Detection of Oxidative Clustered DNA Lesions in X-Irradiated ... - BioOne

RADIATION RESEARCH

167, 207–216 (2007)

0033-7587/07 $15.00 䉷 2007 by Radiation Research Society. All rights of reproduction in any form reserved.

Detection of Oxidative Clustered DNA Lesions in X-Irradiated Mouse Skin Tissues and Human MCF-7 Breast Cancer Cells Esha Gollapalle,a Rong Wang,b Ronke Adetolu,a Doug Tsao,a Dave Francisco,a George Sigounasb and Alexandros G. Georgakilasa,1 a

Biology Department, East Carolina University, and b Department of Internal Medicine, Brody School of Medicine, Hematology/Oncology Section, East Carolina University, Greenville, North Carolina 27858

is the deficiency of cellular repair mechanisms against diverse types of oxidative DNA damage (3–6). This pool of oxidative DNA damage can contain a wide variety of single or clustered DNA lesions, including single-strand breaks (SSBs) oxidized bases and/or apurinic-apyrimidinic (abasic, AP) sites (7). Of the different repair pathways, base excision repair (BER) is generally believed to be the primary defense against clustered DNA lesions other than doublestrand breaks (DSBs) formed by endogenous sources of oxidative stress and/or exogenous sources such as ionizing radiation (8–10). Bistranded clustered DNA lesions (i.e., two or more DNA lesions within a short DNA fragment of 1–10 bp on opposing DNA strands, including AP sites, oxidized purines/pyrimidines or SSBs) are readily induced by low doses of ionizing radiation and a variety of chemicals (11–13). They are hypothesized to challenge the cellular repair machinery, producing cytotoxic and mutagenic effects. Endogenous clustered DNA lesions accumulate in some human cells, pointing to a possible role of clusters in the promotion of genetic instability and mutagenesis (14, 15). Using DNA repair proteins isolated from E. coli, Sutherland et al. developed an assay for the quantification of oxidative clustered DNA lesions in cells (11). This assay was based on the fact that the repair enzymes (DNA glycosylases and AP endonucleases) involved in the BER pathway had functional activity in vitro, i.e., on isolated DNA carrying clustered lesions. Once these enzymes detect the lesion in a cluster, they excise the defective base and cleave the DNA strand through their intrinsic lyase activity (DNA glycosylases) or directly cleave the DNA (e.g. AP endonucleases) and create an SSB in each strand, resulting in the formation of a DSB that can be measured using agarose gel DNA electrophoresis and number average length analysis (16, 17). DSBs formed postirradiation are in addition to those directly induced by radiation, and they correspond to the number of clusters measured as described previously. Furthermore, putrescine has been used for the detection of very closely spaced abasic (AP) sites (1–5 bp apart), which are poorly detected by Nfo AP endonuclease (18). Finally, the terms ‘‘abasic’’ or ‘‘oxybase’’ clusters re-

Gollapalle, E., Wang, R., Adetolu, R., Tsao, D., Francisco, D., Sigounas, G. and Georgakilas, A. G. Detection of Oxidative Clustered DNA Lesions in X-Irradiated Mouse Skin Tissues and Human MCF-7 Breast Cancer Cells. Radiat. Res. 167, 207–216 (2007). Bistranded oxidative clustered DNA lesions are closely spaced lesions (1–10 bp) that challenge the DNA repair mechanisms and are associated with genomic instability. The endogenous levels of oxidative clustered DNA lesions in cells of human cancer cell lines or in animal tissues remain unknown, and these lesions may persist for a long time after irradiation. We measured the different types of DNA clusters in cells of two human cell lines, MCF-7 and MCF-10A, and in skin obtained from mice exposed to either 12.5 Gy or sham X radiation. For the detection and measurement of oxidative clustered DNA lesions, we used adaptations of number average length analysis, constant-field agarose gel electrophoresis, putrescine, and the repair enzymes APE1, OGG1 (human) and Nth1 (E. coli). Increased levels of all cluster types were detected in skin tissue from animals exposed to radiation at 20 weeks postirradiation. The level of endogenous (no radiation treatment) oxidative clustered DNA lesions was higher in MCF-7 cells compared to nonmalignant MCF-10A cells. To the best of our knowledge, this is the first study to demonstrate persistence of oxidative clustered DNA lesions for up to 20 weeks in animal tissues exposed to radiation and to detect these clusters in human breast cancer cells. This may underscore the biological significance of clustered DNA lesions. 䉷 2007 by Radiation Research Society

