DNA damage responses after exposure to DNA-based products

BACKGROUND: The development of DNA-based therapies holds great promise for the treatment of diseases that remain difficult to manage using conventional pharmaceuticals. Whilst there are considerable data regarding chemical-induced DNA damage, there are limited reports published studying the potential of exogenous DNA to damage genomic DNA. METHODS: To investigate this problem, the differential gene expression (DGE) of DNA repair genes was examined to identify biomarkers, based on the hypothesis that DNA damage, including double-strand breaks (DSBs) and insertional mutagenesis, would be expected to induce biological pathways associated with repair. Human HepG2 cells were exposed to the chemical genotoxins, etoposide (ETOP) and methylmethanesulphonate (MMS), as positive controls, or biological agents (i.e. exogenous DNA with and without the use of transfection complexes or via various viral vectors). Following transfection (6-72 h) the cells were harvested for RNA and DGE was determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The expression of genes involved in the repair of DSBs were significantly increased after treatment with ETOP (>4-fold) or MMS (>5-fold). Transfection using Effectene and ExGen 500 resulted in no significant changes; however, transfection with ExGen 500 resulted in an increase in the expression levels of GADD45 mRNA, consistent with global cellular stress. Viral vectors increased (3-6-fold) expression of genes associated with DSBs and cellular stress responses and, as expected, the effect was the most marked with the retroviral vector. CONCLUSIONS: The DGE profiles observed in HepG2 cells following transduction/transfection suggest that a subset of DNA repair genes may provide novel biomarkers to rapidly detect DNA damage induced by DNA products at the level of the genome, rather than at selected genes.     

DNA damage in thymocytes of mice under combined acute whole body exposure to cadmium ions and gamma-radiation

The effect of the combined acute whole body exposure to cadmium chloride (0.5 mg Cd2+ per kg body weight of animals) and gamma-radiation (1 Gy) on the DNA damage induction in thymocytes and thymic cellularity of mice was studied. It has been shown that CdCl2 solution injection 0.5 h before irradiation reduces the quantity of single-strand DNA breaks and alkali-labile sites in thymocytes 48 h after injection compared to gamma-radiation action only. The observed effect is accompanied by a sharp decrease of the thymic cellularity compared with the separate effects of both cadmium ions and irradiation, which masks the overall genotoxic effect of combined exposure and gives an illusion of cadmiumL ions radioprotective action. Cadmium chloride injection 24 h before irradiation leads to a significant additive increase in the single-strand DNA breaks and alkali-labile sites number as compared to the separate effects of cadmium ions and irradiation alone. At the same time the decrease in the percentage of DNA tightly bound to proteins (DNA-protein cross-links) was noted in comparison with the action of gamma-radiation only. Statistically significant changes in thymic cellularity compared with separate effects of cadmium ions and irradiation were not found. Thus, our research has shown that under a combined action of cadmium ions and gamma-radiation on thymocytes in mice at the applied doses and exposure schemes the additive effects, rather than antagonism or radioprotective effects are observed.     

Evaluating DNA damage in rodent brain after acute 60 Hz magnetic-field exposure

In recent years, numerous studies have reported a weak association between 60 Hz magnetic-field exposure and the incidence of certain cancers. To date, no mechanism to explain these findings has been identified. The objective of the current study was to investigate whether acute magnetic-field exposure could elicit DNA damage within brain cells from both whole brain and cerebellar homogenates from adult rats, adult mice and immature mice. Rodents were exposed to a 60 Hz magnetic field (0, 0.1, 1 or 2 mT) for 2 h. Then, at 0, 2 and 4 h after exposure, animals were killed humanely, their brains were rapidly removed and homogenized, and cells were cast into agarose gels for processing by the alkaline comet assay. Four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. For each species, a significant increase in DNA damage was detected by each of the four parameters in the positive control (2 Gy X rays) relative to the concurrent nonirradiated negative and sham controls. However, none of the four parameters detected a significant increase in DNA damage in brain cell homogenates from any magnetic-field exposure (0- 2 mT) at any time after exposure. The dose-response and time-course data from the multiple animal groups tested in this study provide no evidence of magnetic-field-induced DNA damage.     

Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication

The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20 degrees C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants.     

