Frequency Of Strand Breaks Research Articles

Role of H4K16 acetylation in 53BP1 recruitment to double-strand break sites in in vitro aged cells

Increased frequency of DNA double strand breaks (DSBs) with aging suggests an age-associated decline in DSB repair efficiency, which is also influenced by the epigenetic landscape. H4 acetylation at lysine 16 (H4K16Ac) has been related to DSB repair since deacetylation of this mark is required for efficient 53BP1 recruitment to DSBs. Although age-associated changes in H4K16Ac levels have been studied, their contribution to age-related DSB accumulation remains unknown. In vitro aged Human Dermal Fibroblasts (HDFs) display lower levels of H4K16A that correlate with reduced recruitment of 53BP1 to basal DSBs. Following DNA damage induction, early passage (EP) cells suffered from a transient H4K16 deacetylation that allowed proper 53BP1 recruitment to DSBs. In contrast, to reach this specific and optimum level, aged cells responded by increasing their overall lower H4K16Ac levels. Induced hyperacetylation of late passage (LP) cells using trichostatin A increased H4K16Ac levels but did not ameliorate 53BP1 recruitment. Instead, deacetylation induced by MOF silencing reduced H4K16Ac levels and compromised 53BP1 recruitment in both EP and LP cells. Age-associated decrease of H4K16Ac levels contributes to the repair defect displayed by in vitro aged cells. H4K16Ac responds to DNA damage in order to reach a specific, optimum level that allows proper 53BP1 recruitment. This response may be compromised with age, as LP cells depart from lower H4K16Ac levels. Variations in H4K16Ac following the activation of the DNA damage response and aging point at this histone mark as a key mediator between DNA repair and age-associated chromatin alterations.

Precision digital mapping of endogenous and induced genomic DNA breaks by INDUCE-seq

Understanding how breaks form and are repaired in the genome depends on the accurate measurement of the frequency and position of DNA double strand breaks (DSBs). This is crucial for identification of a chemical’s DNA damage potential and for safe development of therapies, including genome editing technologies. Current DSB sequencing methods suffer from high background levels, the inability to accurately measure low frequency endogenous breaks and high sequencing costs. Here we describe INDUCE-seq, which overcomes these problems, detecting simultaneously the presence of low-level endogenous DSBs caused by physiological processes, and higher-level recurrent breaks induced by restriction enzymes or CRISPR-Cas nucleases. INDUCE-seq exploits an innovative NGS flow cell enrichment method, permitting the digital detection of breaks. It can therefore be used to determine the mechanism of DSB repair and to facilitate safe development of therapeutic genome editing. We further discuss how the method can be adapted to detect other genomic features.

Genotoxic effects of electromagnetic field radiations from mobile phones

The use of wireless communication technology in mobile phones has revolutionized modern telecommunication and mobile phones have become so popular that their number exceeds the global population. Electromagnetic field radiations (EMR) are an integral part of wireless technology, which are emitted by mobile phones, mobile tower antennas, electric power stations, transmission lines, radars, microwave ovens, television sets, refrigerators, diagnostic, therapeutic, and other electronic devices. Manmade EMR sources have added to the existing burden of natural EMR human exposure arising from the Sun, cosmos, atmospheric discharges, and thunder storms. EMR including radiofrequency waves (RF) and extremely low-frequency radiation (ELF) has generated great interest as their short-term exposure causes headache, fatigue, tinnitus, concentration problems, depression, memory loss, skin irritation, sleep disorders, nausea, cardiovascular effects, chest pain, immunity, and hormonal disorders in humans, whereas long-term exposure to EMR leads to the development of cancer. The review has been written by collecting the information using various search engines including google scholar, PubMed, SciFinder, Science direct, EMF-portal, saferemr, and other websites from the internet. The main focus of this review is to delineate the mutagenic and genotoxic effects of EMR in humans and mammals. Numerous investigations revealed that exposure in the range of 0–300 GHz EMR is harmless as it did not increase micronuclei and chromosome aberrations. On the contrary, several other studies have demonstrated that exposure to EMR is genotoxic and mutagenic as it increases the frequency of micronuclei, chromosome aberrations, DNA adducts, DNA single and double strand breaks at the molecular level in vitro and in vivo. The EMR exposure induces reactive oxygen species and changes the fidelity of genes involved in signal transduction, cytoskeleton formation, and cellular metabolism.