INTRODUCTION

Human cells are susceptible to DNA damage in vivo due to exposure to endogenous and environmental oxidizing factors, including endogenous production of reactive oxygen species (ROS) during aerobic cellular metabolism (1, 2). One of the main etiological hypotheses for the promotion of genomic instability, mutagenesis and tumorigenesis 1 Address for correspondence: Biology Department, Howell Science Complex, East Carolina University, Greenville, NC; e-mail: [email protected]

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fer to all clusters containing at least one abasic site or one oxidized base, respectively. Substantial evidence supports the accumulation and persistence of various oxidative nonclustered DNA lesions (e.g. abasic sites or oxidized bases) in human or animal cells and tissues at values ranging from 100–10,000 lesions/ Gbp (2, 19–21). In addition, elevated levels of nonclustered oxidative DNA damage (e.g. 8-oxodG) or DSBs have been detected in rodents or cancer patients exposed to radiation even several weeks (1–24 weeks) postirradiation, possibly indicating an induced chronic oxidative stress (22–26). Furthermore, in breast cancer patients, increased levels of nonclustered oxidative DNA lesions (e.g. 8-oxodG, abasic sites and single-strand breaks) and/or impairment of DNA lesion repair have been reported (27–29). As outlined above, there are no data on the possible accumulation of oxidative clustered DNA lesions in human malignant cells or tissues and in mammalian or human tissues exposed to ionizing radiation. To address this question, we first assessed the level of these lesions in skin tissue from mice exposed or not exposed to 12.5 Gy of X rays. Then we determined the endogenous levels of oxidative clustered DNA lesions in MCF-10A human breast cancer cells and the corresponding nonmalignant MCF-10A breast cells. For the detection and measurement of clusters, we used constant-field agarose gel electrophoresis, quantitative electronic imaging, and number average length analysis and as probes the human repair enzymes APE1 and OGG1, the E. coli Nth1, and putrescine (16, 17, 30). Our results show that oxidative clustered DNA lesions can be detected in nonirradiated mammalian tissues. Exposure of these tissues to ionizing radiation (X rays) leads to an accumulation of DNA clusters that are detected even 20 weeks after irradiation. Furthermore, a bystander effect of radiation on induction of oxidative clustered DNA lesions was observed in skin areas that were well shielded and protected from radiation. Measurement of endogenous oxidative clustered DNA lesions (no radiation treatment) in cells of the human breast cancer line MCF-7 revealed elevated levels compared with the nonmalignant MCF-10A cells. These results raise questions about the possible accumulation of clusters in human malignant tissues or human normal tissues exposed to ionizing radiation. MATERIALS AND METHODS Animal Procedures, Irradiations and Statistical Analysis Female C57BL mice, 7–8 weeks old, obtained from Harlan Laboratories (Indianapolis, IN) were used in this study. Mice were allowed ad libitum access to food and water. Animal procedures were approved by the East Carolina University Animal Care Committee. For the tissue irradiation studies, mice were separated in two groups. Animals in group I were exposed to 12.5 Gy of X radiation while animals in group II were sham-irradiated. Air-breathing mice were restrained in lucite containers and shielded with a 3-mm-thick lead sheeting everywhere except for the thoracic area (from inferior to the neck to just superior to the diaphragm). The lead-shielding methodology used in these studies has been tested

