Induction Of DNA Single-strand Breaks Research Articles

Important effects of neighbouring nucleotides on electron induced DNA single-strand breaks

In this Letter, we present Quantum Mechanics/Molecular Mechanics (QM/MM) calculations on molecules containing a 2-deoxycytidine-3′-monophosphate moiety (3′-dCMPH). In particular, we examine the effect that including neighbouring nucleotides at the Molecular Mechanic (MM) level has on the calculated electron affinities and on the energetic barriers of the C3′–O3′ bond cleavage. Our results demonstrate that the surrounding nucleotides relocate the excess electron from the π ∗ orbital of the base to a diffuse phosphate-centred orbital, leading to the formation of a dipole-bound anion state. Both the electron affinities and the activation energy of C3′–O3′ bond cleavage are strongly increased.

WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

The Werner syndrome helicase/3′-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated ( wrn-kd), and the corresponding wild-type cells ( wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and S-phase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was observed for DNA single- and double-strand break formation in response to topotecan. Topotecan induced DNA single-strand breaks 6 h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA double-strand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA single-strand breaks into DSBs during the resolution of stalled replication forks at topo I–DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.

A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process.

Repair of 2′-C-Cyano-2′-Deoxy-1-β-d-arabino-Pentofuranosylcytosine–Induced DNA Single-Strand Breaks by Transcription-Coupled Nucleotide Excision Repair

The cytosine nucleoside analogue 2'-C-cyano-2'-deoxy-1-beta-d-arabino-pentofuranosylcytosine (CNDAC) causes DNA single-strand breaks after its incorporation into DNA. This investigation sought to determine if DNA excision repair pathways were activated to repair this damage. Neither the base excision repair nor the mismatch repair pathway seemed to be involved. Cells deficient in the CSB protein, which initiates transcription-coupled nucleotide excision repair (NER) pathway (TC-NER), exhibited increased clonogenic sensitivity to CNDAC, whereas cells deficient in XPC, which initiates global genome NER, were slightly resistant relative to wild-type cells. The cells lacking either helicase XPB, which unwinds 5' of the lesion, or endonuclease XPF, which incises 5' to a lesion, exhibited increased clonogenic sensitivity to CNDAC, as did cells lacking the XPF partner protein ERCC1. This sensitization was independent of p53 function. Repletion of XPF restored sensitivity comparable with the wild type. In contrast, cells lacking either XPD, the 3'-helicase, or the 3'-endonuclease XPG were equally as sensitive as wild-type cells. In comparison, cells deficient in XPF were not sensitized to other cytosine nucleoside analogues, troxacitabine and cytarabine. Thus, the single-strand nick caused by CNDAC is recognized and, in part, repaired by the TC-NER pathway. NER proteins that function in the 5' direction relative to the UV-induced lesion also participate in the repair of the CNDAC-induced nick, in contrast to proteins that process on the 3' side of the lesion.

Genotoxicity of visible light (400–800 nm) and photoprotection assessment of ectoin, l-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400–800 nm) and ultraviolet A (UVA)/visible (315–800 nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8 J/cm 2, respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1 mM), ERT (0.5 mM) and mannitol (1.5 mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1 mM), ERT (0.5 mM) and mannitol (1.5 mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.

Immediate biomarker responses to benzo[a]pyrene in polluted and unpolluted populations of the blue mussel ( Mytilus edulis L.) at high-latitudes

Immediate biomarker responses of two high-latitude populations of the blue mussel Mytilus edulis to benzo[a]pyrene (B[a]P) were evaluated. Mussels collected from a clean and a polluted site in southwest Iceland were exposed to the nominal dose of 100 μg B[a]P L − l for 3 h, after 4 days of acclimatization in clean seawater. To test the sensitivity to the toxicant and immediate biological responses, the following biomarkers were used: DNA single strand breaks, heart rate and feeding rate. All the biomarkers revealed differences between the study sites. Irrespective of the origin of the organisms, the short time exposure to the high B[a]P concentration did not induce DNA single strand breaks or significantly affect the feeding rate. However, the heart rate results showed significantly different responses. The mussels from the polluted site (Reykjavík harbour) increased their heart rate when exposed to B[a]P, while no difference was observed between the heart rate values of the individuals from the clean site (Hvassahraun). The mussels seem to sense the pollutant they have been previously exposed to, and their acute response indicates physiological adaptation to the polluted environment. The results indicate limited sensitivity and temporal predictivity, i.e. transient measurable changes of these biomarkers, as well as showing that the background of the organisms should be considered when evaluating short-term biomarker responses to contaminants.