QTL analysis of horticultural traits differentiating the cultivated tomato from the closely related species Lycopersicon pimpinellifolium

Molecular markers were used to map and characterize quantitative trait loci (QTLs) for several characters of agronomic and biological importance in an interspecific backcross of tomato. The parents of the cross were an elite processing inbred Lycopersicon esculentum cv M82-1-7 and the closely related red-fruited wild species L. pimpinellifolium (LA1589). A total of 257 BC₁ plants were grown under field conditions in Ithaca, New York and scored for 19 quantitative traits. A genetic linkage map was constructed for the same population using 115 RFLP, 3 RAPD and 2 morphological markers that spanned 1,279 cM of the tomato genome with an average interval length of 10.7 cM. A minimum of 54 putatively significant QTLs (P<0.001; LOD> 2.4) were detected for all characters with a range of 1–7 QTLs detected per character. Of the total 54 QTLs 11% had alleles with effects opposite to those predicted by the parental phenotypes. The percentage of phenotypic variation associated with single QTLs ranged from 4% to 47%. Multilocus analysis showed that the cumulative action of all QTLs detected for each trait accounted for 12–59% of the phenotypic variation. The difference in fruit weight was controlled largely by a single major QTL (fw2.2). Digenic epistasis was not evident. Several regions of the genome (including the region near sp on chromosome 6) showed effects on more than one trait. Implications for variety improvement and inferences about the domestication of the cultivated tomato are discussed.     

DNA damage in human leukocytes after acute in vitro exposure to a 1.9 GHz pulse-modulated radiofrequency field

Blood cultures from human volunteers were exposed to an acute 1.9 GHz pulse-modulated radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures during the exposure was maintained at 37.0 +/- 0.5 degrees C. DNA damage was quantified in leukocytes by the alkaline comet assay and the cytokinesis-block micronucleus assay. When compared to the sham-treated controls, no evidence of increased primary DNA damage was detected by any parameter for any of the RF-field-exposed cultures when evaluated using the alkaline comet assay. Furthermore, no significant differences in the frequency of binucleated cells, incidence of micronucleated binucleated cells, or total incidence of micronuclei were detected between any of the RF-field-exposed cultures and the sham-treated control at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz pulse-modulated RF-field exposure causes DNA damage in cultured human leukocytes.     

Oxidative stress generated damage to DNA by gastrointestinal exposure to insoluble particles

There is growing concern that gastrointestinal exposure to particles is associated with increased risk of toxicity to internal organs and carcinogenicity. The mechanism of action is related to particle-induced oxidative stress and oxidation of DNA. Observations from animal models indicate that gastrointestinal exposure to single-walled carbon nanotubes (SWCNT), fullerenes C60, carbon black, titanium dioxide and diesel exhaust particles generates oxidized DNA base lesions in organs such as the bone marrow, liver and lung. Oral exposure to nanosized carbon black has also been associated with increased level of lipid peroxidation derived exocyclic DNA adducts in the liver, suggesting multiple pathways of oxidative stress for particle-generated damage to DNA. At equal dose, diesel exhaust particles (SRM2975) generated larger levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in rat liver than carbon black (Printex 90) did, whereas exposure to fullerenes C60 and SWCNT was the least potent. This ranking of samples was also observed for oxidatively damaged DNA in cultured cells. The extent of translocation from the gut is largely unresolved. However, there is evidence indicating that gastrointestinal exposure to particulate matter is associated with oxidative damage to DNA and this might be associated with increased risk of cancer.     

Characterization and possible redox regulation of the purified calmodulin‐dependent NAD+ kinase from Lycopersicon pimpinellifolium

The soluble and calmodulin (CaM)‐dependent NAD⁺ kinase from Lycopersicon pimpinellifolium was previously shown to be largely inactivated in isolated cells exposed to a short‐term NaCl stress (Delumeau, Morère‐Le Paven, Montrichard, Laval‐Martin (2000) Plant Cell & Environment 23, 329–336). Nevertheless, the activity could be restored by adding a high dithiothreitol concentration to the protein extract, suggesting that the salt stress triggers an oxidation of the enzyme which leads to its inactivation. It was then interesting to investigate the effect of thiol‐modifying reagents and disulphide reductants on the activity of L. pimpinellifolium NAD⁺ kinase. A three‐step purification procedure was then established and allowed isolation of the enzyme which exists under two forms: a monomer and a dimer of a 56 kDa subunit, characterized, respectively, by pIs of 6·8 and 7·1. Isolated NAD⁺ kinase had a high affinity for CaM, half saturation being obtained for 7 ng mL^?1 bovine CaM. The activity of NAD⁺ kinase was strongly inhibited by thiol‐modifying reagents and oxidized glutathione. NAD⁺ kinase was also found to be air‐inactivated, the residual activity being stimulated by disulphide reductants. The most efficient of them is reduced thioredoxin from Escherichia coli which induced a five‐fold increase in activity and restored 80% of the initial activity. These results which can be related to those previously observed in vivo suggest that the activity of the L. pimpinellifolium NAD⁺ kinase, besides its dependence on CaM, is also dependent on the reduction state of the protein which could be regulated by the thioredoxin h/NADP‐thioredoxin reductase system.     

