Niacin deficiency increases spontaneous and etoposide-induced chromosomal instability in rat bone marrow cells in vivo

Poly(ADP-ribose) polymerase (PARP) binds to DNA single and double strand breaks and uses NAD in the synthesis of poly(ADP-ribose) (pADPr). Niacin deficiency in rats decreases bone marrow NAD(+) and limits pADPr synthesis in response to DNA damage, while pharmacological supplementation with nicotinic acid (NA) increases bone marrow NAD(+) and pADPr. The purpose of this study was to determine if niacin status alters the extent of DNA damage and chromosomal instability before and after treatment with the chemotherapy drug etoposide (ETO). Genotoxicity was evaluated using the comet, micronucleus and sister chromatid exchange (SCE) assays. Male Long-Evans rats were fed niacin deficient (ND), or pair-fed (PF) niacin replete (30mg niacin/kg) or NA supplemented (4g niacin/kg) diets for 3 weeks. Rats were gavaged with ETO (1-25mg/kg) suspended in corn oil or an equal volume of vehicle (CON). Comet analysis demonstrated that ETO-induced DNA damage (mean tail moment (MTM) and proportion of cells with significant damage) was greater in bone marrow cells from ND rats, compared to PF or NA rats. Surprisingly, niacin deficiency alone caused 6.2- and 2.8-fold increases in spontaneous micronucleus formation and SCE frequency, respectively. As expected, ETO treatment increased the level of micronuclei (MN) and SCEs in all diet groups; however, the absolute increases were greater in ND bone marrow. These data show that niacin is required for the maintenance of chromosomal stability and may facilitate DNA repair in vivo, in a tissue that is sensitive to niacin depletion and impaired pADPr metabolism. Pharmacological intakes of niacin do not appear to be further protective compared to adequate intakes. Niacin supplementation may help to protect the bone marrow cells of cancer patients with compromised nutritional status from the side effects of genotoxic chemotherapy drugs.     

Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay – A methodological overview

There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results.     

Comet assay-based methods for measuring DNA repair <Emphasis Type="Italic">in vitro</Emphasis>; estimates of inter- and intra-individual variation

DNA repair is one of the important determinants of susceptibility to cancer. It is therefore useful to be able to measure DNA repair capacity in samples from population studies. Our aim was, first, to develop a simple comet-based in vitro assay for nucleotide excision repair (NER), similar to that already in use for base excision repair (BER), and then to apply these in vitro assays to lymphocyte samples collected on several occasions from healthy subjects, to gain an impression of the degree of intra- and inter-individual variability. The in vitro assay consists of an incubation of lymphocyte extract with substrate nucleoid DNA from cells pretreated with specific damaging agent; either photosensitiser plus light to induce 8-oxoguanine, for BER, or short wavelength ultraviolet light irradiation for NER. In the new NER assay, which requires magnesium but not adenosine triphosphate, there was significant accumulation of UV-dependent incisions during a 30-min incubation of extract with DNA. We found significant correlations between individual repair rates from samples taken on different occasions; i.e. individuals have a characteristic repair capacity. There was also significant variation between individuals, to the extent of about fourfold for BER and tenfold for NER. There was no correlation between BER and NER rates. The BER and NER assays are simple to perform and can provide valuable information in molecular epidemiological studies in which DNA instability is an endpoint.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3′ ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5′ phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5′ extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido–pyrimidine–DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).     

Nucleotide excision repair functions in the removal of chromium-induced DNA damage in mammalian cells