Cell proliferation and hepatocarcinogenesis in rat initiated by diethylnitrosamine and promoted by phenobarbital: Potential roles of early DNA damage and liver metallothionein expression

Cell proliferation plays an important role in multistage chemical carcinogenesis. Again, several reports demonstrated that upregulation of metallothionein (MT) expression is associated with increased cell proliferation that may contribute to the pathogenesis of preneoplastic phenotype to frank malignancy. In this study, we evaluated the roles of early DNA damage, altered expressions of liver MT and Ki-67 nuclear antigen, and altered hepatic levels of zinc (Zn) and copper (Cu) on cell proliferation and the progression of hepatocarcinogenesis through premalignant, late premalignant and malignant transformation phases in male Sprague–Dawley rats. We have further studied the association between MT expression and cell proliferation in hepatocarcinogenesis. There was substantial induction of DNA single-strand breaks (SSBs) ( P < 0.001) and development of hepatocellular premalignant lesions along with significant decrease in hepatic levels of Zn and increase in Cu content following a single, necrogenic, intraperitoneal (i.p.) injection (200 mg/Kg body weight) of diethylnitrosamine (DEN) at week 4 of the experimental protocol. Moreover, DEN + phenobarbital (PB)-treatment significantly elevated MT-, Ki-67-, and BrdU-immunoexpressions along with their immunolabeling indices. Furthermore, positive correlations between MT- and Ki-67- labeling ( P = 0.0006) at various time intervals, as well as, between MT immunoreactivity and 5’-bromo-2’-deoxyuridine-labeling index (BrdU-LI) ( P = 0.0007) indicate that, MT expression might be associated with Ki-67 expression and cell proliferation thereby. The study suggests that DEN treatment may lead to alteration of Zn and Cu levels resulting in early DNA damage along with elevation of MT expression that may ultimately lead to hepatic cell proliferation. The results thus provide evidence in support of the role of MT as a potential positive regulator of cell growth during the early stages of hepatocellular transformation in rats.

Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM 10)

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM 10 (particulate matter < 10 μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50 μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2 h cell exposure to EOMs, 24 h treatment interval was optimal for DNA adduct determination. No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample. Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m 3), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice ≪ Prague < Sofia. Based on these data we suppose that two endpoints DNA breakage and DNA adduction are sensitive in vitro biomarkers for estimation of genotoxic activity of organic complex mixture associated with airborne particles. On the other hand, the measurement of protein p53 up-regulation manifested some limitations; therefore it cannot be used as a reliable endpoint for in vitro genotoxicity assessment.

Differential involvement of reactive oxygen species in apoptosis induced by two classes of selenium compounds in human prostate cancer cells

Selenium is a promising chemopreventive agent for prostate cancer, possibly via an induction of apoptosis. Earlier studies have shown that selenite induces DNA single strand breaks (SSBs), reactive oxygen species (ROS), p53 Ser-15 phosphorylation and caspase-dependent and -independent apoptosis, whereas a methylselenol precursor methylseleninic acid (MSeA) induces caspase-mediated apoptosis regardless of p53 status. Here we address three main questions: What types of ROS are induced by selenite vs. MSeA in LNCaP (p53 wild type, androgen-responsive) and DU145 (mutant p53, androgen-independent) prostate cancer cells? Does ROS generation depend on androgen signaling? What are the relationships among ROS, DNA SSBs, p53 and caspases? We show that selenite (5 microM) induced superoxide and hydrogen peroxide in LNCaP cells much more than in DU145 cells and the ROS generation was not affected by physiological androgen stimulation. MSeA (10 microM) induced apoptosis without either type of ROS in both cell lines. In LNCaP cells, we established superoxide as a primary mediator for selenite-induced DNA SSBs, p53 activation and caspase-mediated apoptosis. Furthermore a p53-dominant negative mutant attenuated selenite-induced ROS, leading to a proportionate protection against apoptosis. The results support the p53-mitochondria axis in a feedback loop for sustaining superoxide production to lead to efficient caspase-mediated apoptosis by selenite. In contrast, caspase-mediated apoptosis induced by MSeA does not involve ROS induction. Since p53 is frequently mutated or deleted in prostate cancer and many other cancers, our results suggest that genotoxic vs. nongenotoxic classes of selenium may exert differential apoptosis efficacy depending on the p53 status of the cancer cells.