extensively, and dosimetric analysis indicated that there are negligible dose to the shielded areas. The mice were irradiated with a Siemens Stabilipan X irradiator (250 kVp, 15 mA) at a dose rate of 0.6 Gy/min. Twenty weeks after irradiation, mice of all groups were killed humanely with a high dose of anesthetic. The skin was subsequently removed and stored in ⫺80⬚C for analysis of DNA damage. For the irradiation of isolated DNA, control (unirradiated) tissue DNA was extensively dialyzed against 20 mM potassium phosphate buffer, pH 7.4 and ␥-irradiated at 50 ng/␮l using a 137Cs ␥-ray source (Radiation Facility, Brody School of Medicine, ECU) at a dose rate of 0.57 Gy/min. Paired Student’s t tests were used to evaluate the differences between cluster yields in the different groups. Cell Culture The MCF-7 human breast cancer cells and the non-tumorigenic MCF10A cells were both obtained from ATCC (Manassas, VA). MCF-7 cells were grown in RPMI 1640 medium (Hyclone, Logan, UT) with 5% fetal bovine serum (FBS: Hyclone) at 5% CO2 (31). MCF-10A cells were grown in DMEM/F12 medium (1:1) (Mediatech Cellgro, Herndon, VA) with 10% horse serum (Gibco-Invitrogen, Carlsbad, CA), insulin (10 ␮g/ ml, Sigma), EGF (20 ng/ml, Sigma, St. Louis, MO), hydrocortisone (0.5 ␮g/ml, Sigma) and cholera toxin (100 ng/ml, Calbiochem-EMD Biosciences, San Diego, CA) in 95% air/5% CO2, following the recommendations of the Karmanos Institute. Isolation of DNA A High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN) was used for all DNA isolations from tissues or cells without the use of phenol. This kit is stated by the manufacturer to yield DNA fragments in the range of 6–28 kbp. In our case, the size of the rendered DNA fragment was below 23 kbp (⬃21 kbp) as additionally assessed by pulsed-field gel electrophoresis [PFGE: 6 V/cm, 8 h, 1% agarose (BioRad Molecular Biology Grade) gel, pulses 0.1–1 s, run in 0.5⫻ TBE] performed in a Bio-Rad CHEF DR-II apparatus in 0.5⫻ TBE (data not shown). Tissues were removed from the ⫺80⬚C freezer and 30–40 mg of whole skin tissue was processed. After an initial overnight lysis in 300 ␮l of Proteinase K (Roche) solution of high radical scavenging capacity [5 mg/ml in tissue lysis buffer (4 M urea, 200 mM Tris, 20 mM NaCl, 200 mM EDTA, pH 7.4)] at 37⬚C, DNA was extracted according to the manufacturer’s instructions. Special attention was given to using freshly prepared and autoclaved buffers to avoid induction of additional DNA damage and to maintaining the maximum temperature in the final extraction step below 60⬚C instead of the 70⬚C as recommended by the manufacturer. The column was centrifuged for 1 min at ⬃5000g). The purity and concentration of the isolated DNA was measured using a standard UV spectrophotometry procedure (32). The isolated DNA was dissolved in standard TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5), divided into aliquots and stored at ⫺20⬚C until further use. Hereafter and for clarity we define control tissue DNA to be that which is isolated from animals not exposed to radiation or sham-irradiated and irradiated tissue DNA to be that which is isolated from animals and tissue directly exposed to X rays. This irradiated tissue DNA was used for all enzyme titrations (see below). For the isolation of DNA from cells, approximately 100,000 cells were resuspended in 200 ␮l PBS and lysed with proteinase K lysis solution (as described above) at 37⬚C for 3 h. The DNA derived from cells was isolated as described above. Detection and Measurement of Bistranded Oxidative Clustered DNA Lesions For the measurement of bistranded DNA lesions, the methodology described previously was used with some modifications (17, 18). The repair enzymes APE1 (human), OGG1 (human) and Nth1 (E. coli) were used as enzymatic probes. All enzymes were purchased from New England Biolabs (Beverly, MA) and had been tested extensively against any non-

DNA DAMAGE CLUSTERS IN MOUSE TISSUES AND HUMAN BREAST CANCER CELLS

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FIG. 1. Frequencies of DSBs and oxypurine oxidative clustered DNA lesions in DNA isolated from control mouse tissue induced by ␥ rays in 20 mM phosphate buffer. Lines, least-squares linear fits. Large open symbols are means of three independent experiments (SEMs are shown when larger than points; small symbols are independent points. Yields for DSB and cluster induction derived from the linear fittings of the data are 2.75 ⫾ 0.24 DSBs/Mbp per gray, 4.33 ⫾ 0.14 Fpg clusters/Mbp per gray, and 4.63 ⫾ 0.45 OGG1 clusters/Mbp per gray.

specific or nuclease activity by the company (personal communication with Dr. Guthrie, New England Biolabs). Furthermore, validation studies in our laboratories indicated that these enzymes show no significant nonspecific activity and that in DNA isolated from control mouse tissue and irradiated with ␥ rays they detect levels of clusters similar to those reported previously by Sutherland et al. and Georgakilas et al. (18, 33). Finally, it should be noted that the human enzymes APE1 and OGG1 were used for the first time in this study for the detection of clustered DNA lesions. For the detection of bistranded oxidative clustered DNA lesions, 20 ng of isolated DNA (⬃1 ␮l) were mixed and incubated on ice for 30 min with 7 ␮l of the appropriate enzyme reaction buffer: APE1 (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, pH 7.9), OGG1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 pH 7.9), Fpg (10 mM Tris-HCl, 10 mM MgCl2 pH 7.0), Endo IV (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2 pH 7.9), and Nth1 (20 mM Tris-HCl, 1 mM EDTA pH 8.0). Before the addition of enzyme, 1 ␮l of 10⫻ DDT (10 mM, Sigma) was added to the samples. After 5 min incubation on ice, 1 ␮l (1–3 U) of the appropriate enzyme was added, mixed and incubated for 15 min on ice and then placed at 37⬚C for 1 h (total reaction volume 10 ␮l). The reaction was stopped by adding 5 ␮l of ice-cold native stop solution (50% glycerol, 100 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol) and incubating on ice for 30 min prior to electrophoresis. The DNA solutions were loaded and left to equilibrate for 10 min, and electrophoresis was performed using neutral constantfield agarose gel electrophoresis (37): 1% agarose (Molecular Biology Agarose, Bio-Rad), 50 V, 3 h in 0.5⫻ TBE, pH 7.6. Gels were stained using ethidium bromide and destained as described previously (17). An electronic image was obtained using a FluorChem娂 8800 imaging system (Alpha Innotech, San Leandro, CA). Electronic images for each gel lane were processed using QuantiScan (BioSoft, Ferguson, MO), and a densitogram was obtained. DNA standards (␭-HindIII digest) were used to obtain the corresponding dispersion curve with Origin 6.1 (OriginLab, Northampton, MA). The number average lengths (Ln) for each sample were calculated using the equations described by Sutherland et al. (17). The frequencies of DSBs and different types of oxidative clustered DNA lesions (abasic or oxidized base clusters) were measured based on Ln values and using number average length analysis (17). For each enzymetreated sample (⫹ lane), an accompanying control sample (⫺ lane) was prepared by following the same steps without addition of the enzyme.

Finally, the amount of each enzyme needed to give complete cleavage of its sensitive sites was determined by titrating 20 ng of irradiated tissue DNA with increasing amounts (units) of each enzyme. The amount of each enzyme required to give complete cleavage of oxypurine or abasic clusters using E. coli enzymes (Endo IV, Fpg or Nth1) or human enzymes (OGG1) was determined by titrating irradiated DNA (15 Gy) in phosphate buffer with increasing enzyme amounts (0– 5 U) as described above; 3 U Endo IV (Nfo) and 4 U OGG1, Fpg or Nth1 were chosen. To detect more closely spaced abasic DNA clusters, putrescine was used as described previously (18). Titrating irradiated tissue DNA (20 ng) with increasing concentrations of putrescine (Sigma), a concentration of 10 mM (in doubly distilled water) was found to offer the greatest efficiency in the detection of putrescine clusters and was therefore used in all subsequent experiments. The putrescine-treated (⫹ lanes) and nontreated samples (⫺ lanes) were analyzed as described above. For simplicity, we include the APE1 or putrescine clusters in the family of abasic clusters, the OGG1 clusters as oxypurine clusters, and Nth1 clusters as oxypyrimidine clusters, as suggested previously for the corresponding E. coli enzymes (14).

RESULTS

In this study, we assessed the presence of clustered DNA lesions in tissue obtained from either control animals or animals exposed to ionizing radiation. Since some adaptations of the methods published previously (16) for the detection of clusters were used, we performed a series of experiments to validate these approaches. Figure 1 shows the radiation-induced DSBs and oxypurine clusters detected by Fpg (E. coli) or OGG1 (human) enzymes in isolated control mouse tissue-derived DNA ␥-irradiated in phosphate buffer. From the slopes of the linear fittings, the average yields were derived for each type of cluster. Human OGG1 and E. coli Fpg detected similar levels of clusters. Comparable results were obtained when other human and E. coli en-

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zymes such as APE1, Nth1 and Endo IV were used (data not shown). Figure 2 shows two representative gels used for the assessment of oxidative clustered DNA lesions in mouse skin tissue DNA without (Fig. 2A) or with (Fig. 2B) exposure to ionizing radiation. Treatment of control tissue DNA with various enzymes did not result in a significant increase in DNA fragmentation (Fig. 2A). This indicates the presence of low levels of endogenous enzyme-specific clusters in these tissues. In the case of irradiated tissue-derived DNA, the size of the DNA incubated with Nth1 decreased significantly compared to nonenzymatically treated samples (lane 5), indicating a higher level of clusters in irradiated tissues (Fig. 2B, lanes 2–4). In the same gel, we included from skin areas DNA from irradiated animals but not directly exposed to radiation (lanes 6, 7). In this case, treatment with Nth1 (lane 6) revealed additional DNA fragmentation and DNA size reduction, suggesting lower levels of Nth1 clusters in these samples compared to the irradiated ones. Titration experiments and using irradiated tissue DNA as the substrate indicated that the optimal number of clusters were detected by 1 U APE1, 2 U OGG1 and 2 U Nth1 (Fig. 2C and D). Furthermore, the human enzymes APE1 and OGG1 detected ⬃10% more abasic or oxypurine clusters compared to their E. coli-derived homologs Endo IV (also known as Nfo) and Fpg (data not shown). To evaluate the possible role of stress on the formation of oxidative clustered DNA lesions in animals, we performed experiments by subjecting the mice to the whole irradiation procedure without an actual radiation exposure. The levels of endogenous oxidative clustered DNA lesions in tissue DNA from control and sham-irradiated animals were assessed using three repair enzymes (APE1, OGG1, Nth1) and putrescine (Fig. 3). There was no significant difference in oxidative clustered DNA lesion levels between the control and sham-irradiated animals (P ⬎ 0.05). The average frequencies of endogenous oxidative clustered DNA lesions in control skin tissue DNA ranged from 600 to 900 clusters/Gbp (or 0.6–0.9 clusters/Mbp). Since background endogenous levels of oxidative clustered DNA lesions were not found to change when the animals undergo sham irradiation, these experiments can serve as an additional control for our studies. Based on endogenous oxidative clustered DNA lesions found in control skin tissue, we explored the possible cluster accumulation in tissue of skin areas either exposed directly to a localized dose of 12.5 Gy of X rays or protected with a lead shield. As shown in Fig. 4, analysis of tissue DNA directly exposed to radiation revealed significantly elevated levels for all different oxidative clustered DNA lesions compared with the background levels observed in Fig. 3 (P ⬍ 0.001). In addition, measurement of oxidative clustered DNA lesion frequencies in skin tissue from the same irradiated animals but shielded with lead revealed values significantly higher than background levels (Fig. 3) (P ⬍ 0.001) but lower than those seen in tissues directly ex-

posed to radiation (Fig. 4) (P ⬍ 0.01). In relative values, and for the irradiated samples, the ratios we found for all different clusters are: 1 APE1 cluster:⬃1.05 putrescine clusters:⬃1.04 OGG1 clusters:⬃1.02 Nth1 clusters. Similar ratios within means of error have been found for nonexposed tissues. Measurement of DSBs for the irradiated samples revealed insignificant levels of DSBs (⬃3 DSBs/Gbp). Previous data have suggested increased levels of oxidative stress and defective repair of 8-hydroxyguanine in MCF-7 cells (39, 40). Therefore, we measured the endogenous oxidative clustered DNA lesion frequencies in DNA isolated from cells of the human breast cell lines MCF-7 (malignant) and MCF-10A (nonmalignant). We found that the levels of all different oxidative clustered DNA lesions, especially the OGG1 clusters, in MCF-7 cells were increased compared to MCF-10A cells (P ⬍ 0.001) (Fig. 5). The different oxidative clustered DNA lesion ratios for MCF-7 and MCF-10A cells were found to be similar (within margins of error) to that for irradiated tissue DNA: 1 APE1 cluster:⬃1.04 putrescine clusters:⬃1.07 OGG1 clusters:⬃1.06 Nth1 clusters. DISCUSSION

Significant stress for all cells is produced by reactive oxygen species (ROS) that are unavoidably formed as byproducts of endogenous metabolism or after exposure to environmental oxidizing agents. This pool of potentially mutagenic and carcinogenic DNA alterations is confronted primarily by the base excision repair (BER) pathway (9). The different types of bistranded or unistranded clustered DNA lesions appear to be the most challenging lesions for the cell to repair (12, 41, 42). Attempted repair of these complex lesions can lead to significant delay of the repair machinery and generation of persistent SSBs and/or DSBs as repair intermediates (43–45). Since endogenous clusters have been shown to accumulate in some normal human cells of different tissue origin (14, 15), we first explored the possibility of detecting oxidative clustered DNA lesions in animal tissue exposed and not exposed to ionizing radiation. In addition and as a first step in addressing the question of possible accumulation of clusters in human malignant tissue, i.e. breast tumors, we measured the level of endogenous clusters in transformed and malignant human mammary epithelial cells. We validated our approach by detecting the levels of DSBs and oxypurine clusters [expected to have the higher values of all clusters (33)] in control mouse DNA induced by ␥ rays. Our findings (Fig. 1) are in agreement with others obtained under similar irradiation conditions (18, 33). For T7 DNA irradiated in phosphate buffer, the aforementioned studies suggest a yield of 2.4 DSBs/Mbp per gray and 5.2 Fpg clusters/Mbp per gray in comparison with our yields of ⬃2.7 DSBs/Mbp per gray and ⬃4.3 Fpg clusters/Mbp per gray. Finally based, on the yields of ⬃4 Nfo clusters/Mbp per gray and ⬃1.5 Nth1 clusters/Mbp per gray reported by the same au-


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FIG. 2. Detection of oxidative clustered DNA lesion in DNA isolated from mouse tissues. Panels A and B: Electronic images of electrophoretic DNA from skin tissue from nonirradiated mouse and animals exposed to 12.5 Gy of X rays. Lanes 1 and 8: Molecular weight standards: ␭-HindIII DNA digest. Panel A: 10–20 ng of DNA was (⫹ lanes) or was not (⫺ lanes) treated with 0.5–2 U of different repair enzymes for detection of endogenous clusters (⫹); lanes 2–3: OGG1, lanes 4–5: Nth1, lanes 6–7: APE1. Panel B: DNAs from skin tissue exposed (lanes 2–5) and not exposed (lanes 6–7) to X radiation (12.5 Gy) were treated with increasing amounts of E. coli Nth1 for detection of clusters (lanes 2–5: 0–2 U) (⫹). Lanes 6, 7: DNA not exposed to radiation from nonirradiated animals treated (⫹) and not treated (⫺) with 2 U of Nth1. Panel C: Titration results for irradiated tissue DNA (20 ng) cleavage by increasing amounts of human APE1 (䡬) for the detection of abasic clusters. Panel D: Titration of the same irradiated DNA (20 ng) with increasing amounts of human OGG1 (䡬) and E. coli Nth1 (#) for the detection of oxybase clusters. Open symbols show average values of two independent experiments. Error bars are standard errors and in some cases are smaller than the corresponding symbol. The curves, solid for human enzymes and dashed for E. coli, were fitted to the points by eye.