Some hexavalent chromium (Cr(VI))-containing compounds are human lung carcinogens. While ample information is available on the genetic lesions produced by Cr, surprisingly little is known regarding the cellular mechanisms involved in the removal of Cr-DNA adducts. Nucleotide excision repair (NER) is a highly versatile pathway that is responsive to a variety of DNA helix-distorting lesions. Binary Cr-DNA monoadducts do not produce a significant degree of helical distortion. However, these lesions are unstable due to the propensity of Cr(III) to form DNA adducts (DNA interstrand crosslinks, DNA-protein/amino acid ternary adducts) which may serve as substrates for NER. Therefore, the focus of this study was to determine the role of NER in the processing of Cr-DNA damage using normal (CHO-AA8) and NER-deficient [UV-5 (XP-D); UV-41 (ERCC4/XP-F)] hamster cells. We found that both UV-5 and UV-41 cells exhibited an increased sensitivity towards Cr(VI)-induced clonogenic lethality relative to AA8 cells and were completely deficient in the removal of Cr-DNA adducts. In contrast, repair-complemented UV-5 (expressing hamster XPD) and UV-41 (expressing human ERCC4) cells exhibited similar clonogenic survival and removed Cr-DNA adducts to a similar extent as AA8 cells. In order to extend these findings to the molecular level, we examined the ability of Cr(III)-damaged DNA to induce DNA repair synthesis in cell extracts. Repair synthesis was observed in reactions using extracts derived from AA8, or repair-complemented, but not NER-deficient cells. Cr(III)-induced repair resynthesis was sensitive to inhibition by the DNA polymerase δ/ε inhibitor, aphidicolin, but not 2′,3′-dideoxythymidine triphosphate (ddTTP), a polymerase β inhibitor. These results collectively suggest that NER functions in the protection of cells from Cr(VI) lethality and is essential for the removal of Cr(III)-DNA adducts. Consequently, NER may represent an important mechanism for preventing Cr(VI)-induced mutagenesis and neoplastic transformation.     

Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: Comparison with quercetin

Oxidative DNA damage has been described as an important type of damage that occurs in neuronal cells, with severe implications in many neurodegenerative diseases and in aging. We have previously reported the protection of four new synthetic nitrogen compounds (FMA4, FMA7, FMA762 and FMA796) against oxidative stress conditions. In this work, we studied their effects on oxidative DNA damage induced in rat pheochromocytoma (PC12) cells, using the Comet assay, and compared them with a natural antioxidant, quercetin. Among the compounds tested, FMA762 and FMA796 were the most effective in preventing tert-butylhydroperoxide (t-BHP)-induced formation of DNA strand breaks and in improving the cells’ capacity to repair this kind of damage. These effects were similar to the ones of quercetin, a flavonoid with known antioxidant activity. Moreover, contrarily to quercetin, they increased the repair capacity of oxidised bases induced with the photosensitiser Ro 19-8022. This effect seems to be mediated by an increase in DNA repair enzymes activity, assessed by the in vitro BER assay, but no regulation at the expression of OGG1 and APE1 genes was detected. In addition to other properties previously found for the nitrogen compounds, they now prove their effectiveness against oxidative stress-induced DNA damage in the neuronal cell model used.     

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.     

Genotoxic action of Luna Experience-SC 400 fungicide on rat bone marrow

Abstract

Background: Fungicides describe all chemicals used to control fungi that infect plants. Luna Experience SC-400 is a new line of fungicide that consist of Fluopyram and Tebuconazole.

Objective: In this study, We investigated the genotoxicty and cytotoxicty of Luna Experience-SC 400 using comet assay, micronucleus test and polychromatic erythrocytes number in rat bone marrow. The present study is the first report indicating the effects of genotoxic and cytotoxic of Luna experience SC-400 on rat bone marrow cells.

Material and Methods: We used three different doses (5mg/kg, 10mg/kg, 20mg/kg) of Luna Experience SC 400 at 48 h intervals during 30 days by gavage in rats.Genotoxicity was evaluated using comet assay and micronucleus test and cytotoxicity was measured the PCE/NCE rate in rat bone marrow.

Results: Based on these experimental results, we report that Luna Experience-SC 400 fungicide presents genotoxic and cytotoxic potential on rat bone marrow. There is a significant difference between negative control group and all the doses of Luna Experience-SC 400 (p?<?0.05) for comet assay and micronucleus. Even moderate and high doses of fungicides seem to have reached the values of almost positive control group for Genetic Damage Index (GDI) and Damaged Cell Percentage (DCP). In this study, we also investigated the PCE/NCE rate. Fungicide caused a decrease in the level of significant in the PCE/NCE ratio (p?<?0.05).

