Level Of Single-strand Breaks Research Articles

Light-based methods for whole blood bacterial inactivation enabled by a recirculating flow system.

Light of certain wavelengths can be used to inactivate pathogens. Whole blood is opaque; thus, the penetration of light is reduced. Here, we overcame this limitation using a thin transparent tube that is illuminated from all angles. Three light-based techniques were evaluated: photodynamic therapy (PDT) using a 660-nm light and antibody-photosensitizer conjugates, ultraviolet, and violet light. We observed a reduction of 55-71% of Staphylococcus aureus after 5h of exposure (starting concentration 107 CFUmL-1 ) and an 88-97% reduction in methicillin-resistant Staphylococcus aureus (MRSA) (starting 104 CFUmL-1 ). An 83-92% decrease for S.aureus and 98-99.9% decrease for MRSA were observed when combined with an immunocapture approach. Complete blood count with differential analysis did not reveal any significant changes in the blood cell numbers. Genotoxicity studies showed that violet and ultraviolet did not induce any significant level of single strand breaks and alkali labile sites in the peripheral blood mononuclear cells (PBMC). In contrast, ultraviolet did induce a very low level of cyclobutane pyrimidine dimers, a UV damage indicator. PDT generated a significant level of single strand breaks and 8-oxoGua in these cells. The approaches showed promise for whole blood pathogen inactivation with minimal collateral damage to PBMC.

DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro.

Mutations in the human Cu/Zn superoxide dismutase type-1 (hSOD1) gene are common in familial amyotrophic lateral sclerosis (fALS). The pathophysiology has been linked to, e.g., organelle dysfunction, RNA metabolism and oxidative DNA damage conferred by SOD1 malfunction. However, apart from metabolically evoked DNA oxidation, it is unclear whether severe genotoxicity including DNA single-strand breaks (SSBs) and double-strand breaks (DSBs), originates from loss of function of nuclear SOD1 enzyme. Factors that endogenously interfere with DNA integrity and repair complexes in hSOD1-mediated fALS remain similarly unexplored. In this regard, uncontrolled activation of transposable elements (TEs) might contribute to DNA disintegration and neurodegeneration. The aim of this study was to elucidate the role of the fALS-causing hSOD1G93A mutation in the generation of severe DNA damage beyond well-characterized DNA base oxidation. Therefore, DNA damage was assessed in spinal tissue of hSOD1G93A-overexpressing mice and in corresponding motor neuron-enriched cell cultures in vitro. Overexpression of the hSOD1G93A locus did not change the threshold for severe DNA damage per se. We found that levels of SSBs and DSBs were unaltered between hSOD1G93A and control conditions, as demonstrated in post-mitotic motor neurons and in astrocytes susceptible to replication-dependent DNA breakage. Analogously, parameters indicative of DNA damage response processes were not activated in vivo or in vitro. Evidence for a mutation-related elevation in TE activation was not detected, in accordance with the absence of TAR DNA binding protein 43 (TDP-43) proteinopathy in terms of cytoplasmic mislocation or nuclear loss, as nuclear TDP-43 is supposed to silence TEs physiologically. Conclusively, the superoxide dismutase function of SOD1 might not be required to preserve DNA integrity in motor neurons, at least when the function of TDP-43 is unaltered. Our data establish a foundation for further investigations addressing functional TDP-43 interaction with ALS-relevant genetic mutations.

Abstract A22: Association of breast cancer risk and concentrations of tissue estrogens to single strand breaks in DNA