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra

Dual-energy CT provides enhanced diagnostic power with similar or even reduced radiation dose as compared to single-energy CT. Its principle is based on the distinct physical properties of low and high energetic photons, which, however, may also affect the biological effectiveness and hence the extent of CT-induced cellular damage. Therefore, a comparative analysis of biological effectiveness of dual- and single-energy CT scans with focus on early gene regulation and frequency of radiation-induced DNA double strand breaks (DSBs) was performed. Blood samples from three healthy individuals were irradiated ex vivo with single-energy (80 kV and 150 kV) and dual-energy tube voltages (80 kV/Sn150kV) employing a modern dual source CT scanner resulting in Volume Computed Tomography Dose Index (CTDIvol) of 15.79–18.26 mGy and dose length product (DLP) of 606.7–613.8 mGy*cm. Non-irradiated samples served as a control. Differential gene expression in peripheral blood mononuclear cells was analyzed 6 h after irradiation using whole transcriptome sequencing. DSB frequency was studied by 53BP1 + γH2AX co-immunostaining and microscopic evaluation of their focal accumulation at DSBs. Neither the analysis of gene expression nor DSB frequency provided any evidence for significantly increased biological effectiveness of dual-energy CT in comparison to samples irradiated with particular single-energy CT spectra. Relative to control, irradiated samples were characterized by a significantly higher rate of DSBs (p < 0.001) and the shared upregulation of five genes, AEN, BAX, DDB2, FDXR and EDA2R, which have already been suggested as radiation-induced biomarkers in previous studies. Despite steadily decreasing doses, CT diagnostics remain a genotoxic stressor with impact on gene regulation and DNA integrity. However, no evidence was found that varying X-ray spectra of CT impact the extent of cellular damage.

Twist1 induces chromosomal instability (CIN) in colorectal cancer cells.

Twist1 is a basic helix-loop-helix transcription factor, essential during early development in mammals. While Twist1 induces epithelial-to-mesenchymal transition (EMT), here we show that Twist1 overexpression enhances nuclear and mitotic aberrations. This is accompanied by an increase in whole chromosomal copy number gains and losses, underscoring the role of Twist1 in inducing chromosomal instability (CIN) in colorectal cancer cells. Array comparative genomic hybridization (array CGH) analysis further shows sub-chromosomal deletions, consistent with an increased frequency of DNA double strand breaks (DSBs). Remarkably, Twist1 overexpression downmodulates key cell cycle checkpoint factors—Bub1, BubR1, Mad1 and Mad2—that regulate CIN. Mathematical simulations using the RACIPE tool show a negative correlation of Twist1 with E-cadherin and BubR1. Data analyses of gene expression profiles of patient samples from The Cancer Genome Atlas (TCGA) reveal a positive correlation between Twist1 and mesenchymal genes across cancers, whereas the correlation of TWIST1 with CIN and DSB genes is cancer subtype-specific. Taken together, these studies highlight the mechanistic involvement of Twist1 in the deregulation of factors that maintain genome stability during EMT in colorectal cancer cells. Twist1 overexpression enhances genome instability in the context of EMT that further contributes to cellular heterogeneity. In addition, these studies imply that Twist1 downmodulates nuclear lamins that further alter spatiotemporal organization of the cancer genome and epigenome. Notwithstanding their genetic background, colorectal cancer cells nevertheless maintain their overall ploidy, while the downstream effects of Twist1 enhance CIN and DNA damage enriching for sub-populations of aggressive cancer cells.

Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD

BackgroundThe C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated.Methods and resultsUsing DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls.ConclusionsCollectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

A common genomic code for chromatin architecture and recombination landscape.

Recent findings established a link between DNA sequence composition and interphase chromatin architecture and explained the evolutionary conservation of TADs (Topologically Associated Domains) and LADs (Lamina Associated Domains) in mammals. This prompted us to analyse conformation capture and recombination rate data to study the relationship between chromatin architecture and recombination landscape of human and mouse genomes. The results reveal that: (1) low recombination domains and blocks of elevated linkage disequilibrium tend to coincide with TADs and isochores, indicating co-evolving regulatory elements and genes in insulated neighbourhoods; (2) double strand break (DSB) and recombination frequencies increase in the short loops of GC-rich TADs, whereas recombination cold spots are typical of LADs and (3) the binding and loading of proteins, which are critical for DSB and meiotic recombination (SPO11, DMC1, H3K4me3 and PRMD9) are higher in GC-rich TADs. One explanation for these observations is that the occurrence of DSB and recombination in meiotic cells are associated with compositional and epigenetic features (genomic code) that influence DNA stiffness/flexibility and appear to be similar to those guiding the chromatin architecture in the interphase nucleus of pre-leptotene cells.

