Protective effect of melatonin against in vitro iron ions and 7 mT 50 Hz magnetic field-induced DNA damage in rat lymphocytes

We have previously shown that simultaneous exposure of rat lymphocytes to iron ions and 50Hz magnetic field (MF) caused an increase in the number of cells with DNA strand breaks. Although the mechanism of MF-induced DNA damage is not known, we suppose that it involves free radicals. In the present study, to confirm our hypothesis, we have examined the effect of melatonin, an established free radicals scavenger, on DNA damage in rat peripheral blood lymphocytes exposed in vitro to iron ions and 50Hz MF. The alkaline comet assay was chosen for the assessment of DNA damage. During pre-incubation, part of the cell samples were supplemented with melatonin (0.5 or 1.0mM). The experiments were performed on the cell samples incubated for 3h in Helmholtz coils at 7mT 50Hz MF. During MF exposure, some samples were treated with ferrous chloride (FeCl2, 10microg/ml), while the rest served as controls. A significant increase in the number of cells with DNA damage was found only after simultaneous exposure of lymphocytes to FeCl2 and 7mT 50Hz MF, compared to the control samples or those incubated with FeCl2 alone. However, when the cells were treated with melatonin and then exposed to iron ions and 50Hz MF, the number of damaged cells was significantly reduced, and the effect depended on the concentration of melatonin. The reduction reached about 50% at 0.5mM and about 100% at 1.0mM. Our results indicate that melatonin provides protection against DNA damage in rat lymphocytes exposed in vitro to iron ions and 50Hz MF (7mT). Therefore, it can be suggested that free radicals may be involved in 50Hz magnetic field and iron ions-induced DNA damage in rat blood lymphocytes. The future experimental studies, in vitro and in vivo, should provide an answer to the question concerning the role of melatonin in the free radical processes in the power frequency magnetic field.     

Free radicals-mediated induction of oxidized DNA bases and DNA-protein cross-links by nickel chloride

Using the comet assay, we showed that nickel chloride at 250-1000 microM induced DNA damage in human lymphocytes, measured as the change in comet tail moment, which increased with nickel concentration up to 500 microM and then decreased. Observed increase might follow from the induction of strand breaks or/and alkali-labile sites (ALS) by nickel, whereas decrease from its induction of DNA-DNA and/or DNA-protein cross-links. Proteinase K caused an increase in the tail moment, suggesting that nickel chloride at 1000 microM might cross-link DNA with nuclear proteins. Lymphocytes exposed to NiCl(2) and treated with enzymes recognizing oxidized and alkylated bases: endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), displayed greater extent of DNA damage than those not treated with these enzymes, indicating the induction of oxidized and alkylated bases by nickel. The incubation of lymphocytes with spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by nickel. The pre-treatment with Vitamin C at 10 microM and Vitamin E at 25 microM decreased the tail moment of the cells exposed to NiCl(2) at the concentrations of the metal causing strand breaks or/and ALS. The results obtained suggest that free radicals may be involved in the formation of strand breaks or/and ALS in DNA as well as DNA-protein cross-links induced by NiCl(2). Nickel chloride can also alkylate DNA bases. Our results support thesis on multiple, free radicals-based genotoxicity pathways of nickel.     

Acute exposure to a 60 Hz magnetic field increases DNA strand breaks in rat brain cells

Acute (2 h) exposure of rats to a 60 Hz magnetic field (flux densities 0.1, 0.25, and 0.5 mT) caused a dose-dependent increase in DNA strand breaks in brain cells of the animals (assayed by a microgel electrophoresis method at 4 h postexposure). An increase in single-strand DNA breaks was observed after exposure to magnetic fields of 0.1, 0.25, and 0.5 mT, whereas an increase in double-strand DNA breaks was observed at 0.25 and 0.5 mT. Because DNA strand breaks may affect cellular functions, lead to carcinogenesis and cell death, and be related to onset of neurodegenerative diseases, our data may have important implications for the possible health effects of exposure to 60 Hz magnetic fields. Bioelectromagnetics 18:156–165, 1997. © 1997 Wiley-Liss, Inc.     