DNA damage and micronucleus induction in human leukocytes after acute in vitro exposure to a 1.9 GHz continuous-wave radiofrequency field

Human blood cultures were exposed to a 1.9 GHz continuous-wave (CW) radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) of 0.0, 0.1, 0.26, 0.92, 2.4 and 10 W/kg were achieved, and the temperature within the cultures during a 2-h exposure was maintained at 37.0 +/- 0.5 degrees C. Concurrent negative (incubator) and positive (1.5 Gy (137)Cs gamma radiation) control cultures were run for each experiment. DNA damage was quantified immediately after RF-field exposure using the alkaline comet assay, and four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. No evidence of increased primary DNA damage was detected by any parameter for RF-field-exposed cultures at any SAR tested. The formation of micronuclei in the RF-field-exposed blood cell cultures was assessed using the cytokinesis-block micronucleus assay. There was no significant difference in the binucleated cell frequency, incidence of micronucleated binucleated cells, or total incidence of micronuclei between any of the RF-field-exposed cultures and the sham-exposed controls at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz CW RF-field exposure causes DNA damage in cultured human leukocytes.     

DNA damage and apoptosis in the immature mouse cerebellum after acute exposure to a 1 mT, 60 Hz magnetic field.

Several recent studies have reported that whole-body exposure of rodents to power frequency magnetic fields (MFs) can result in DNA single- and double-strand breaks in the brains of these animals. The current study was undertaken to investigate whether an acute 2h exposure of a 1 mT, 60 Hz MF could elicit DNA damage, and subsequently apoptosis, in the brains of immature (10-day-old) mice. DNA damage was quantitated at 0, 2, 4, and 24h after exposure using the alkaline comet assay. Apoptosis was quantitated in the external granule cell layer (EGCL) of the immature mouse cerebellum at 0 and 24h after exposure to MF by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. While increased DNA damage was detected by tail ratio at 2h after MF exposure, no supporting evidence of increased DNA damage was detected by the other parameters. In addition, no similar differences were observed using these parameters at any of the other post-exposure times. No increase in apoptosis was observed in the EGCL of MF-exposed mice, when compared to sham mice. Taken together, these results do not support the hypothesis that acute MF exposure causes DNA damage in the cerebellums of immature mice.     

Reactive oxygen species and DNA damage after ultrasound exposure

The aim of this work was to detect the formation of hydrogen peroxide and hydroxyl radicals after ultrasound (US) exposure and test the hypothesis that reactive oxygen species induced by ultrasound can contribute to DNA damage. Formation of reactive oxygen species was observed in incubated medium after sonication with 1 MHz continuous ultrasound at the intensities of 0.61-2.44 W/cm2. Free radicals and hydrogen peroxide produced by ultrasound exposure of cells can lead to DNA damage. Comet assay was used to assess the effect of ultrasound on the level of nuclear DNA damage. The nucleated erythrocytes from fish were exposed in vitro to ultrasound at the same intensities and frequency. It was noticed that ultrasound in all used intensities induced DNA damage. The effect was not eliminated by the addition of catalase, which indicates that DNA damage was not caused by hydrogen peroxide only. The results showed that the DNA damage can be repair and this mechanism was the most effective after 30 and 60 min after sonication. Furthermore, the ultrasound-induced DNA damage in the presence of sonosensitizer (Zn- and AlCl-phthalocyanine) was studied. It was noticed that phthalocyaniens (Pcs) alone or with ultrasound did not induce significant changes in the level of DNA damage.     

Cell number, cell size and hormone levels in semi-isogenic mutants of Lycopersicon pimpinellifolium differing in fruit size

Tomato fruit growth parameters, cell number and cell size, and hormone levels [IAA, abscisic acid (ABA), zeatin (Z)/zeatin riboside (ZR), isopentenyladenosine (i-Ado)/isopentenlyadenine (i-Ade)], in the wild-type ( Lycopersicon pimpinellifolium Mill.) and a semi-isogenic mutant (mutant III) differing in fruit size were investigated during fruit development. An image-processing system was used for the determination of cell number and single cell size per fruit and hormone levels were measured by radioimmuno-assay (RIA). The bigger fruits of mutant III showed higher cell numbers throughout fruit development and cells enlarged faster than in wild-type fruits. During the first 10 days of fruit growth, the main cell division period after fertilization, high concentrations of cytokinins were found, these being correlated with high cell division activity. There were only slight differences in IAA and ABA levels in the different sized fruits. The results emphasized the importance of the cell number per fruit at anthesis as a determining factor of final fruit size in tomatoes. A possible relationship between cytokinins and subsequent fruit development is discussed.     

Assessment of DNA damage and repair in Mycobacterium terrae after exposure to UV irradiation

AIM: Ultraviolet (UV) irradiation for drinking water treatment was examined for inactivation and subsequent dark and photo-repair of Mycobacterium terrae. METHODS AND RESULTS: UV sources tested were low pressure (monochromatic, 254 nm) and medium pressure (polychromatic UV output) Hg lamps. UV exposure resulted in inactivation, and was followed by dark or photo-repair experiments. Inactivation and repair were quantified utilizing a molecular-based endonuclease sensitive site (ESS) assay and conventional colony forming unit (CFU) viability assay. Mycobacterium terrae was more resistant to UV disinfection compared to many other bacteria, with approximately 2-log reduction at a UV fluence of 10 mJ cm(-2) ; similar to UV inactivation of M. tuberculosis. There was no difference in inactivation between monochromatic or polychromatic UV lamps. Mycobacterium terrae did not undergo detectable dark repair. Photo-repair resulted in recovery from inactivation by approximately 0.5-log in less than 30 min for both UV lamp systems. CONCLUSIONS: Mycobacterium terrae is able to photo-repair DNA damage within a short timeframe. The number of pyrimidine dimers induced by UV light were similar for Escherichia coli and M. terrae, however, this similarity did not hold true for viability results. SIGNIFICANCE AND IMPACT OF THE STUDY: There is no practical difference between UV sources for disinfection or prevention of DNA repair for M. terrae. The capability of M. terrae to photo-repair UV damage fairly quickly is important for wastewater treatment applications where disinfected effluent is exposed to sunlight. Finally, molecular based assay results should be evaluated with respect to differences in the nucleic acid content of the test micro-organism.     

The protective effect and mechanism of soybean oil and its extracts on DNA damage in human ECV304 cells exposed to UV-C

The degree of DNA damage in the human endothelial cell line ECV304 exposed to UV-C, with or without the presence of soybean oil (SBO), was assessed by the Comet assay. After 5-min exposure to UV-C, the %Tail DNA in the ECV304 cells ranged from 0% to 20% for SBO treatment groups and from 50% to 70% for the control group. The result indicated a strong protective effect of SBO against UV-C-induced DNA damage. To clarity the mechanism of this protective effect of SBO, the methanol extract of SBO (MESO) was analyzed and its capacity against UV-C-induced DNA damage was evaluated. Gas chromatography mass spectrometry (GC-MS) analysis confirmed that MESO contained many antioxidants including n-3-polyunsaturated fatty acid (n-3-PUFA), tocopherols and phytosterols. Comet assay revealed that the MESO was also active in reducing the DNA damage dose-dependently (P<0.0001) vs. control in the ECV304 cells. Therefore, we concluded that these potential antioxidants may be responsible for the scavenge of oxidative radicals induced by UV-C irradiation. This study suggested that dietary SBO, which is abundant of antioxidants, may reduce the content or impact of reactive oxygen species (ROS) and lower the risk of diseases caused by ROS.     

Genetic analysis of traits distinguishing outcrossing and self-pollinating forms of currant tomato,Lycopersicon pimpinellifolium (Jusl.) Mill

The evolution of inbreeding is common throughout the angiosperms, although little is known about the developmental and genetic processes involved. Lycopersicon pimpinellifolium (currant tomato) is a self-compatible species with variation in outcrossing rate correlated with floral morphology. Mature flowers from inbreeding and outcrossing populations differ greatly in characters affecting mating behavior (petal, anther, and style lengths); other flower parts (sepals, ovaries) show minimal differences. Analysis of genetic behavior, including quantitative trait locus (QTL) mapping, was performed on representative selfing and outcrossing plants derived from two contrasting natural populations. Six morphological traits were analyzed: flowers per inflorescence; petal, anther, and style lengths; and lengths of the fertile and sterile portions of anthers. All traits were smaller in the selfing parent and had continuous patterns of segregation in the F(2). Phenotypic correlations among traits were all positive, but varied in strength. Quantitative trait locus mapping was done using 48 RFLP markers. Five QTL total were found involving four of the six traits: total anther length, anther sterile length, style length, and flowers per inflorescence. Each of these four traits had a QTL of major (>25%) effect on phenotypic variance.