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FIG. 3. Levels of different types of endogenous oxidative clustered DNA lesions detected in DNA isolated from nonirradiated mouse tissues: control and sham-irradiated. Abasic clusters were detected using APE1 (panel A) and putrescine (panel B). Oxypurine and oxypyrimidine clusters were detected using OGG1 (panel C) and Nth1 (panel D). Solid symbols represent individual experiments. Open symbols show average values. Error bars are standard errors and in some cases are smaller than the corresponding symbol.

thors, for a dose of 10 Gy we can calculate a frequency of ⬃40 Nfo clusters/Mbp and ⬃15 Nth1 clusters/Mbp. Again, these numbers are in very good agreement with our data (Fig. 1). Based on this validation we performed a series of experiments to calculate the possible accumulation of oxidative clustered DNA lesions in mouse tissue after exposure to ionizing radiation. Our results show measurable endogenous levels of different oxidative clustered DNA lesions in nonirradiated mouse skin tissues (Fig. 3) at a steady state of ⬃0.6–0.9 cluster/Mbp. These cluster frequencies are higher than that reported for DNA isolated from human skin primary cell cultures (20–40 clusters/Gbp or 0.02–0.04 cluster/Mbp) (15). However, a completely different isolation and enzyme treatment method was followed by embedding DNA in agarose plugs and using transverse alternating-field electrophoresis for DNA analysis (15). On the other hand, previous data for levels of total endogenous isolated oxidative lesions suggest higher frequencies (aba-

sic sites: 10–60 lesions/Mbp and 8-oxodG: 0.1–4 lesions/ Mbp) for tissue DNA of mouse, rat or human origin (2, 46, 47). The specific number is in excellent agreement with our cumulative frequency (⬃3/Mbp) of OGG1, Nth1 and APE1 clusters. Overall, our reported oxidative clustered DNA lesion estimates are about ⬃10-fold lower than estimates of the total number of lesions per Mbp reported in several published studies (see above), which is consistent with the idea that substantial numbers of lesions are in the form of clusters rather than isolated lesions. Exposure of the animals to 12.5 Gy of X rays resulted in a significant elevation of background (endogenous) oxidative clustered DNA lesion levels as long as 20 weeks after the initial exposure (Fig. 4). Previous data on the processing of abasic clusters in human monocytes suggested the persistence of some closely spaced clusters as long as 2 weeks after irradiation with 5 Gy of ␥ rays (12). Based on Monte Carlo damage simulation models (48), a dose of


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FIG. 4. Levels of different types of oxidative clustered DNA lesion detected in DNA isolated from tissues in mice irradiated with 12.5 Gy of X rays: exposed and unexposed neighboring tissues. Abasic clusters were detected using APE1 (panel A) and putrescine (panel B). Oxypurine and oxypyrimidine clusters were detected using human OGG1 (panel C) and Nth1 (panel D). Solid symbols represent individual experiments. Open symbols show average values. Error bars are standard errors and in some cases are smaller than the corresponding symbol.

⬃12 Gy of X rays is expected to induce at the maximum ⬃9200 non-DSB clusters/cell or ⬃1500 clusters/Gbp (average human genome size ⬃3.2 Gbp). Assuming that the half-time for cluster removal is less than 2.67 days, none of the clusters formed by the 12-Gy dose of radiation is expected to remain unrepaired after 20 weeks (personal communication with Dr. R. D. Stewart, Purdue University). Two additional processes that are expected to remove persistent clusters are ‘‘cluster splitting’’ through the process of DNA replication and cell death (12). Consequently, it seems likely that the difference in the number of clusters observed in irradiated and control samples is due to the formation (regeneration) of clusters after irradiation rather than to residual clusters formed by the initial radiation dose. The increased numbers of clusters observed at 20 weeks may be a manifestation of chronic oxidative stress induced in the animals after the initial exposure to radiation. Radiation has been shown to produce chronic oxidative stress

[for a review see ref. (24)]. Reaction oxygen species (ROS) and reactive nitrogen species (RNS) are regularly formed and degraded by all aerobic organisms. Generation and removal of ROS/RNS are in balance in the presence of effective antioxidants and antioxidant enzymes. Any increase in the ratio of ROS-RNS to antioxidant defenses can create cellular stress and a consequent initiation of mitochondrial or other cellular changes like DNA repair impairment that in turn can lead to a cascade of irreversible damage. Several indicators of oxidative stress have been detected in in vitro models after irradiation as well as in different tissues after irradiation of rodents (24). The relatively elevated levels of oxidative clustered DNA lesions in the nonexposed tissues in the irradiated animals (Fig. 4) might also be the result of bystander effects, i.e. the induction of DNA damage in cells not directly hit by radiation (49). Many studies suggest elevated levels of oxidative DNA lesions like 8-oxoG in human cancer patients after radiotherapy (22, 50) or in mice