Genotoxic and teratogenic effect of freshwater sediment samples from the Rhine and Elbe River (Germany) in zebrafish embryo using a multi-endpoint testing strategy.

The embryotoxic potential of three model sediment samples with a distinct and well-characterized pollutant burden from the main German river basins Rhine and Elbe was investigated. The Fish Embryo Contact Test (FECT) in zebrafish (Danio rerio) was applied and submitted to further development to allow for a comprehensive risk assessment of such complex environmental samples. As particulate pollutants are constructive constituents of sediments, they underlay episodic source-sink dynamics, becoming available to benthic organisms. As bioavailability of xenobiotics is a crucial factor for ecotoxicological hazard, we focused on the direct particle-exposure pathway, evaluating throughput-capable endpoints and considering toxicokinetics. Fish embryo and larvae were exposed toward reconstituted (freeze-dried) sediment samples on a microcosm-scale experimental approach. A range of different developmental embryonic stages were considered to gain knowledge of potential correlations with metabolic competence during the early embryogenesis. Morphological, physiological, and molecular endpoints were investigated to elucidate induced adverse effects, placing particular emphasis on genomic instability, assessed by the in vivo comet assay. Flow cytometry was used to investigate the extent of induced cell death, since cytotoxicity can lead to confounding effects. The implementation of relative toxicity indices further provides inter-comparability between samples and related studies. All of the investigated sediments represent a significant ecotoxicological hazard by disrupting embryogenesis in zebrafish. Beside the induction of acute toxicity, morphological and physiological embryotoxic effects could be identified in a concentration-response manner. Increased DNA strand break frequency was detected after sediment contact in characteristic non-monotonic dose-response behavior due to overlapping cytotoxic effects. The embryonic zebrafish toxicity model along with the in vivo comet assay and molecular biomarker analysis should prospectively be considered to assess the ecotoxicological potential of sediments allowing for a comprehensive hazard ranking. In order to elucidate mode of action, novel techniques such as flow cytometry have been adopted and proved to be valuable tools for advanced risk assessment and management.

DNA-PKcs Inhibition Sensitizes Cancer Cells to Carbon-Ion Irradiation via Telomere Capping Disruption

Heavy-ion irradiation induces a higher frequency of DNA double strand breaks (DSBs) which must be properly repaired. Critical shortening of telomeres can trigger DNA damage responses such as DSBs. Telomeres are very sensitive to oxidative stress such as ionizing radiation. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the central component in the non-homologous end joining (NHEJ) repair complex and participates in telomere maintenance. Therefore, it is expected to enhance the cell killing effect of heavy-ion irradiation via DNA-PKcs inhibition. To test this hypothesis, cellular radiosensitivity was measured by the clonal genetic assay. DNA damage repair was relatively quantified by long PCR. Apoptosis was quantified by flow-cytometric analysis of annexin V/PI double staining, and senescence was analyzed by galactosidase activity. Telomere length was semi-quantified by real-time PCR. P53 and p21 expression was determined by western blotting. Our data demonstrated that MCF-7 and HeLa cells with DNA-PKcs inhibition were more susceptible to carbon-ion irradiation than Those without DNA-PKcs inhibition. Even though NHEJ was inhibited by the DNA-PKcs specific inhibitor, NU7026, most DNA damage induced by carbon-ion irradiation was repaired within 24 hours after irradiation in both cell lines. However, potential lethal damage repair (PLDR) could not restore cellular inactivation in DNA-PKcs inhibited cells. MCF-7 cells showed extensive senescence and accelerated telomere length reduction, while HeLa cells underwent significant apoptosis after irradiation with NU7026 incubation. In addition, both cell lines with shorter telomere were more susceptible to carbon-ion radiation. Our current data suggested that DNA-PKcs inhibition could enhance cellular sensitivity to carbon-ion radiation via disturbing its functional role in telomere end protection. The combination of DNA-PKcs inhibition and carbon-ion irradiation may be an efficient method of heavy-ion therapy.