Evaluation of in vitro Genotoxic Potential of Kojic Acid in Human Lymphoblastoid Cells

Although kojic acid is used as a cosmetic agent for skin whitening, information of its genotoxicity in in vitro assay is much complicated. In order to evaluate its genotoxic potentials in vitro, we conducted comet assay in regular and acellular versions, chromosome aberration assay, and TK mutation assay in human lymphoblastoid cells. Positive results were obtained in all of the comet, chromosome aberration, and TK mutation assays at almost identical concentration in both TK6 and WTK1 cells. In the acellular comet assay, kojic acid led to positive responses at pH12 and pH13, suggesting that it induced DNA single strand breaks (SSBs). In the TK mutation assay, kojic acid increased the fraction of normal growing but not slowly growing mutants, suggesting that observed gene mutations are due to point mutations within the TK locus but not gross structural changes that can form chromosome aberrations observable by a microscopy.

DNA Lesion and Hprt Mutant Frequency in Rat Lymphocytes and V79 Chinese Hamster Lung Cells Exposed to Cadmium

Cadmium is a potential carcinogenic environmental and occupational pollutant. A wide variety of mutagens have been shown to cause DNA damage, but it is not yet clear whether the DNA damage is relative to inducement of mutations. DNA damage and the formation of mutations at the hypoxanthine guanine phosphoribosyl trans ferase (HPRT) induced by cadmium chloride (CdCl(2)) were investigated with rat lymphocytes and V79 Chinese hamster lung cells. The hprt mutant frequency (MF) assay was used as the method to measure gene mutation in the rat lymphocytes and V79 cells exposed to CdCl(2), and comet assay analysis was performed to detect DNA lesion and repair in CdCl(2)-induced V79 cells. The results showed that CdCl(2) treatment caused a strong genotoxic effect and a marginal effect on the frequency of gene mutations. The hprt mutant frequencies in the rat lymphocytes and V79 cells exposed to CdCl(2) were statistically higher than those of the negative control. There was statistical significance in TL, TD and percentage of comet cell with tails. CdCl(2) treatment can induce DNA single-strand breaks. There was a dose-dependent increase between CdCl(2) and DNA lesion. After cells were treated with CdCl(2) and hydrogen peroxide (H(2)O(2)), the TL and TD declined with repair time increasing, which indicated that DNA damages were repaired gradually. However, DNA repair with treatment of CdCl(2) was slower than that of H(2)O(2) in V79 cells, which suggests that CdCl(2) affected DNA repair of damaged cells. The study also showed that the hprt MF and comet assay can be used for genotoxicity testing of heavy metals. DNA damage detected with the comet assay may be relative to mutagenesis.

Causes of DNA single-strand breaks during reduction of chromate by glutathione in vitro and in cells

Carcinogenic chromates induce DNA single-strand breaks (SSB) that are detectable by conventional alkali-based assays. However, the extent of direct breakage has been uncertain because excision repair and hydrolysis of Cr–DNA adducts at alkaline pH also generate SSB. We examined mechanisms of SSB production during chromate reduction by glutathione (GSH) and assessed the significance of these lesions in cells using genetic approaches. Cr(VI) reduction was biphasic and the formation of SSB occurred exclusively during the slow reaction phase. Catalase or iron chelators completely blocked DNA breakage, as did the use of GSH purified by a modified Chelex procedure. Thus, the direct intermediates of GSH–chromate reactions were unable to cause SSB unless activated by H 2O 2. SSB repair-deficient XRCC1 −/− and proficient XRCC1 + EM9 cells had identical survival at doses causing up to 60% clonogenic death and accumulation of 1 mM Cr(VI). However, XRCC1 −/− cells displayed higher lethality in the more toxic range and the depletion of GSH made them hypersensitive even to moderate doses. Elevation of cellular catalase or GSH levels eliminated survival differences between XRCC1 −/− and XRCC1 + cells. In summary, formation of toxic SSB in cells occurs at relatively high chromate doses, requires H 2O 2, and is suppressed by high GSH concentrations.

Ros-Induced Histone Modifications and their Role in Cell Survival and Cell Death

Much is known about the distal DNA damage repair response. In particular, many of the enzymes and auxiliary proteins that participate in DNA repair have been characterized. In addition, knowledge of signaling pathways activated in response to DNA damage is increasing. In contrast, comparatively less is known of DNA damage-sensing molecules or of the specific alterations to chromatin structure recognized by such DNA damage sensors. Thus, precisely how chromatin structure is altered in response to DNA damage and how such alterations regulate DNA repair processes remain important unanswered questions. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after double-strand break formation, extends over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci. We have shown that reactive oxygen species (ROS)-induced DNA single-strand breaks induce the incorporation of 32P specifically into histone H3. ADP-Ribosylation of histones may stimulate local chromatin relaxation to facilitate the repair process, and, indeed, histone ribosylation preceded DNA damage-induced histone H3 phosphorylation. However, H3 phosphorylation occurred concomitant with overall chromatin condensation, as revealed by decreased sensitivity of chromatin to digestion by micrococcal nuclease and by DAPI staining of nuclei. Inhibitors of the ERK and p38MAPK pathways and inhibition of poly(ADP-ribose) polymerase all reduced ROS-induced H3 phosphorylation, chromatin condensation, and cell death. Precisely how changes in the post-translational modification of histone H3 regulate the survival response remains unclear. Attempts to determine the precise site of histone H3 phosphorylation, putative histone H3 kinases, and histone H3 interacting proteins are underway.

Genotoxic potential of quinolone antimicrobials in the in vitro comet assay and micronucleus test

The purpose of this study was to examine the genotoxicity of quinolone antimicrobials. We investigated the genotoxic potential of eight quinolones, namely nalidixic acid (NA), pipemidic acid (PPA), oxolinic acid (OA), piromidic acid (PA), enoxacin (ENX), ofloxacin (OFLX), norfloxacin (NFLX) and ciprofloxacin (CPFX), by the in vitro alkaline single-cell gel electrophoresis (comet) assay at pH > 13. WTK-1 cells (mutant p53) were treated with each of the eight quinolones at 62.5–1000 μg/mL for 2, 4 and 20 h. NFLX and CPFX significantly induced DNA damage concentration-dependently after 4 and 20 h treatment, but this damage was recoverable. On the other hand, DNA was not damaged in the cells treated with six other quinolones. In the cells treated with NFLX and CPFX for 20 h, DNA migration was compared by the comet assay at pH 10, 12.1 and >13. The comet assay both at pH 12.1 and >13 showed increased DNA migration, but there was no positive response in the comet assay at pH 10. In the in vitro micronucleus (MN) test, WTK-1 cells were treated with each of four quinolones (NA, PPA, NFLX and CPFX) at 15.63–125 μg/mL for 20 h. NFLX significantly increased MNs in the cells, but no changes were noted in the cells treated with three other quinolones. These results suggest that NFLX and CPFX induced DNA single strand breaks (SSBs), and that NFLX-induced SSBs resulted in chromosome aberrations.

Aloe-emodin induced DNA damage through generation of reactive oxygen species in human lung carcinoma cells

The DNA aggregation was found in aloe-emodin-induced H460 cell apoptosis in this study. Aloe-emodin (40 μM)-induced DNA single strand breaks were observed by comet assay. Aloe-emodin induced decreases in the mRNA of DNA repair enzymes such as hMTH1, hOGG1 and APE. Although the activity of the radical-scavenging enzyme SOD was enhanced by aloe-emodin, the effects of aloe-emodin on H460 cell apoptosis were suspected to result from the prooxidant. These results suggest that aloe-emodin induced DNA damage through generation of reactive oxygen species in human lung carcinoma cells.