Interaction of Microwaves and a Temporally Incoherent Magnetic Field on Single and Double DNA Strand Breaks in Rat Brain Cells

The effect of a temporally incoherent magnetic field noise on microwave-induced DNA single and double strand breaks in rat brain cells was investigated. Four treatment groups of rats were studied: microwave-exposure (continuous-wave 2450-MHz microwaves, power density 1 mW/cm², average whole-body specific absorption rate of 0.6 W/kg), noise-exposure (45 mG), microwave + noise-exposure, and sham-exposure. Animals were exposed to these conditions for 2h. DNA single- and double-strand breaks in brain cells of these animals were assayed 4h later using a microgel electrophoresis assay. Results show that brain cells of microwave-exposed rats had significantly higher levels of DNA single- and double-strand breaks when compared with sham-exposed animals. Exposure to noise alone did not significantly affect the levels (i.e., they were similar to those of the sham-exposed rats). However, simultaneous noise exposure blocked microwave-induced increases in DNA strand breaks. These data indicate that simultaneous exposure to a temporally incoherent magnetic field could block microwave-induced DNA damage in brain cells of the rat.     

Single strand DNA breaks in rat brain cells exposed to microwave radiation

This investigation concerns with the effect of low intensity microwave (2.45 and 16.5 GHz, SAR 1.0 and 2.01 W/kg, respectively) radiation on developing rat brain. Wistar rats (35 days old, male, six rats in each group) were selected for this study. These animals were exposed for 35 days at the above mentioned frequencies separately in two different exposure systems. After the exposure period, the rats were sacrificed and the whole brain tissue was dissected and used for study of single strand DNA breaks by micro gel electrophoresis (comet assay). Single strand DNA breaks were measured as tail length of comet. Fifty cells from each slide and two slides per animal were observed. One-way ANOVA method was adopted for statistical analysis. This study shows that the chronic exposure to these radiations cause statistically significant (p<0.001) increase in DNA single strand breaks in brain cells of rat.     

Effect of 900-, 1800-, and 2100-MHz radiofrequency radiation on DNA and oxidative stress in brain

Ubiquitous and ever increasing use of mobile phones led to the growing concern about the effects of radiofrequency radiation (RFR) emitted by cell phones on biological systems. The aim of this study is to explore whether long-term RFR exposure at different frequencies affects DNA damage and oxidant-antioxidant parameters in the blood and brain tissue of rats. 28 male Sprague Dawley rats were randomly divided into four equal groups (n = 7). They were identified as Group 1: sham-control, Group 2: 900 MHz, Group 3: 1800 MHz, and Group 4: 2100 MHz. Experimental groups of rats were exposed to RFR 2 h/day for 6 months. The sham-control group of rats was subjected to the same experimental condition but generator was turned off. Specific absorption rates (SARs) at brain with 1 g average were calculated as 0.0845 W/kg, 0.04563 W/kg, and 0.03957, at 900 MHz, 1800 MHz, and 2100 MHz, respectively. Additionally, malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), total antioxidant status (TAS), and total oxidant status (TOS) analyses were conducted in the brain tissue samples. Results of the study showed that DNA damage and oxidative stress indicators were found higher in the RFR exposure groups than in the sham-control group. In conclusion, 900-, 1800-, and 2100-MHz RFR emitted from mobile phones may cause oxidative damage, induce increase in lipid peroxidation, and increase oxidative DNA damage formation in the frontal lobe of the rat brain tissues. Furthermore, 2100-MHz RFR may cause formation of DNA single-strand breaks.     

Sesamol as a potential radioprotective agent: in vitro studies

Protection against radiation-induced DNA strand breaks is an important aspect in the design and development of a radioprotector. In this study, the radioprotective efficacy of sesamol, a natural antioxidant, was investigated in aqueous solution of plasmid DNA (pBR322) and compared with that of melatonin, a known antioxidant-based radioprotector. Thermal denaturation studies on irradiated calf thymus DNA were also carried out with sesamol and melatonin. Sesamol demonstrated greater radioprotective efficacy in both plasmid DNA and calf thymus DNA. To assess the radical scavenging capacity of sesamol and melatonin, 2-deoxyribose degradation, DPPH and ABTS assays were performed. Sesamol exhibited more scavenging capacity compared to melatonin. In vitro studies with V79 cells showed that sesamol is 20 times more potent than melatonin. It is proposed that the greater radioprotective efficacy of sesamol could be due to its greater capacity for scavenging of free radicals compared to melatonin. The results will be helpful in understanding the mechanisms and development of sesamol as a radioprotector.     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.     

Extremely low frequency electromagnetic fields as effectors of cellular responses in vitro: possible immune cell activation

There is presently an intense discussion if electromagnetic field (EMF) exposure has consequences for human health. This include exposure to structures and appliances that emit in the extremely low frequency (ELF) range of the electromagnetic spectrum, as well as emission coming from communication devices using the radiofrequency part of the spectrum. Biological effects of such exposures have been noted frequently, although the implication for specific health effects is not that clear. The basic interaction mechanism(s) between such fields and living matter is unknown. Numerous hypotheses have been suggested, although none is convincingly supported by experimental data. Various cellular components, processes, and systems can be affected by EMF exposure. Since it is unlikely that EMF can induce DNA damage directly, most studies have examined EMF effects on the cell membrane level, general and specific gene expression, and signal transduction pathways. In addition, a large number of studies have been performed regarding cell proliferation, cell cycle regulation, cell differentiation, metabolism, and various physiological characteristics of cells. Although 50/60 Hz EMF do not directly lead to genotoxic effects, it is possible that certain cellular processes altered by exposure to EMF indirectly affect the structure of DNA causing strand breaks and other chromosomal aberrations. The aim of this article is to present a hypothesis of a possible initial cellular event affected by exposure to ELF EMF, an event which is compatible with the multitude of effects observed after exposure. Based on an extensive literature review, we suggest that ELF EMF exposure is able to perform such activation by means of increasing levels of free radicals. Such a general activation is compatible with the diverse nature of observed effects. Free radicals are intermediates in natural processes like mitochondrial metabolism and are also a key feature of phagocytosis. Free radical release is inducible by ionizing radiation or phorbol ester treatment, both leading to genomic instability. EMF might be a stimulus to induce an "activated state" of the cell such as phagocytosis, which then enhances the release of free radicals, in turn leading to genotoxic events. We envisage that EMF exposure can cause both acute and chronic effects that are mediated by increased free radical levels: (1) Direct activation of, for example macrophages (or other cells) by short-term exposure to EMF leads to phagocytosis (or other cell specific responses) and consequently, free radical production. This pathway may be utilized to positively influence certain aspects of the immune response, and could be useful for specific therapeutic applications. (2) EMF-induced macrophage (cell) activation includes direct stimulation of free radical production. (3) An increase in the lifetime of free radicals by EMF leads to persistently elevated free radical concentrations. In general, reactions in which radicals are involved become more frequent, increasing the possibility of DNA damage. (4) Long-term EMF exposure leads to a chronically increased level of free radicals, subsequently causing an inhibition of the effects of the pineal gland hormone melatonin. Taken together, these EMF induced reactions could lead to a higher incidence of DNA damage and therefore, to an increased risk of tumour development. While the effects on melatonin and the extension of the lifetime of radicals can explain the link between EMF exposure and the incidence of for example leukaemia, the two additional mechanisms described here specifically for mouse macrophages, can explain the possible correlation between immune cell system stimulation and EMF exposure.     

Ex-vivo and in vitro protective effects of kolaviron against oxygen-derived radical-induced DNA damage and oxidative stress in human lymphocytes and rat liver cells

The present study reports the protective effects of kolaviron, a Garcinia biflavonoid from the seeds of Garcinia kola widely consumed in some West African countries against oxidative damage to molecular targets ex-vivo and in vitro. Treatment with hydrogen peroxide (H2O2) at a concentration of 100 micromol/L increased the levels of DNA strand breaks and oxidized purine (formamidopyrimidine glycosylase (FPG) and pyrimidine (endonuclease III (ENDO III) sites) bases in both human lymphocytes and rat liver cells using alkaline single cell gel electrophoresis (the comet assay). Kolaviron was protective at concentrations between 30-90 micromol/L and decreased H2O2-induced DNA strand breaks and oxidized bases. Neither alpha-tocopherol nor curcumin decreased H2O2-induced DNA damage in this assay. In lymphocytes incubated with Fe3+/GSH, Fe3+ was reduced to Fe2+ by GSH initiating a free radical generating reaction which induced 11.7, 6.3, and 4.9 fold increase respectively in strand breaks, ENDO III and FPG sensitive sites compared with control levels. Deferoxamine (2 mmol/L), an established iron chelator significantly inhibited GSH/Fe3+-induced strand breaks and oxidized base damage. Similarly, kolaviron at 30 and 90 micromol/L significantly attenuated GSH/Fe3+-induced strand breaks as well as base oxidation. Kolaviron (100 mg/kg bw) administered to rats for one week protected rat liver cells against H2O2-induced formation of strand breaks, ENDO III, and FPG sensitive sites, Fe3+/EDTA/ascorbate-induced malondialdehyde formation and protein oxidation using gamma-glutamyl semialdehyde (GGS) and 2-amino-adipic semialdehyde (AAS) as biomarkers of oxidative damage to proteins. We suggest that kolaviron exhibits protective effects against oxidative damage to molecular targets via scavenging of free radicals and iron binding. Kolaviron may therefore be relevant in the chemoprevention of oxidant-induced genotoxicity and possibly human carcinogenesis.     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)     

In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA-protein cross-links

Lead is present in the natural and occupational environment and is reported to interact with DNA, but the mechanism of this interaction is not fully understood. Using the alkaline comet assay we showed that lead acetate at 1-100 microM induced DNA damage in isolated human lymphocytes measured the change in the comet tail length. At 1 and 10 microM we observed an increase in the tail length, whereas at 100 microM a decrease was seen. The former effect could follow from the induction of DNA strand breaks and/or alkali-labile sites (ALS), the latter from the formation of DNA-DNA and/or DNA-protein cross-links. No difference was observed between tail length for the alkaline and pH 12.1 versions of the assay, which indicates that strand breaks and not ALS are responsible for the tail length increase induced by lead. The neutral version of the test revealed that lead acetate induced DNA double-strand breaks at all concentrations tested. The presence of spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) did not influence the level of DNA damage induced by lead. Post-treatment of the lead-damaged DNA (at 100 microM treatment concentration) by endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II, an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage. Proteinase K caused an increase in comet tail length, suggesting that lead acetate might cross-link DNA with nuclear proteins. Vitamin A, E, C, calcium chloride and zinc chloride acted synergistically on DNA damage evoked by lead. The results obtained suggest that lead acetate may induce single-strand breaks (SSB) and double-strand breaks (DSB) in DNA as well as DNA-protein cross-links. The participation of free radicals in DNA-damaging potential of lead is not important and it concerns other reactive species than could be trapped by DMPO or PBN.     

Interaction of amoxicillin with DNA in human lymphocytes and H. pylori-infected and non-infected gastric mucosa cells

Amoxicillin is a penicillin derivative belonging to a group of beta-lactam antibiotics used in Helicobacter pylori eradication. Clinical application of amoxicillin is underlined by its antibacterial activity, but little is known about its interaction with DNA of human cells. Using the alkaline comet assay we investigated the genotoxicity of amoxicillin in human peripheral blood lymphocytes as well as in H. pylori-infected and non-infected human gastric mucosa cells. To assess the role of reactive oxygen species in the genotoxicity of amoxicillin we employed a set of antioxidant and free radical scavengers, including Vitamins C and E, melatonin and the nitrone spin trap N-tert-butyl-alpha-phenyl-nitrone (PBN). Amoxicillin-induced DNA damage was completely repaired after 60 min. The vitamins, melatonin and the spin trap decreased the extent of the damage. The cells exposed to amoxicillin and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized bases displayed greater extent of DNA damage than those not treated with these enzymes. H. pylori non-infected gastric mucosa cells exposed to hydrogen peroxide repaired their DNA in a 60 min incubation, but the infected cells were not able to do so. The action of DNA repair enzymes, the vitamins, melatonin and PBN indicated that amoxicillin-induced oxidative DNA damage. The drug did not induce DNA strand breaks in isolated pUC19 plasmid DNA. Our results suggest that amoxicillin can induce DNA damage in human lymphocytes and gastric mucosa cells and this effect may follow from the production of reactive oxygen species. Cellular activation of the drug is needed to induce DNA damage. Free radical scavengers and antioxidants may be used to assist H. pylori eradication with amoxicillin to protect DNA of the host cells. Our results suggest also that H. pylori infection may alter gastric mucosa cells response to DNA-damaging agents and in this way contribute to initiation/promotion of cancer transformation of these cells induced by external or internal carcinogens.     

Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes

Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.2 to 2 microM induced DNA damage in human leukemic K562 and BV173 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes and leukemic CCRF-CEM cells without the expression of BCR/ABL. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the circular plasmid relaxation assay. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. K562 cells were unable to repair H(2)O(2)-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with Vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Therefore, the mechanism of the anti-leukemic action of STI571 may involve not only the inhibition of BCR/ABL, but also DNA damage in the cells expressing this fusion protein. DNA damage induced by STI571 may follow from oxidative and alkylative base modifications.     

Radiofrequency (microwave) radiation exposure of mammalian cells during UV-induced DNA repair synthesis

The effect of continuous-wave (CW) and pulsed-wave (PW) radiofrequency radiation (RFR) in the microwave range on UV-induced DNA repair has been investigated in MRC-5 normal human diploid fibroblasts. RFR exposure at power densities of 1 (or 5) and 10 mW/cm2 gave a maximum specific absorption rate (SAR) (at 10 mW/cm2) of 0.39 +/- 0.15 W/kg for 350 MHz RFR, 4.5 +/- 3.0 W/kg for 850 MHz RFR, and 2.7 +/- 1.6 W/kg for 1.2 GHz RFR. RFR exposures for 1 to 3 h at 37 degrees C, in either continuous-wave or pulsed-wave modes, had no effect on the rate of repair replication label incorporated into preexisting UV-damaged DNA. RFR exposures (PW), with a constant medium temperature of 39 degrees C at 350 and 850 MHz during the repair period after UV damage, also had no effect. Assay for induction of repair synthesis by RFR exposure alone in non-UV irradiated cells was negative for the 350-, 850-, and 1200-MHz CW and PW RFR at 37 degrees C and the 350- and 850-MHz PW RFR at 39 degrees C. RFR does not induce DNA repair under these exposure conditions. In preliminary experiments--with the tissue culture medium maintained at 39 degrees C and RFR exposures (PW) at the frequencies of 350, 850, and 1200 MHz--no effect on incorporation of [3H]thymidine into DNA undergoing semiconservative synthesis was observed.     

The effect of anoxia on anthracycline-induced DNA damage in the RPMI-6410 human lymphoblastoid cell line

The alkaline elution procedure developed by Kohn and co-workers was used with the RPMI-6410 cultured human lymphoblastoid cell line to examine the hypothesis that anthracycline-induced DNA strand scission is mediated by oxygen- or superoxide-derived free radicals. Hypoxia was induced by gassing with nitrogen containing 5% carbon dioxide and less than 4 ppm oxygen. