp. 465-468) modified the method to suit in situ conditions using Chinese hamster eggs that were irradiated for a different duration. The agarose embedded cells were lysed under mild alkaline conditions, and after DNA unwinding the slides were neutralized and stained with acridine orange. The ratio of green (dsDNA) to red (ssDNA) fluorescence was the index for DNA damage. This method was modified as the first “alkaline comet assay” by Singh et al. (1988, pp. 184-191), in which embedded blood cells in agarose were lysed and brought to alkaline conditions to enable unwinding, and then were electrophoresed. The slides were then neutralized and stained with ethidium bromide for observation under a fluorescent microscope. The migration of broken DNA strands appeared as comets originating from the nucleus.

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Another approach particularly used for carcinogen analysis was immunological and chemical detection of the modified bases/nucleosides and carcinogen (or a metabolite thereof)-DNA adducts, referred to as damage-associated located biomarkers. In the immunological method (Santella 1999, pp. 733-739), two classes of antigens were used to immunize for the production of mono- and polyclonal antibodies. Modified DNA with unusual oxidized bases was electrostatically complexed to a methylated carrier protein such as keyhole limpet hemocyanin. The second category of antigens was the carcinogen-DNA adducts coupled to the same carrier protein. For example, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene-bound DNA-carrier protein is used to immunize and develop polyclonal antibodies, which also helped detect DNA damage due to polycyclic aromatic hydrocarbon. Radioimmunoassays, competitive ELISA, and immunohistochemistry were the standard methods to examine the damaged DNA. Because of a lack of reference, the methods suffer from not being quantitative method. Using this method DNA damage in WBC by 4-aminobiphenyl present in cigarette smoke, 8-methoxysporalane as a chemotherapeutic drug, and 8-hydroxy- or 8-oxo-deoxyguanosine (8-OHdG) a nucleoside product of oxidative damage could be evaluated. Analysis of one such biomarker, 8-OHdG, has been a routine measure for DNA damage in leukocytes (Marczynski et al. 2002, pp. 274-277). A direct chemical analysis of 8-OHdG in the urine samples, after releasing it from DNA (hydrolysates) was possible using Electrochemical-HPLC and UV detection. The total DNA was denatured at high temperature and digested by the nuclease to release the 8-OHG. A positive correlation was established between the amount of 8-OHdG and DNA damage tested from the comet assay. In another study (Schraufstltter et al. 1988, pp. 1040), the 8-OHdG formation was correlated with the fluorimetric detection of damaged DNA as above, and a high degree of positive correlation was seen.

Extensive DNA breakage characterizes apoptosis in the cells. The 3’-OH termini of the DNA breaks are labeled with biotinylated dUTP (b-dUTP) either by exogenous terminal deoxynucleotidyl transferase (TdT) assay or by E. coli DNA polymerase nick translation (NT) assay (Gorczyca, Gong & Darzynkiewthe icz 1993, pp. 1945-1951). In NT assay, pre-counted leukocytes were fixed with formaldehyde and washed with ethanol. Nick translation was done with polymerase and all dNTP’s except b-dUTP replaced dTTP. Biotin conjugated labeling was stained with avidin-fluorescein isethionate. In TdT assay (subsequently modified as TUNEL assay), post-fixed cells were treated with TdT enzyme and b-dUTP, besides the usual NTP’s. The staining was done as in NT assay. The fragmented or otherwise damaged DNA would take the stain and can be monitored under a fluorescence microscope. A background chromosomal staining was performed with fluorescent propidium iodide. With these procedures, it was possible to make tissue-specific assessments and even quantification of the DNA damage. These methods were fast and very accurate to microscopically observe the DNA damage as only apoptotic cells with damaged DNA would take the green fluorescent stain.

How DNA damage occurs: Numerous factors are attributed to the DNA damage, which include base transitions (purines to pyrimidines and vice versa) and base modifications like oxidation, halogenation, alkylation, nitration, glycosylation, etc. Further, any intercalating agents like ethidium bromide which distorts the physical helical structure of DNA can eventually lead to frame-shift mutations and misreading of the DNA codes. High energy radiations like X-ray and γ-ray physically distort the DNA and produce dsDNA breaks. UV either generates ROS or directly transforms the adjacent thymine or cytosine bases into dimmers which are misread during replication. Heavy metals are responsible for generating different ROS for direct oxidative damage to DNA.

HaCaT and eukaryotic cells’ characteristics and which one is more sensitive to DNA damage: HaCaT a is human keratinocyte cell line commonly used to study carcinogen and UV/H2O2 oxidative effects. Compare to other cell lines and tissues like blood, these cells are more robust to counter oxidative DNA damage. In a study (Haycock et al. 2000, 15639-15636), it was established that peroxides and lipid hydroperoxides induce the glutathione peroxidase activity, which is capable of reducing the ROS level below the threshold needed for DNA damage. Further, it has been shown that these cells tend to actively take dehydroascorbic acid in and reduce to ascorbate (Vitamin C) as a measure to quench peroxides (Savini, Duflot & Avigliano, 2000). Another reason for relatively high ROS tolerance has been ascribed to the level of protection from oxidants derived from phospholipids (Kuno et al. 2008, 1628-1636). Up-regulation of a Type-II platelet-activating factor acetylhydrolase enzyme has been shown to hydrolyze the oxidized fatty acids originating from phospholipids. ROS can also affect gene expression, mediated through altering the signal transduction pathways and transcriptional factors. In HaCaT cells, it has been shown that H2O2 activates transcriptional factor NFκB (Li & Karin 1999, 1137-1143), which in turn suppresses transcription of several genes directly responsible for the proliferative activity and prevent UV-induced oncogenic progression (Manna et al. 2005, pp. 1676-1684).

Multistage carcinogenesis: According to Hemminki & Mutanen (2001, pp. 11–18), cancer is a multistage disease and is the summation effect of many genes that interact in orderly manner in a polygenic pathway (A→B→C→). Heterogenous and allelic pathways (like A→B→K→ and A1→A2→) may also occur. These genes, fundamentally categories-risky- and low risk genes, attribute to primarily six essential alterations on way to malignancy: self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, prolonged angiogenesis, and tissue invasion and metastasis. Heredity non-polyposis colorectal cancer (HNPCC) and breast cancer (BRCA1 and 2) are examples of high-risk genes in which the prevalence of a single trait in a family substantially increases cancer risks, and they may also induce almost any kind of cancer. Low-risk genes are less prevalent in populations and are mostly ascribed to carcinogen metabolism, hormone receptors, and DNA repair process. These genes modulate the carcinogenic process by way of immunosuppression and may lead to specific cancers like non-Hodgkin’s lymphoma, cervix cancer, squamous cell skin cancer, etc.

Chemical Carcinogenesis: These cancers are associated with occupation and lifestyle. Over 200 different chemicals are known to initiate human cancer (Luch 2005, pp. 113-125) and a great majority of them require metabolic activity to transform to putative carcinogen. The predominant ones are Aminoazo dyes, Aromatic amines/amides Hydrocarbons, Metals, Natural carcinogens, N-Nitroso compounds, Olefins, Paraffins, and even some anti-cancer drugs. Xenobiotic substances are metabolized through cytochrome P450-monooxygenases, glutathione-S-transferase, sulfotransferases, etc to genotoxic compounds. DNA-binding and mutations in the cancer susceptible genes viz. TP53, KRAS, etc. are the primary cause of genotoxic manifestations and this happens at the level of DNA damage and impaired repair, and oxidative damage. The non-genotoxic carcinogens like 2,3,7,8-tetrachlorodibenzo-p-dioxin do not bind to or damage DNA but affect indirectly the expression of several cell-cycle related genes.

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Oxidative DNA Damage: Oxygen radicals the produced during reduction of O2 can attack DNA nucleosides and induce strand breaks. Moreover radicals can also oxidize lipids and the resultant oxidized fatty acids can further react with DNA to form adducts (Marnett 2000, pp. 361-370). The most common oxidized nucleoside in oxidative reaction is 8-OHdG, and the most effective radical is the hydroxyl radical (OH.). Hydroxyl radical is generated through two reactions:

Fenton reaction; Fe2+ + H2O2 → intermediate complexes → Fe3+ + OH- + OH.,

and splitting of H2O2 in presence of UV; H-O-O-H → OH. + OH.

Another free radical produced from O2 is superoxide anion (O2.), which reacts with nitrite oxide radical (NO.) to form a covalently bonded peroxynitrite by a reaction;

NO. + O2. → ONOO(Halliwell 2006, 312-322).

Interestingly, ONOOoxidizes DNA bases and can also produce OH. for further oxidation. Due to inflammatory reactions, macrophages produce nitric oxide and superoxide radicals, which can diffuse to other tissues and damage DNA. 8-OHdG not only causes aberrations in DNA replication, but also G→T transitions especially in the oncogenes, tumor suppressor genes and, apoptosis-inducing genes like p53 (Ambs et al. 1999, pp. 86-88). 8-Oxoadenine, thymine glycol, 5-hydroxyuracil, and uracil glycol are the other oxidized DNA bases that cause extensive mutagenesis. Another round of DNA damage occurs indirectly from lipid peroxidation. Polyunsaturated fatty acid residues in membrane phospholipids are highly sensitive to oxidation. Lipid hydroperoxides are the primary oxidation products reacting with metals to form more reactive epoxides and aldehydes. Malondialdehyde is the major aldehyde, which reacts with DNA to produce dG, dA, and dC adducts, and this causes G→T, A→G, and C→T transitions and extensive mutations, and sometimes even double transitions (CC→TT) (Reid & Loeb 1993, 3904-3907).

Comet Assay

Development: Since the first report of Singh et al. (1988, pp. 184-191) on a simple protocol of single cell gel electrophoresis (alkaline comet assay), there have been several modifications and adaptations of this method. Olive, Banath & Durand (1990) modified the method (neutral comet assay) by performing electrophoresis at near neutral pH. Superior results were obtained from alkaline assays because it enabled detection of dsDNA and ssDNA breaks and also rthe evealed presence of alkali labile sites. The neutral comet on the other hand primarily detected dsDNA breaks. A gel-bound comet assay was developed by McNamee (n.d.), in which gel bound films replaced the slides. Singh et al. (2002, pp. 555-560) modified the original alkaline comet assay to suit the detection of DNA single strand breaks. This precautionary modification was to avoid traces of iron present in water which by itself breaks the dsDNA by adding 8-hydroxyquinoline to chelate iron. Further, Singh et al. (2003, pp. 1420-1430) developed a modification of neutral comet assay to determine DNA double-strand breaks. In this tightly condensed chromatin in samples like sperm cells were lysed as usual and before electrophoresis proteinase, K pre-treatment was given to soften the chromatin before any oxidant’s treatment and electrophoresis. Another modification was to treat DNA with lesion-specific repair endonuclease prior to electrophoresis. As presented by Collins et al. (1997, pp. 139-147), for facilitating ssDNA brakes at sites where modified bases are produced, lesion specific enzymes like endonuclease III and formamidopyrimidine glycosylase were used. The most notable modification has been to apply the comet assay for apoptosis detection. As shown by Singh (2000, pp. 328-337), the steps up to lysis, alkaline treatment and neutralization were same to that of alkaline comet, but no electrophoresis was conducted and cells were stained with Yoyo-1 dye, rather than ethidium bromide. The dye distinguished the apoptotic nuclei from intact or the necrotic ones under fluorescence microscope.

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There were problem with storage of blood cells and cryopreservation led to autonomous damage due to freeze thaw. To ascertain the effect of freezing conditions the lymphocyte suspension in RPMI cell culture medium was stored at room temperature, at 4oC and -20oC (Anderson et al. 1997, pp. 115-125). It was revealed that up to 4 d of storage there was no change in DNA damage at 4oC and room temperature, but at -20oC there was marked adverse effect on the sample. After 4 d the samples kept at room temperature or 4oC gave inconsistent results; in one batch DNA damage (ssDNA comet) was more seen at room temperature and in other just the opposite. Basically, storing for over 4 d will attract a number of endogenous DNA nucleases which will cut the strands. Besides it depends on the DNA repair activity functional at which temperature that the DNA strand will be retrieved back. This balance of damaging and repair would decide which temperature would be harmful to DNA. There would also be physical damage to the cells particularly to osmolarity and membrane integrity. For e.g. RBC’s would haemolyze and red hemoglobin will be oozed out leaving the cells dehydrated for any consequent damage to physical integraty of DNA. Such conditions would also affect the enzymes repairing the DNA. Finally, microbial contamination to the samples will add to DNase action to destruct the DNA. At -20oC, there would be freezing of water and crystallization would partially cause dehydration and in turn loss of water with physical damage of water crystals would render severe damage to DNA. Some respite can be given by adding glycerol to the sample but this test has not been done. The problem of cryopreservation was rectified by mixing samples with a cryo preserving agent, DMSO and a cell culture medium RPMI together with fetal calf serum and progressively freezing to -80oC (Hininger et al. 2004, 76). The samples can be stored for at least 4 months if not repeatedly freeze-thawed. It is not advised to freeze at such low temperatures without above additions as this may cause dehydration and water crystal related physical damage to DNA. After thawing the cell viability was always checked by tryptan blue exclusion technique before proceeding for comet assay. The DNA repair activity of such cryopreserved samples gets affected while thawing. It was found that frozen cells’ DNA tends to repair slowly compared to the fresh samples at room temperature. This means the comet results would somewhat differ even under cryopreservation.

A recent advancement in comet assay has been Comet-fluorescence in situ hybridization (FISH) developed by Bock et al. (1999, pp. 207-217) and later compiled by Spivak, Cox & Hanawalt (2009, pp. 44-50). In this method, alkaline comet is used as convention to detect single strand breaks. It is believed that comet “heads” contain intact chromatin with undamaged loops and supercoiled structures, whereas comet “tails” primarily are the relaxed DNA loops with one or more breaks. A gene-specific FISH fluorescent oligonucleotide probe recognizes a particular gene. Depending on where the FISH spots appear, one can judge whether the particular gene is damaged (spot in the tail) or intact (spot in head).

Methodology: The alkaline Comet assay can be easily applied for blood cells to evaluate the changes in DNA intactness attributed to several factors. One such method devised by Hininger et al. (2004) describes the steps of blood sample collection, preparation of cells and comet assay and result analysis including statistical treatments. Blood is collected in heparin tubes to prevent clotting and to this a mixture of DMSO and RPMI 1600 cell culture medium is added. The samples were progressively frozen to -80oC and stored at this temperature. Storage for 4 months is possible. For the assays, cells were thawed at 37oC and cells were harvested by centrifugation and washed with Ca2+ and Mg2+-free PBS to set a count of 20,000 cells per µl. This blood cell suspension was mixed low melting point agarose at 37oC and poured on a pre-coated (with high melting point agarose) microscope slide. For lysis, buffered sarcosine, Triton X-100, NaCl and DMSO was added, and afterwards for alkaline unwinding of DNA NaOH and EDTA solution was added. Electrophoresis in same solution was carried out except that in place of gel platforms, 12-14 slides were placed in a series. Illumination was restricted to avoid any further damage to DNA. After electrophoresis slides were neutralized in Tris buffer, stained with ethidium bromide and examined under a fluorescence microscope attached to CCD camera and a PC with comet Analysis software. Results are expressed as percentage DNA in the tail or in Tail Moment (tail DNA/total DNA) or tail length (distance from head center to end of the tail in µm). Fifty cells per sample were viewed and the values averaged. Student’s t-test pair or some non-parametric analysis of variance is used to measure the effect of dependent variables. Despite great utility and ease of application, there are also some limitations in this method, namely practical difficulty in collecting 50 cells for single analysis, inability to get viable single cell preparations specially of apoptotic/necrotic cells, inability to get information on fragmented DNA size, there is no internal reference to measure the extend of DNA damage, since membrane and mitochondrial damage also contribute to DNA damage the genotoxicity of any agent can be falsely interpreted, DNA repair like excision repair introduces ssDNA cuts and can be falsely interpreted as actual ssDNA damage (Olive & Banath, 2006, pp. 24-25).

To verify the reliability of this assay particularly for biomonitoring of human subjects of different groups, the variation within an experiment, variation between different assays of same sample at different times, different data analysis methods, intra individual variation with time etc. were carried out with at least 900 individuals (Collins et al. 1997, pp. 139-146) and the test revealed high degree of reliability of the assay. There have been numerous other studies on validation aspect of comet assay in terms of age, geographical regions, occupational variations, smoking vs. non-smoking individuals, and it was found that overall the assay led to some mid-point attributes and some reference values for correct assessment of the variations (Møller 2006, pp. 84-104; Betti et al. 1995; Schmid et al. 2007, pp. 180-187).