Abstract Introduction: Single-strand breaks (SSB) in DNA are discontinuities in one strand of the DNA and are usually accompanied by loss of a single base and by damaged 5- or 3-termini at the site of the break. If not repaired rapidly or appropriately, chromosomal SSBs pose a serious threat to genetic stability and cancer development. We hypothesize that if the presence of single strand DNA breaks can be quantified directly, it will provide a means of detecting a process that puts cells at high risk of developing cancer. The association between a quantitative measure of SSB and other measures of breast cancer risk was determined. Subjects: 206 postmenopausal and 99 premenopausal, healthy women with intact, healthy bilateral breasts, without implants or history of radiation, willing to undergo a random fine-needle aspiration (rFNA) of the breast within 3.5 months of a normal mammogram were recruited to the study. Exclusions: use of tamoxifen, raloxifene, or aromatase inhibitor within 2 years of participation or oral contraceptives or other hormone treatments within 3 months of study enrollment. Participants completed personal and medical history questionnaires. Blood for hormone levels and random fine needle aspirates of the breast (rFNA) were collected following a breast exam. Methods: rFNA of the breast of women at unspecified risks for breast cancer were analyzed for SSB by a nick translation procedure. SSB levels digital two-dimensional breast density of the entire breast (PBD), mRNA of genes associated with DNA damage, and breast steroid concentrations by a LC/MS/MS procedure. Results: Based on the cpm in the purified sample, the specific activity of the 3H-dCTP, and quantity of DNA in the sample, the incorporation of 3H-dCTP ranged from 0.03 to 8.59 pmol/µg DNA. The β-coefficients for the relationships between SSB and measures of breast cancer risk were determined by a multiple regression procedure. PBD adjusted for age was associated with SSB in postmenopausal women (P = 0.007) but was not associated with SSB in premenopausal women. Further adjustment for BMI reduced the PBD relationship to SSB by 35% but adjustment for BMI. APEX1 was not significantly associated with SSB. XRCC1 mRNA was negatively associated with SSB in premenopausal women (p = 0.016), and was not altered by adjustment for age. The antioxidant functions NRF-1 and SOD2 were both significantly negatively associated with SSBs, P = 0.001 and 0.045, respectively, and both were decreased by less than 5% after adjustment for age. Breast tissue concentrations of 4-hydroxyestradiol exceeded those of estradiol, were correlated with tissue estradiol, and were significantly (P = 0.011) negatively related to SSB levels. Breast tissue concentrations of estradiol, estrone, 4-hydroxyestrone, and androstenedione were not significantly related to SSB. Conclusions: The most likely mechanism by which 4-OHE2 or 2-OHE1 could protect against formation of SSBs in the breast is by their antioxidative properties. 4-OHE2 and other catechol estrogens are capable of undergoing redox reactions, cycling between the catechol structure, semiquinone radical, and o-quinone. They are also capable of forming metal complexes, sequestering metals, preventing them from undergoing redox reactions. In addition, 4-OHE2 has a binding affinity relative to that of estradiol of 1.51; that of 2-OHE1 is 1.02 Therefore, the catechol estrogens have activity similar to or greater than that of estradiol in terms of activation of NRF-1 and Mn SOD, both of which were elevated in association with lower SSB levels. We conclude that SSBs measured by the nick translation procedure are associated with, but not redundant with, measures of breast cancer risk and with deficiencies of both DNA damage responses and antioxidant mechanisms. Concentrations of 4-hydroxyestradiol in breast tissue may serve an antioxidant function and may be protective. Citation Format: Mathavi Sahadevan, Oukseub Lee, Miguel Muzzio, Belinda Phan, Lisa Jacobs, Nagi Khouri, Jun Wang, Hong Hu, Vered Stearns, Robert T. Chatterton. Association of breast cancer risk and concentrations of tissue estrogens to single strand breaks in DNA. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A22.

Analysis of heat-labile sites generated by reactions of depleted uranium and ascorbate in plasmid DNA

The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single-strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate, cisplatin, and chromic chloride were compared with those formed by reaction of uranyl acetate and ascorbate. Uranyl ion in the presence of ascorbate produced U-DNA adducts that converted to SSB on heating. Piperidine, which acted on DNA methylated by methyl methanesulfonate to convert methyl-DNA adducts to SSB, served in the opposite fashion as U-DNA adducts by decreasing the level of SSB. The observation that piperidine also decreased the gel shift for metal-DNA adducts formed by monofunctional cisplatin and chromic chloride was interpreted to suggest that piperidine served to remove U-DNA adducts. Radical scavengers did not affect the formation of uranium-induced SSB, suggesting that SSB arose from the presence of U-DNA adducts and not from the presence of free radicals. A model is proposed to predict how U-DNA adducts may serve as initial lesions that convert to SSB or AP sites. The results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding the mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposure in humans.

DNA crosslinks, DNA damage and repair in peripheral blood lymphocytes of non-small cell lung cancer patients treated with platinum derivatives

Lung cancer is the leading cause of cancer-related mortality in the world. Chemotherapy has been the mainstay of treatment for advanced non-small cell lung cancer (NSCLC) and platinum-based derivatives have been shown to improve overall survival. The aim of the present study was to investigate the DNA damage [single strand breaks (SSBs) and DNA crosslinks] and DNA repair in peripheral blood lymphocytes in patients with NSCLC treated with platinum derivatives using modified comet assay. Twenty patients in the final (4th) stage of NSCLC and 10 age-corresponding healthy controls participated in the study. Alkaline comet assay was performed according to the appropriate protocol. The DNA base excision repair (BER) activity of the controls was significantly higher compared to that of cancer patients, and the activity of DNA nucleotide excision repair (NER) was almost at the same level both in controls and patients. We observed changes in the amount of SSBs and DNA crosslinks during the course of chemotherapy. We found a significantly higher level of SSBs immediately after administration of chemotherapy. Similarly, we found the highest incidence of DNA crosslinks immediately or 1 day after chemotherapy (compared to measurement before chemotherapy). Moreover, we compared the levels of DNA repair in patients who survived chemotherapy with those in patients who died in the course of chemotherapy: the activity of BER was higher in the case of surviving patients, while the levels of NER were essentially the same. The data arising from the present study confirm the findings of other studies dealing with DNA damage and repair in cancer patients treated with chemotherapy. Moreover, our results indicated that despite the fact that cisplatin-DNA adducts are removed by the NER pathway, BER may also play a role in the clinical status of patients and their survival.

Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells

Exogenous and endogenous damage to DNA is constantly challenging the stability of our genome. This DNA damage increase the frequency of errors in DNA replication, thus causing point mutations or chromosomal rearrangements and has been implicated in aging, cancer, and neurodegenerative diseases. Therefore, efficient DNA repair is vital for the maintenance of genome stability. The general notion has been that DNA repair capacity decreases with age although there are conflicting results. Here, we focused on potential age-associated changes in DNA damage response and the capacities of repairing DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in human peripheral blood mononuclear cells (PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ-H2AX response, and DSB repair capacity in a study population consisting of 216 individuals from a population-based sample of twins aged 40-77 years. Age in this range did not seem to have any effect on the SSB parameters. However, γ-H2AX response and DSB repair capacity decreased with increasing age, although the associations did not reach statistical significance after adjustment for batch effect across multiple experiments. No gender differences were observed for any of the parameters analyzed. Our findings suggest that in PBMCs, the repair of SSBs is maintained until old age, whereas the response to and the repair of DSBs decrease.

Abstract 5369: Prostate cancer radiosensitization through PARP-1 hyperactivation

Abstract The clinical experimental agent, beta-lapachone (Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether beta-lapachone may warrant clinical evaluation as a radiosensitizer. Beta-lapachone killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species (ROS), irreversible DNA single strand breaks (SSBs), PARP-1 hyperactivation, NAD+/ATP depletion, and µ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), beta-lapachone radiosensitized NQO1+ prostate cancer cells, under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSBs, gamma-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of µ-calpain-induced programmed cell death. Radiosensitization by beta-lapachone was blocked by the NQO1 inhibitor, dicoumarol, or the PARP-1 inhibitor, DPQ. In a mouse xenograft model of prostate cancer, beta-lapachone synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in ∼60% of human prostate tumors evaluated relative to adjacent normal tissue, where beta-lapachone might be efficacious alone or in combination with radiation. Our findings offer a rationale for clinical assessment of beta-lapachone (Arq501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation. Since similar data have been found for all NQO1 endogenously over-expressing cancers, including nonsmall cell lung, pancreatic and breast cancer, this strategy should have a broad use for many cancers and in combination with many DNA damaging agents used for cancer therapy. This work was supported by NIH grant 2 R01 CA102792 to DAB Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5369. doi:10.1158/1538-7445.AM2011-5369

Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.

Prostate Cancer Radiosensitization through Poly(ADP-Ribose) Polymerase-1 Hyperactivation

The clinical experimental agent, β-lapachone (β-lap; Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether β-lap may warrant clinical evaluation as a radiosensitizer. β-Lap killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species, irreversible DNA single-strand breaks (SSB), poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, NAD(+)/ATP depletion, and μ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), β-lap radiosensitized NQO1(+) prostate cancer cells under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSB level, γ-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of μ-calpain-induced programmed cell death. Radiosensitization by β-lap was blocked by the NQO1 inhibitor dicoumarol or the PARP-1 inhibitor DPQ. In a mouse xenograft model of prostate cancer, β-lap synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in ∼60% of human prostate tumors evaluated relative to adjacent normal tissue, where β-lap might be efficacious alone or in combination with radiation. Our findings offer a rationale for the clinical utilization of β-lap (Arq 501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation.

UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria ( Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in ϕX174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and alkylative DNA damages and mutation.

Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination–Defective Cells

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers.

Effects of one-day reperfusion after transient forebrain ischemia on circulatory system in the rat

Although ischemia/reperfusion injury remains incompletely understood, it appears that reactive oxygen species produced mainly during postischemic recirculation play a critical role. The present study examined the impact of forebrain ischemia and subsequent one-day reperfusion on several blood parameters. We determined glutamate concentration in whole blood, measured Cu/Zn- and Mn-SOD (superoxide dismutase) activity in blood cells as well as plasma, and investigated the prevalence of single and double strand breaks of lymphocyte DNA. The results of our experiment showed that the concentration of glutamic acid in whole blood was increased by about 25%. Antioxidant activity of total SOD and Cu/Zn-SOD was reduced in blood cells and plasma. Mn-SOD activity in blood cells was not affected by ischemic insult and one-day reperfusion, but we detected its significantly lower activity in samples of plasma. We observed a weakly reduced level of double and a significantly elevated level of single strand breaks of lymphocyte DNA. In conclusion, one day of recovery after the ischemic attack failed to return peripheral circulatory system to physiological conditions. Reduced antioxidant capacity in the blood and an elevated level of excitotoxic amino acid glutamate may cause lymphocyte DNA damage, and probably contribute to insufficient postischemic recovery of brain tissue.

DNA single- and double-strand breaks by alkaline- and immuno-comet assay in lymphocytes of workers exposed to styrene

Occupational exposure to styrene was studied in 34 workers employed in the production of fiberglass-reinforced plastic sheets and compared to 29 unexposed healthy controls. We evaluated genotoxic effects induced by occupational styrene exposure in lymphocytes by alkaline version of the comet assay to detect single-strand breaks (SSBs), DNA oxidation products (formamido pyrimidine glycosilase (Fpg)- and endonuclease (Endo III)-sensitive sites) and DNA repair kinetics studies, as well as the neutral version of comet assay for DNA double-strand breaks (DSBs). An innovative aspect of this study was the use of immuno-comet assay, a new technique that recognizes DSBs with specific antibody by DAPI/FITC method. The battery of parameters included markers of external and internal exposure. Exposed workers showed significant high levels of SSBs ( p < 0.0001) and DSBs ( p < 0.0001) in neutral- and immuno-comet assay. A drastic decrease in DNA repair activity as compared to controls was observed (180 min vs. 35 min). Styrene workplace concentration significantly correlated with alkaline comet parameters (TM, p = 0.013; TI, p = 0.008), in negative with TL ( p = 0.022), and with DNA-base oxidation (TM Endo III, p = 0.048 and TI Endo III, p = 0.028). There was a significant negative correlation between urinary metabolites (MA + PGA) and TM Endo III ( p = 0.032) and TI Endo III ( p = 0.017).

ATR-FTIR spectroscopy detects alterations induced by organotin(IV) carboxylates in MCF-7 cells at sub-cytotoxic/-genotoxic concentrations

The environmental impact of metal complexes such as organotin(IV) compounds is of increasing concern. Genotoxic effects of organotin(IV) compounds (0.01 μg/ml, 0.1 μg/ml or 1.0 μg/ml) were measured using the alkaline single-cell gel electrophoresis (comet) assay to measure DNA single-strand breaks (SSBs) and the cytokinesis-block micronucleus (CBMN) assay to determine micronucleus formation. Biochemical-cell signatures were also ascertained using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. In the comet assay, organotin(IV) carboxylates induced significantly-elevated levels of DNA SSBs. Elevated micronucleus-forming activities were also observed. Following interrogation using ATR-FTIR spectroscopy, infrared spectra in the biomolecular range (900 cm-1 – 1800 cm-1) derived from organotin-treated MCF-7 cells exhibited clear alterations in their biochemical-cell fingerprint compared to control-cell populations following exposures as low as 0.0001 μg/ml. Mono-, di- or tri-organotin(IV) carboxylates (0.1 μg/ml, 1.0 μg/ml or 10.0 μg/ml) were markedly cytotoxic as determined by the clonogenic assay following treatment of MCF-7 cells with ≥ 1.0 μg/ml. Our results demonstrate that ATR-FTIR spectroscopy can be applied to detect molecular alterations induced by organotin(IV) compounds at sub-cytotoxic and sub-genotoxic concentrations. This biophysical approach points to a novel means of assessing risk associated with environmental contaminants.PACS codes: 87.15.-v, 87.17.-d, 87.18.-h

Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells

Ionizing radiation (IR) induces two classes of complex DNA damage, double-strand breaks (DSBs) and non-DSB bi-stranded oxidative clustered DNA lesions (OCDLs). OCDLs may consist of single strand breaks (SSBs), oxidized purines/pyrimidines and abasic sites within 5–10 bp. These significant biological lesions are hypothesized to challenge the repair machinery and carry a high mutagenic potential. MSH2, a classical DNA mismatch repair protein, has been also implicated in other repair pathways associated with DSB and base lesion processing. MSH2 mutations have been identified in acute lymphoblastic leukemia (ALL) patients as well as in other types of cancers. Our research model involves two precursors B (pre-B) ALL human cell lines, NALM-6 cells, homozygous null for MSH2, and wild type 697 cells. Using a modified version of neutral and alkaline single cell gel electrophoresis (SCGE) with Escherichia coli repair enzymes as damage probes, the processing capacity of single strand breaks (SSBs), DSBs and OCDLs was assessed in NALM-6 and 697 cells exposed to a radiotherapy relevant γ-ray dose of 5 Gy. Using reverse transcriptase PCR and Western blotting we verified the complete lack of expression of MSH2 in the NALM-6 cells at the transcriptional and translational level. No differences were measured between NALM-6 and 697 cells in the induction levels of SSBs, DSBs and OCDLs after exposure to γ-rays. However, 697 cells repaired each lesion more efficiently with significant differences observed after 1–3 h post-irradiation. Lastly, our results indicate a significantly higher population of apoptotic 697 cells compared to NALM-6 cells 6–24 h post-irradiation. Our studies suggest that MSH2 is probably involved in the processing of the biologically significant clustered DNA damages as well as the execution of apoptosis induced by ionizing radiation.

Induced genome instability and bystander effect caused by the radiation originating from Chornobyl catastrophe

Many environmental factors (ultra-violet and ionizing radiation (IR), emotional stress, infections, hyperthermia, tumor toxins, strong medical products, intensive noise and vibration, etc.) can have toxic influence on cellular genome. Named agents can induce the development of oxidizing stress with activation of early regulatory and late structural genes. As a result, there appear de novo chromosome aberrations and genetic mutations with high frequency during many generations in certain part of cells. Only in some cases this is the cause of increased cellular death rate by apoptosis. These different effects of irradiation are known as radiation induced instability of genome which is associated with epigenetic changes of an oxidation-reduction metabolism in descendants of irradiated cells. The augmentation of DNA sensitivity to oxyradicals and other genome toxins and strengthening of the generation of active forms of oxygen is typical. Moreover, the irradiated cells cause in not irradiated cells-neighbours changes typical for irradiated cells (such as: increase of oxygen active forms, frequencies sisterly chromatid exchanges, so called «Bystander effect», BsEf) through cytokines and other factors of the protein nature. We investigated the influence of irradiation (dose 5 Sv during 16 hours) in female mice Balb/c (LD 50/30 5,85 Sv) and C57BL/6 (LD 50/30 6,70 Sv). We studied the levels of single strand breaks of DNA (SSB DNA ) and BsEf. BsEf is characterized by the ability of irradiated animals cells (taken from various tissues and organs, such as peripheral blood, spleen, liver and brain) to induce increased quantity of SSB DNA in the cells taken from not irradiated animals. The analysis was done during first hours, in one and three weeks after exposure with help of DNA labeled by fluorescent dye Picogreen technique. Subsequently the speed of its unwinding was estimated. Damaging influence of the irradiated cells on not irradiated ones was investigated after addition to them the culture medium from irradiated cells. At the irradiated animals cells levels of SSB DNA in the first day after irradiation grew in all kinds of researched cells. In C57BL/6 mice (which are more resistant to irradiation) the level of SSF DNA was much lower and the culture medium from the cells obtained from irradiated animals practically did not possess ability to induce increase of SSB DNA in not irradiated cells. Conclusion: in line of mice, more resistant to irradiation, the levels of expression SSB DNA and Bystander effect was much lower.