Conclusion: Our in vivo study suggests that the gavage exposure to Luna experience SC 400 used in the present investigation may be genotoxic and cytotoxic in rat bone marrow in view of these findings. Because this findings is first report represented in the pesticide biology, it is important to carry out more investigations using various cytogenetic tests under different experimental conditions to definitively resolve the the possible genotoxic and cytotoxic risk associated with new generation pesticides-fungicides.     

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.     

DNA damage and repair in Helicobacter pylori-infected gastric mucosa cells

Helicobacter pylori is a common human pathogen and its infection is believed to contribute to gastric cancer. Impaired DNA repair may fuel up cancer transformation by the accumulation of mutation and increased susceptibility to exogenous carcinogens. To evaluate the role of infection of H. pylori in DNA damage and repair we determined: (1) the level of endogenous basal, oxidative and alkylative DNA damage, and (2) the efficacy of removal of DNA damage induced by hydrogen peroxide and the antibiotic amoxicillin in the H. pylori-infected and non-infected GMCs. DNA damage and the efficacy of DNA repair were evaluated by the alkaline single cell gel electrophoresis (comet assay). Specific damage to the DNA bases were assayed with the DNA repair enzymes formamidopyrimidine-DNA glycosylase (Fpg) recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The level of basal and oxidative DNA in the infected GMCs was higher than non-infected cells. H. pylori-infected GMCs displayed enhanced susceptibility to hydrogen peroxide than control cells. There was no difference between the efficacy of DNA repair in the infected and non-infected cells after treatment with hydrogen peroxide and amoxicillin. Our results indicate that H. pylori infection may be correlated with oxidative DNA damage in GMCs. Therefore, these features can be considered as a risk marker for gastric cancer associated with H. pylori infection and the comet assay may be applied to evaluate this marker.     

Effect of Wortmannin on the repair profiles of DNA double-strand breaks in the whole genome and in interstitial telomeric sequences of Chinese hamster cells

The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was applied to analyze the effect of Wortmannin (WM) in the rejoining kinetics of ionizing radiation-induced DNA double-strand breaks (DSBs) in the whole genome and in the long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cell lines. The results indicate that the ITRS blocks from wild-type Chinese hamster cell lines, CHO9 and V79B, exhibit a slower initial rejoining rate of ionizing radiation-induced DSBs than the genome overall. Neither Rad51C nor the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) activities, involved in homologous recombination (HR) and in non-homologous end-joining (NHEJ) pathways of DSB repair respectively, influenced the rejoining kinetics within ITRS in contrast to DNA sequences in the whole genome. Nevertheless, DSB removal rate within ITRS was decreased in the absence of Ku86 activity, though at a lower affectation level than in the whole genome, thus homogenizing both rejoining kinetics rates. WM treatment slowed down the DSB rejoining kinetics rate in ITRS, this effect being more pronounced in the whole genome, resulting in a similar pattern to that of the Ku86 deficient cells. In fact, no WM effect was detected in the Ku86 deficient Chinese hamster cells, so probably WM does not add further impairment in DSB rejoining than that resulted as a consequence of absence of Ku activity. The same slowing effect was also observed after treatment of Rad51C and DNA-PKcs defective hamster cells by WM, suggesting that: (1) there is no potentiation of the HR when the NHEJ is impaired by WM, either in the whole genome or in the ITRS, and (2) that this impairment may probably involve more targets than DNA-PKcs. These results suggest that there is an intragenomic heterogeneity in DSB repair, as well as in the effect of WM on this process.     

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK_cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.     

The use of comet assay in measuring DNA damage and repair efficiency in child,adult, and old age populations

In the present study, we used the Comet assay to estimate basal DNA damage in three distinct populations aged 5–10, 40–50, and 60–70 years old. The DNA damage induced by hydrogen peroxide and γ-irradiation in the lymphocytes of these populations, as well as their repair activity, was also studied. Finally, we measured apoptosis and necrosis after the effect of these agents. Our results indicate that the older population (60–70 years old) showed higher basal levels of DNA damage and was more sensitive to the effects of the DNA-damaging agents than the adult one (40–50 years old), who, in turn, was more sensitive than the younger population (5–10 years old). A decline of the repair efficiency with age to the DNA damage induced by the two agents was also observed. Apoptosis and necrosis were also affected by age.     