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FIG. 5. Levels of different types of endogenous oxidative clustered DNA lesion detected in DNA isolated from malignant MCF-7 cells and nonmalignant MCF-10A cells. Abasic clusters were detected using APE1 (panel A) and putrescine (panel B). Oxypurine and oxypyrimidine clusters were detected using OGG1 (panel C) and Nth1 (panel D). Solid symbols represent individual experiments. Open symbols show average values. Error bars are standard errors and in some cases are smaller than the corresponding symbol.

exposed to radiotherapy doses detected several weeks (1– 24) postirradiation (23, 25). All of these studies, in agreement with our results, suggest an induction of chronic oxidative stress and/or DNA repair efficiency reduction due to radiation exposure. Comparing the endogenous oxidative clustered DNA lesion levels in MCF-7 human breast cancer cells with MCF10A cells (non-malignant origin), significantly higher levels (P ⬍ 0.01) of these lesions were found in the cancer cells (Fig. 5). Since culture conditions may affect cell survival and cell growth, MCF-7 and MCF-10A cells were also cultured under similar conditions and using the same medium (DMEM/F12). We found that the type of culture medium did not affect the endogenous levels of clusters detected in MCF-7 cells (data not shown). The previously reported endogenous cluster frequencies in T7 viral DNA of 0.1–0.45 cluster/Mbp (38) are in excellent agreement with ours (Fig. 5). The involvement of DNA damage in the evolution of cancer has been debated (3). Several studies have associ-

ated breast cancer with increased oxidative DNA damage levels and/or repair deficiencies (27, 29, 51). Also, it has been shown that MCF-7 cells are defective in the processing of 8-oxoG (39, 40) and are BRCA1 hemizygous, very weakly expressing the BRCA1 gene (52). We believe that the increased oxidative clustered DNA lesion levels found in MCF-7 cells are associated with some or all the above repair deficiencies. The lower endogenous oxidative clustered DNA lesion levels in MCF-7 cells compared with tissue DNA (Figs. 2 and 4) may be related to the different tissue origin (15) and/or associated repair capacity as well as to the overall status of oxidative stress in each tissue. Finally, putrescine treatment was unable to detect significantly higher levels of abasic clusters than the human APE1 in all treated samples, including the irradiated ones. This result contradicts the previous increased efficiency of putrescine (compared to E. coli Endo IV) to detect abasic clusters in irradiated T7 DNA (18). This difference may be due to the different types of abasic clusters detected by


APE1 and/or different types and complexity of clusters existing in the DNA substrates used in this study. It may be that in the case of endogenous clusters or the ones induced by chronic oxidative stress in a tissue, the highly dense clusters detected by putrescine constitute a relative small population. This may also explain the relatively similar levels of oxidative clustered DNA lesions detected in our studies, i.e. the complexity and different classes of DNA lesions directly induced by radiation (Fig. 1), may be greater than those induced endogenously as a result of chronic oxidative stress (Figs. 3–5). It has been suggested that clustered DNA damage is refractory to cellular repair mechanisms, can be persistent, and can accumulate at higher levels in cells or tissues. Our studies provide evidence that oxidative clustered DNA lesions can accumulate at detectable levels in mammalian tissues exposed to an X-ray dose of 12.5 Gy or in human breast cells of malignant origin. In addition, exposure of tissues to ionizing radiation, as in radiotherapy, can lead to an even greater accumulation of clustered DNA damage, probably due to induction of chronic oxidative stress and regeneration of clusters. High levels of clusters can contribute significantly to an increased mutation rate and genomic instability. Based on these results, future studies must investigate cluster accumulation in malignant human tissues or human tissues (normal and malignant) exposed to ionizing radiation. ACKNOWLEDGMENTS The authors would like to thank Dr. R. D. Stewart (Purdue University) and Dr. B. M. Sutherland (Brookhaven National Laboratory) for helpful discussions and suggestions. This work has been supported by start-up funds provided to Dr. Georgakilas by the Biology Department of East Carolina University and a Research/Creative Activity Grant to Dr. Georgakilas by Eastern Carolina University. Received: April 19, 2006; accepted: October 2, 2006

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