Effect of Gum Arabic on Oxidative Stress and Inflammation in Adenine–Induced Chronic Renal Failure in Rats

Inflammation and oxidative stress are known to be involved in the pathogenesis of chronic kidney disease in humans, and in chronic renal failure (CRF) in rats. The aim of this work was to study the role of inflammation and oxidative stress in adenine-induced CRF and the effect thereon of the purported nephroprotective agent gum arabic (GA). Rats were divided into four groups and treated for 4 weeks as follows: control, adenine in feed (0.75%, w/w), GA in drinking water (15%, w/v) and adenine+GA, as before. Urine, blood and kidneys were collected from the rats at the end of the treatment for analysis of conventional renal function tests (plasma creatinine and urea concentration). In addition, the concentrations of the pro-inflammatory cytokine TNF-α and the oxidative stress markers glutathione and superoxide dismutase, renal apoptosis, superoxide formation and DNA double strand break frequency, detected by immunohistochemistry for γ-H2AX, were measured. Adenine significantly increased the concentrations of urea and creatinine in plasma, significantly decreased the creatinine clearance and induced significant increases in the concentration of the measured inflammatory mediators. Further, it caused oxidative stress and DNA damage. Treatment with GA significantly ameliorated these actions. The mechanism of the reported salutary effect of GA in adenine-induced CRF is associated with mitigation of the adenine-induced inflammation and generation of free radicals.

Internal exposure to carcinogenic polycyclic aromatic hydrocarbons and DNA damage

In the August issue of the Archives, Kafferlein et al. report no association between occupational exposures to polycyclic aromatic hydrocarbons (PAHs) and deoxyribo nucleic acid (DNA) damage on the individual level (Kafferlein et al. 2012). Internal PAH exposure, assessed in a variety of occupations by means of urinary 3-hydroxy benzo[a]pyrene (3-OH-B[a]P) and 1and 2-naphtol ( P OHNaph), was unrelated with DNA strand break frequencies and the levels of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo), perhaps with the exception of a small group of converter workers where an uncertain correlation between P OHNaph and 8-oxo-dGuo was observed. Authors discuss reasons for this lack of association suggesting to focus on the individual exposure circumstances. These include possible different routes of exposure and co-exposures and a better understanding and assessment of the kinetics and dynamics of the endpoints. We report here the lack of correlations on the individual level in a group of coke oven non-smoker workers between other endpoints of exposure such as urinary 1-hydroxypyrene (1-OHP) and lymphocyte anti-benzo[a]pyrene diolepoxide DNA adduct (anti-B[a]PDE-DNA) and of genotoxic effect such as lymphocyte micronuclei (MN) frequency. Similar lack of correlations observed when using rather different endpoints call for a discussion on possible common explanations. Whereas co-exposures can be thoroughly investigated, we suspect that the kinetics and dynamics of both exposure and effect endpoints for PAHs are far to be understood. Meanwhile, we propose here that both the results of Kafferlein et al. 2012 and ours could be largely explained by the variable routes of exposure occurring among and within occupational settings.

Mechanisms and impacts of chromosomal translocations in cancers

Chromosomal aberrations have been associated with cancer development since their discovery more than a hundred years ago. Chromosomal translocations, a type of particular structural changes involving heterologous chromosomes, have made a critical impact on diagnosis, prognosis and treatment of cancers. For example, the discovery of translocation between chromosomes 9 and 22 and the subsequent success of targeting the fusion product BCR-ABL transformed the therapy for chronic myelogenous leukemia. In the past few decades, tremendous progress has been achieved towards elucidating the mechanism causing chromosomal translocations. This review focuses on the basic mechanisms underlying the generation of chromosomal translocations. In particular, the contribution of frequency of DNA double strand breaks and spatial proximity of translocating loci is discussed.

Internal exposure to carcinogenic polycyclic aromatic hydrocarbons and DNA damage: a null result in brief

We previously reported DNA strand break frequencies and adduct levels of 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxo-dGuo) in 171 blood samples of coke oven, refractory, graphite electrode, and converter workers exposed to polycyclic aromatic hydrocarbons (PAH). Construction workers (n = 48) were used as controls (Marczynski et al. 2009). Internal dose was assessed by the determination of 1-hydroxypyrene (1-OHP) and the sum of five hydroxyphenanthrenes ( P OHPhe) in post-shift urine samples. Our results clearly indicated increased DNA damage in exposed workers on a group level. The results of the dose–response associations on the individual level, however, did not reveal quantitative correlations between measured exposures and effects. We have discussed several potential reasons for the observed results. In brief, internal exposure and effect was based on single time points (post-shift samples), and kinetics for the formation of 8-oxo-dGuo and DNA strand breaks after exposure to chemical carcinogens such as PAH are not known in detail in humans. The individual exposure circumstances such as varying routes of exposures (dermal vs. inhalation) and potential co-exposures to dust or other chemical compounds at the workplace may have contributed to the dose–response association rather than exposure to PAH alone (Courter et al. 2007). Although PAH can undergo redox cycling and are capable of inducing oxidative DNA damage (Park et al. 2008), DNA strand breaks and 8-oxo-dGuo in blood are general biomarkers of oxidative stress, thus being pathwayspecific rather than substance-specific. Finally, PAH are a complex mixture consisting of carcinogenic and non-carcinogenic compounds and exist in varying profiles in different industries and the environment (Rehwagen et al. 2005; Unwin et al. 2006). 1-OHP and P OHPhe are metabolites of pyrene and phenanthrene, thus representing noncarcinogenic PAH rather than carcinogenic PAH. In order to narrow down the reasons for the lacking association between exposures to PAH and DNA damage on the individual level, we re-analyzed the role of carcinogenic PAH. For this purpose, we focused on benzo[a]pyrene (B[a]P) and naphthalene. B[a]P is a complete carcinogen and often used as a positive control in bioassays when studying genotoxicity and carcinogenicity of chemical compounds (IARC 2010). In addition, B[a]P has been shown to have the highest enzyme inducing efficiency in the lung and liver of rodents, thus self-enhancing the formation of reactive electrophilic and carcinogenic intermediates during its metabolism (Elovaara et al. 2007; Skupinska et al. 2007). Naphthalene has been shown to be carcinogenic in rodents at high concentrations after inhalation (NTP 2000). Available biomarkers to assess H. U. Kafferlein (&) B. Marczynski J. Angerer H.-P. Rihs B. Pesch T. Bruning Institut fur Pravention und Arbeitsmedizin der Deutschen Gesetzlichen Unfallversicherung, Institut der Ruhr Universitat Bochum (IPA), Burkle-de-la-Camp Platz 1, 44789 Bochum, Germany e-mail: kaefferlein@ipa-dguv.de

Effect of gum Arabic on oxidative stress and inflammation in adenine–induced chronic renal failure in rats

Inflammation and oxidative stress are known to be involved in the pathogenesis of chronic kidney disease in humans, and in surgically‐induced chronic renal failure (CRF) in rats. Evidence for the involvement of these processes in adenine‐induced CRF in rats is lacking. The aim of this work was to study the role of inflammation and oxidative stress in adenine‐induced CRF, and the effect thereon of the purported nephroprotective agent gum arabic (GA). Rats were divided into four groups and treated for 4 weeks as follows: control, adenine in the feed (0.75%, w/w), GA in drinking water (15%,w/v) and adenine + GA, as before. Urine, blood and kidneys were collected from the rats at the end of the treatment for analysis of conventional renal function tests (plasma creatinine and urea concentration and urinary NAG activity). In addition, the concentrations of several pro‐inflammatory cytokines and indices of oxidative stress, renal apoptosis, superoxide formation and DNA double strand break frequency detected with immunohistochemistry for γ‐H2AX were also conducted in the kidney tissue, plasma and urine. Adenine (0.75% in the feed for 4 weeks) caused significant increases (P &lt; 0.001) in the concentrations of urea and creatinine in plasma, significantly decreased the creatinine clearance (P &lt; 0.01) and induced significant increases in the concentration of the measured inflammatory mediators. Treatment with GA significantly ameliorated these actions, suggesting that the mechanism of the reported salutary effect of GA in adenine‐induced CRF is associated with mitigation of the adenine‐induced inflammation and generation of free radicals.

Defective nonhomologous end joining blocks B-cell development in FLT3/ITD mice

AbstractWe have generated an FLT3/ITD knock-in mouse model in which mice with an FLT3/ITD mutation develop myeloproliferative disease (MPD) and a block in early B-lymphocyte development. To elucidate the role of FLT3/ITD signaling in B-cell development, we studied VDJ recombination in the pro-B cells of FLT3/ITD mice and discovered an increased frequency of DNA double strand breaks (DSBs) introduced by the VDJ recombinase. Early pro-B cells from FLT3/ITD mice were found to have a lower efficiency and decreased accuracy of DSB repair by nonhomologous end joining (NHEJ), which is required for rejoining DSBs during VDJ recombination. Reduced NHEJ repair probably results from reduced expression of Ku86, a key component of the classic DNA-PK-dependent NHEJ pathway. In compensation, early pro-B cells from FLT3/ITD cells mice show increased levels of the alternative, and highly error-prone, NHEJ pathway protein PARP1, explaining the increase in repair errors. These data suggest that, in early pro-B cells from FLT3/ITD mice, impairment of classic NHEJ decreases the ability of cells to complete postcleavage DSB ligation, resulting in failure to complete VDJ recombination and subsequent block of B-lymphocyte maturation. These findings might explain the poor prognosis of leukemia patients with constitutive activation of FLT3 signaling.

Involvement of p21Waf1/Cip1 cleavage during roscovitine-induced apoptosis in non-small cell lung cancer cells

Roscovitine, a cyclin-dependent kinases (CDKs) inhibitor, has been reported to have anti-tumor effects in some cancer cell lines by inducing apoptosis. However, the exact underlying mechanisms are not fully understood. Here, we report that roscovitine induces expression and cleavage of the universal CDK inhibitor p21Waf1/Cip1 in non-small cell lung cancer (NSCLC) A549 cells in a dose-dependent manner. Western blots of roscovitine-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band that was not detected in control cultures. CDK2 activity and PCNA expression were repressed with increasing dose of roscovitine. Accompanying these molecular changes was a progressive arrest of G2 phase and decreasing of 5-bromo-2-deoxyuridine (Brdu) incorporation of S phase cells. Caspase-3 inhibitor z-DEVD-fmk almost completely abolished roscovitine-induced apoptosis, as well as the appearance of 15 kDa band, indicating that p21Waf1/Cip1 cleavage was mediated by caspase-3 activity. Furthermore, this band was predominant in the floating apoptotic cells, while weakened in the adherent cells which were vital and pre-apoptotic. We also showed that roscovitine induced an enhanced expression of gamma-H2AX, which was blocked by caspase-3 inhibition, suggesting that p21Waf1/Cip1 cleavage may interfere with DNA repair, leading to increased frequency of double strand breaks (DSBs) and enhanced apoptosis. Here we show, for the first time, that p21Waf1/Cip1 cleavage, which is mediated by caspase-3 activity, is involved in roscovitine-induced apoptosis.

Assessment of DNA damage and plasma catalase activity in healthy term hyperbilirubinemic infants receiving phototherapy

Phototherapy (PT) is the most widely used form of treatment for unconjugated hyperbilirubinemia. One possible harmful consequence of PT is of a genetic nature. High levels of bilirubin may lead to oxidative damage in newborns: photochemical reactions may produce toxic photoproducts, probably peroxides. In order to investigate this hypothesis further under in vivo conditions, DNA strand-break frequency was examined by means of the comet assay in peripheral lymphocytes of icteric newborns undergoing PT treatment, and the levels of catalase, an antioxidant enzyme, were determined. We analyzed 20 term non-hemolitic hyperbilirubinemic jaundiced neonates before PT (‘before PT’ group) and just prior to ending PT (‘after PT’ group) and compared comet scores of these patients with those of 20 healthy term neonates who all had bilirubin levels in the physiological range. Comet scores (tail length, tail moment and %DNA in tail) of the group ‘before phototherapy’ were 23.5 ± 16.3, 7.41 (0.97–40.7), 33.0 ± 12.1, respectively and scores of after phototherapy group were 3.2 ± 1.8, 0.29 (0.3–3.2), 10.7 ± 3.7, respectively. Comet scores of the control group were 3.0 ± 2.9, 0.25 (0.03–3.22), 10.9 ± 4.5, respectively. Comet scores and plasma catalase activities in hyperbilirubinemic newborns were significantly higher before phototherapy, compared with the values after phototherapy and in the control groups ( p < 0.001). There was no statistical difference between the ‘after phototherapy’ group and the controls ( p > 0.05). These results indicate that high serum bilirubin level has genotoxic effects as is evident from the high rate of DNA strand-breaks in jaundiced newborns. Also PT does not cause an increase in DNA oxidation or induce the genotoxic effects of bilirubin. The counteracting effect of higher catalase activities in hyperbilirubinemic newborns may be responsible for the inactivating toxic and DNA-damaging effects of PT.