High mobility group (HMG) proteins: Modulators of chromatin structure and DNA repair in mammalian cells

It has been almost a decade since the last review appeared comparing and contrasting the influences that the different families of High Mobility Group proteins (HMGA, HMGB and HMGN) have on the various DNA repair pathways in mammalian cells. During that time considerable progress has been made in our understanding of how these non-histone proteins modulate the efficiency of DNA repair by all of the major cellular pathways: nucleotide excision repair, base excision repair, double-stand break repair and mismatch repair. Although there are often similar and over-lapping biological activities shared by all HMG proteins, members of each of the different families appear to have a somewhat ‘individualistic’ impact on various DNA repair pathways. This review will focus on what is currently known about the roles that different HMG proteins play in DNA repair processes and discuss possible future research areas in this rapidly evolving field.     

Comparative study of DNA damage and repair in head and neck cancer after radiation treatment

We compared DNA damage and the efficacy of its repair after genotoxic treatment with γ-radiation of lymphocytes and tissue cells isolated from patients with squamous cell carcinoma of head and neck (HNSCC) and healthy donors. Thirty-seven subjects with HNSCC and 35 healthy donors were enrolled in the study. The extent of DNA damage including oxidative lesions and efficiency of the repair were examined by alkaline comet assay. HNSCC cancer cells were more sensitive to genotoxic treatment and displayed impaired DNA repair. In particular, lesions caused by γ-radiation were repaired less effectively in metastasis of HNSCC than in healthy controls. The differences in radiation sensitivity of cancer and control cells suggested that DNA repair might be critical for HNSCC treatment. We conclude that γ-radiation might be considered as an effective therapeutic strategy for head and neck cancers, including patients in advanced stage of the disease with clear evidence of metastasis.     

Functional aspects of PARylation in induced and programmed DNA repair processes: Preserving genome integrity and modulating physiological events

To cope with the devastating insults constantly inflicted to their genome by intrinsic and extrinsic DNA damaging sources, cells have evolved a sophisticated network of interconnected DNA caretaking mechanisms that will detect, signal and repair the lesions. Among the underlying molecular mechanisms that regulate these events, PARylation catalyzed by Poly(ADP-ribose) polymerases (PARPs), appears as one of the earliest post-translational modification at the site of the lesion that is known to elicit recruitment and regulation of many DNA damage response proteins.     

Werner syndrome (WRN) DNA helicase and base excision repair (BER) factors maintain endothelial homeostasis

The accelerated ageing disease Werner Syndrome (WRN) is characterized by pronounced atherosclerosis. Here, we investigated the influence of WRN downregulation on the functionality of non-replicating human endothelial cells. RNAi-mediated downregulation of WRN reduces cell motility and enhances the expression of factors regulating adhesion, inflammation, hemostasis and vasomotor tone. Moreover, WRN influences endothelial barrier function and Ca²⁺-release, while cell adhesion, Dil-acLDL-uptake and the mRNA expression of NO-synthases (eNOS, iNOS) remained unaffected. Regarding motility, we propose that WRN affects Rac1/FAK/ß1-integrin-related mechanisms regulating cell polarity and directed motility. Since oxidative DNA base damage contributes to aging and atherosclerosis and WRN affects DNA repair, we investigated whether downregulation of base excision repair (BER) factors mimics the effects of WRN knock-down. Indeed, downregulation of particular WRN-interacting base excision repair (BER) proteins (APE1, NEIL1, PARP1) imitates the inhibitory effect of WRN on motility. Knock-down of OGG1, which does not interact with WRN, does not influence motility but increases the mRNA expression of E-selectin, ICAM, VCAM, CCL2 and VEGFR and stimulates adhesion. Thus, individual BER factors themselves differently impact endothelial cell functionality and homeostasis. Impairment of endothelial activities caused by genotoxic stressor (tBHQ) remained largely unaffected by WRN. Summarizing, both WRN, WRN-associated BER proteins and OGG1 promote the maintenance of endothelial cell homeostasis, thereby counteracting the development of ageing-related endothelial malfunction in non-proliferating endothelial cells.