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.

High red meat diets induce greater numbers of colonic DNA double-strand breaks than white meat in rats: attenuation by high-amylose maize starch

Human population studies show that dietary red and processed, but not white, meats are associated with increased risk of colorectal cancer but dietary fibre appears to be protective. We examined whether dietary cooked red or white meat had differential effects on colonic DNA damage in rats and if resistant starch (RS), a dietary fibre component, provided protection. Rats were fed diets containing approximately 15, 25 or 35% of cooked beef or chicken, both with or without 20% high-amylose maize starch (HAMS) as a source of RS, for 4 weeks. DNA single-strand breaks (SSB) and double-strand breaks (DSB) were measured in isolated colonocytes (by comet assay) along with apoptosis levels, colonic mucus thickness and large bowel short-chain fatty acids (SCFA). Both red and white meat increased colonocyte SSB and DSB dose dependently but damage was substantially greater with red meat. Dietary HAMS prevented these increases. Apoptotic cell numbers were increased dose dependently by red meat irrespective of HAMS feeding, whereas white meat only increased apoptotic cell numbers in the presence of HAMS. Red meat induced greater colonic mucus layer thinning than white meat but HAMS was protective in both cases. HAMS induced increases in large bowel SCFA, including butyrate, and significantly lowered concentrations of phenols and cresols. We have demonstrated that dietary red meat causes greater levels of colonic DNA SSB and DSB than white meat, consistent with the epidemiological data. Dietary RS protects against this damage and also against loss of the mucus barrier, probably through increased butyrate production.

Radio-Protective Effect of 2-Deoxy-D-Glucose in Cervical Cancer Patient’s Lymphocytes Exposedin Vitroas Estimated by the Comet Assay

The main objectives of the present study was two fold, namely, first to evaluate the baseline Single- Strand Breaks (SSBs) frequency in Cervical Cancer patient's and secondly, to study the radio-protective effect of 2- deoxy-D-glucose (2-DG) on such cells when exposed to ionising radiation like that of gamma radiation. The reason for proposing such a study is that the DNA of cancer patients has a general tendency to break, i.e. Concept of Common Fragile Sites. This is not yet clearly understood as to whether it is the cause or effect of the pathological processes and necessitates looking into the base-line SSBs in this type of patients. Secondly, the main line of treatment of Cervical Cancer is that of radiotherapy & / or surgery, therefore there is a need to protect the normal cells surrounding the tumour tissue during the course of radiotherapy. This could be achieved by means of a radio- modulator such as 2-DG, which protects normal cells and sensitises the tumour tissue, thus acting as a double-edged sword. Hence the study was designed to evaluate the SSBs by Comet Assay (Single cell Agarose Gel Electrophoresis - SAGE) using a silver staining method, which does not require the usage of fluorescent microscopy and can be analysed with the aid of a simple light microscopy. The results showed that the Cervical Cancer patient's peripheral blood lymphocytes are not entirely fragile and the radiation exposure leads to increasing levels of DNA SSBs, which is reduced by 2-DG showing that the compound can be used as an adjuvant to radiotherapy.

Influence of Polymorphisms in DNA Repair GenesXPD, XRCC1andMGMTon DNA Damage Induced by Gamma Radiation and its Repair in LymphocytesIn Vitro

DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.