Evaluation of Genotoxic Effects in Human Fibroblasts after Intermittent Exposure to 50 Hz Electromagnetic Fields: A Confirmatory Study

The aim of this investigation was to confirm the main results reported in recent studies on the induction of genotoxic effects in human fibroblasts exposed to 50 Hz intermittent (5 min field on/10 min field off) sinusoidal electromagnetic fields. For this purpose, the induction of DNA single-strand breaks was evaluated by applying the alkaline single-cell gel electrophoresis (SCGE)/comet assay. To extend the study and validate the results, in the same experimental conditions, the potential genotoxicity was also tested by exposing the cells to a 50 Hz powerline signal (50 Hz frequency plus its harmonics). The cytokinesis-block micronucleus assay was applied after 24 h intermittent exposure to both sinusoidal and powerline signals to obtain information on cell cycle kinetics. The experiments were carried out on human diploid fibroblasts (ES-1). For each experimental run, exposed and sham-exposed samples were set up; positive controls were also provided by treating cells with hydrogen peroxide or mitomycin C for the comet or micronucleus assay, respectively. No statistically significant difference was detected in exposed compared to sham-exposed samples in any of the experimental conditions tested (P > 0.05). In contrast, the positive controls showed a statistically significant increase in DNA damage in all cases, as expected. Accordingly, our findings do not confirm the results reported previously for either comet induction or an increase in micronucleus frequency.

Nickel compound-induced DNA single-strand breaks in chromosomal and nuclear chromatin in human blood lymphocytes in vitro: Role of oxidative stress and intracellular calcium

The effects of nickel sulfate, and soluble forms of nickel carbonate hydroxide (NiCH), nickel subsulfide, and nickel oxide on delayed induction of DNA single-strand breaks (DNA SSBs) in chromosomal and nuclear chromatin of human blood lymphocytes in culture were studied. After 46 h of initial culture in supplemented RPMI-1640 media at 37 °C, human whole blood lymphocytes in culture were exposed to low concentrations (0–15 μM) of different nickel (Ni) compounds for 2 h, whereas only RPMI-1640 medium served as control. Immediately after 2 h of such exposure, both control and Ni-treated cells were washed with the same medium and incubated further in fresh complete RPMI-1640 culture medium for another 24 h. After a total 70 h of incubation, cells were then arrested at metaphase. Two hours later, the induction of DNA SSBs involving both metaphase chromosomal and interphase nuclear chromatin was measured using the method of electron microscopy in situ end-labeling. The metaphase chromosomal chromatin showed significantly higher DNA SSBs (as measured by an increase in immunogold particles per μm 2 chromatin) due to 15 μM NiCH and NiO when compared to the corresponding control value. Both NiCH and nickel oxide produced significantly higher induction of DNA SSBs than those of nickel subsulfide and nickel sulfate in chromosomal chromatin. The DNA SSBs in chromosomal chromatin were found to be significantly higher than those in nuclear chromatin due to different Ni compounds. Overall, the genotoxic potency seems to be decreased as follows: NiCH > nickel oxide ≥ nickel subsulfide > nickel sulfate. Pretreatment of human blood lymphocytes with either catalase (a H 2O 2 scavenger), or superoxide dismutase (a scavenger of O 2 − radical) or dimethylthiourea (a hydroxyl radical scavenger), or N-acetylcysteine (GSH precursor) significantly reduced DNA SSBs in both chromosomal and nuclear chromatin induced by NiCH, suggesting the involvement of different types of oxidative stress in such genotoxicity. Deferoxamine (a highly specific iron chelator) pretreatment prevented NiCH-induced DNA SSBs in both chromosomal and nuclear chromatin suggesting a role of iron-mediated oxidative stress generating hydroxyl radical in such genotoxicity. Simultaneous treatment with either verapamil (an inhibitor of Ca 2+ through plasma membranes), or dantrolene (an inhibitor of mobilization of [Ca 2+] i from endoplasmic reticulum), or BAPTA (a Ca 2+ chelator) significantly reduced Ni compound-induced DNA SSBs in both chromosomal and nuclear chromatin, suggesting that Ni compound-induced destabilization of calcium homeostasis may also involved in the induction of such DNA SSBs.

Pharmacological Inhibition of Poly(ADP-ribose) Polymerase in Cardiovascular Disorders: Future Directions

Poly(ADP-ribose) polymerase (PARP) activation plays a role in the pathogenesis of various cardiovascular and inflammatory diseases. Reactive oxygen and nitrogen species induce DNA single strand breaks, which serve as obligatory triggers for the activation of PARP. Pharmacological inhibitors of PARP attenuate ischemic and inflammatory cell and organ injury, and this property of the PARP inhibitors can be exploited for the experimental therapy of disease. As several classes of PARP inhibitors move towards clinical development, or have already entered clinical trials, we expect that in the upcoming few years, clinical proof of PARP inhibitors' therapeutic effect will be obtained in human disease. Acute, life-threatening cardiovascular diseases (myocardial infarction, cardiopulmonary bypass in high-risk patients, and other, severe forms of ischemia-reperfusion to other organs including stroke and thoracoabdominal aneurysm repair) represent some of the initial development indications for PARP inhibitors.

A Novel Polypyrimidine Antitumor Agent FdUMP[10] Induces Thymineless Death with Topoisomerase I-DNA Complexes

FdUMP[10], a 10mer of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), the thymidylate synthase inhibitory metabolite of 5-fluorouracil (FU), is most closely correlated with the DNA topoisomerase I (Top1) inhibitor camptothecin in the National Cancer Institute COMPARE analysis, but not with FU. FdUMP[10] exhibits more potent antiproliferative activity than FdUMP or 5-fluoro-2'-deoxyuridine (FdU) and is markedly more active than FU. Camptothecin-resistant P388/CPT45 cells lacking Top1 are cross-resistant to FdUMP[10] as well as to FdUMP, FdU, and the thymidylate synthase inhibitor raltitrexed (Tomudex). FdUMP[10] induces DNA single-strand breaks and cellular Top1-DNA complexes. Such complexes are also observed in response to FdUMP, FdU, raltitrexed, and FU. The FdUMP[10]-induced Top1-DNA complexes are not inhibited by the caspase inhibitor z-VAD-fmk and form independently of apoptotic DNA fragmentation, indicating that they do not correspond to apoptotic Top1-DNA complexes. In biochemical assay, Top1 is directly trapped at uracil and FdU misincorporation sites. We propose that FdUMP[10] damages DNA by trapping Top1 at uracil and FdU misincorporation sites resulting from thymidylate synthase inhibition and thymine depletion.

Alterations of DNA damage-response genes ATM and ATR in pyothorax-associated lymphoma

Pyothorax-associated lymphoma (PAL) is non-Hodgkin's lymphoma that develops from chronic inflammation. Free radicals and oxidative stress generated in the inflammatory lesions could cause DNA damage and thus provide a basis for lymphomagenesis. Ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) genes are responsive genes for DNA damage, therefore potential involvement of these genes in PAL lymphomagenesis was examined in eight PAL cell lines and clinical samples from five cases. ATM mutations were detected in five of eight PAL lines. All but one of these mutations affected the phosphatidylinositol 3-kinase domain, indicating the loss-of-function mutation of ATM gene. Heterozygous mutations of ATR were found in two of eight lines; one a missense and the other a truncation mutation. ATR mutations were also detected in two of five cases in clinical samples from PAL. PAL cells with ATR mutation showed a delay or abrogation in repair for ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) or ultraviolet (UV)-induced DNA single-strand breaks (SSBs), and exhibited a defect in p53 accumulation and failure in cell cycle checkpoint at G1–S phase. These findings showed that mutations of ATR gene result in failure for DNA DSB and SSB repair, suggesting the role of ATM and ATR gene mutations in PAL lymphomagenesis.