Applications: Comet assay is suitable for analysis of a large group or populations, which are at the threat of DNA damage due to genetic disorders and occupational, environmental or lifestyle problems. Genetic biomonitoring of a population exposed to a particular kind of carcinogen/genotoxic agents is a necessity for early prognosis of the consequent diseases/disorders. Dietary supplementation of antioxidants like vitamin C significantly reduces the DNA damaging indexes as revealed from this assay (Kassie, Parzefall & Knasmüler, 2000, 15-20). Further using this test, non-insulin dependent diabetes patients were shown to increased ROS production and vascular complications, including lymphocyte DNA damage, and vitamin C helps to rectify this problem (Sardas et al. 2001, pp. 123-129).

Comet has been applied in number of clinical studies. Citing an example, the leucocytes in patients of Fanconi anaemia are more sensitive to radiation and DNA cross-linking agents like mitomycin C and diepoxybutane than the healthy placebos (Mohseni-Meybodi, Mozdarani & Mozdarani, 2009). A similar investigation also exhibited increased DNA damage in breast cancer patients than the controls (Shahidi, Mozdarani & Bryant, 2007, 263-273). These findings suggest avoidance of radiotherapy and chemotherapy which causes DNA cross-linking in such patients. It is worthwhile to note that cisplatin and some other cytostatic drugs used clinically for the treatment of cancer are effective inducers of DNA-cross-linking and their effects can be judged using comet assay prior to recommending them. Early DNA damage detection in Urinary Bladder transition epithelium by comet assay can prevent bladder cancers (Wang et al. 2007, pp. 51-59). A more sophisticated Comet-FISH assay has been successfully applied to detect colon cancer related oncogenes and tumor suppressor genes, and it was revealed that such genes are oversensitive to oxidative and carcinogen-related DNA breakdown activity (Glei et al. 2007, pp. 279-284). An epidemiological study revealed that DNA damage was over 50% increased in smokers compared to non-smokers and intake of vitamin C in diet substantially reduced this attribute (Hininger et al. 2004, 75-80; Kassie, Parzefall & Knasmüler, 2000, 15-20). In occupational and environmental sectors comet assay has utility in pollutant sensing and management. Presence of lead in water bodies can be correlated with oxidative DNA damage in people living in catchment areas (Arif et al. 2008, pp. 97-101). Occupational exposure to phthalate esters in plastic industry increases urinary phthalate derivatives and ROS production, resulting in DNA damage. This aspect has also been monitored in sperm cells by comet assay (Hauser et al. 2007, pp. 688-695)

Present study: Using comet assay the effect of storage conditions (temperature: room temperature, 4oC, -20oC and -80oC; incubation: immediate, overnight, 72 h, 1 week and 1 month with/without cryoprotecting agent DMSO will be tested on artificial DNA damage. As mentioned, we would expect more artifacts within 4 d period of storage at -20oC over room temperature and at 4oC. Samples beyond this period are expected to be damaged at these preservation temperatures due to osmotic and physical conditions. So most ideally -80oC storage in presence of equal DMSO is expected to provide optimal result (minimum DNA breakdown). Better result is expected if a cell culture medium like RPMI and serum are added to such samples. Samples must be stored in aliquots and one-by-one aliquots should be thawed first in ice and then at room temperature to avoid water crystal’s physical damage to DNA. Theoretically we do not expect any difference in terms of DNA damage between HaCaT keratinocyte and blood cells at given storage conditions because DNA damage is not attributed to oxidative reasons or UV, but rather it is related to dehydration and physical stress, which may be equally prevailing in both cell types.

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Blood samples (5-100 µl) will be mixed with different concentration of low-melting point agarose (0.6-1.2%). The DNA concentration would change with volumes and an optimal DNA that forms a measurable tail had to be established. It is possible that excess material may warrant less apparent DNA damage due to crowding and entangling of ssDNA. The damaged DNA would not pass through nuclear membrane. Moreover, the overt action of endogenous DNA repair enzymes in more dense cells may quickly rectify the damaged sites. We might expect a bell shaped curve if tail parameters are plotted as a function of cell concentration. Few of the samples will be pre-processed with DNA repair enzyme, hOGGl, to elucidate the damage caused due to storage conditions. These results will be compared with the human HaCaT keratinocyte cell lines, considered to be more robust to oxidative damage than human blood cells. The cells will be kept unexposed or exposed to different UV-B doses (1, 5 and 10 min), and will be kept under the storage conditions as depicted for blood cells. The comet assays in these cells may prove that the extent of damage to the DNA would be less severe than the blood sample.

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