Riboflavin‐mediated cellular photoinhibition of cisplatin‐induced oxidative DNA breakage in mice epidermal keratinocytes

Cisplatin is one of the most effective chemotherapeutic agents used in the treatment of various kinds of malignant tumors. A major drawback associated with cisplatin chemotherapy is its cytotoxicity/genotoxicity towards normal tissues. The cytotoxicity has been attributed, in part, to the oxidative stress generated by the drug. Riboflavin is a micronutrient known for its photosensitization characteristics. Owing to the emergence of photodynamic therapy as a modality for the treatment of solid tumors and other accessible lesions in terms of opthalmic, dermatological, cardiovascular and urological diseases, the therapeutic window of riboflavin as a sensitizer has been used to avoid the toxic side effects of cisplatin. Using mice epidermal keratinocytes and comet assay, we show that photochemically activated riboflavin is able to prevent oxidative cellular DNA breakage induced by cisplatin. Irradiation of cells exposed to cisplatin resulted in a dose-dependent increase in the frequency of strand breaks in cellular DNA. Sequential incubation of keratinocytes with riboflavin and cisplatin followed by irradiation led to a significant decrease in strand breakage and reduced the generation of reactive oxygen species. Our results suggest that cisplatin-induced genotoxicity may be blunted or reduced by using riboflavin as a photosensitizer.

Phosphorylation of Sp1 in Response to DNA Damage by Ataxia Telangiectasia-Mutated Kinase

Sp1, a transcription factor that regulates expression of a wide array of essential genes, contains two SQ/TQ cluster domains, which are characteristic of ATM kinase substrates. ATM substrates are transducers and effectors of the DNA damage response, which involves sensing damage, checkpoint activation, DNA repair, and/or apoptosis. A role for Sp1 in the DNA damage response is supported by our findings: Activation of ATM induces Sp1 phosphorylation with kinetics similar to H2AX; inhibition of ATM activity blocks Sp1 phosphorylation; depletion of Sp1 sensitizes cells to DNA damage and increases the frequency of double strand breaks. We have identified serine 101 as a critical site phosphorylated by ATM; Sp1 with serine 101 mutated to alanine (S101A) is not significantly phosphorylated in response to damage and cannot restore increased sensitivity to DNA damage of cells depleted of Sp1. Together, these data show that Sp1 is a novel ATM substrate that plays a role in the cellular response to DNA damage.

Ambient and Biological Monitoring of Exposure and Genotoxic Effects in Mastic Asphalt Workers Exposed to Fumes of Bitumen

Mastic asphalt workers may be exposed to polycyclic aromatic hydrocarbons (PAH) present in bitumen. We conducted a cross-shift study to determine genotoxic effects after exposure to bitumen. For this purpose, external and internal exposure of 202 mastic asphalt workers exposed to bitumen and 55 construction workers without exposure to bitumen was assessed. Exposure by inhalation to fumes of bitumen during the shift was measured by personal ambient monitoring. To assess overall exposure to bitumen (by inhalation and dermal absorption), 1-hydroxypyrene (1-OHP) and the sum of 1-, 2 + 9-,3-,4-hydroxyphenanthrene (OHPhe) were determined in pre- and postshift urine. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) adducts, anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) DNA adducts, DNA strand breaks and alkali-labile sites, and micronucleus frequencies were determined as biomarkers of genotoxic effects in white blood cells (WBC). Concentrations of fumes of bitumen were correlated with a moderate association with 1-OHP and OHPhe after work shift (r s = 0.25, P < 0.001 and r s = 0.36, P < 0.001). Significantly increased 8-oxodGuo adduct levels were observed after shift in both exposure groups (P < 0.0001). Paradoxically, decreased DNA strand break frequencies were observed after shift in both groups (P < 0.05). Postshift values in DNA strand break frequency were associated with 1-OHP (Spearman rank correlation coefficient 0.19, P = 0.01). Significantly more 8-oxodGuo adducts and DNA strand breaks were found in workers exposed to bitumen before and after shift compared with the reference group. However, no dose-dependent association was observed between exposure and genotoxic effects. Nevertheless, the findings indicate that workers exposed to bitumen exerted a higher level of DNA damage (8-oxodGuo and DNA strand breaks) in WBC compared with reference subjects.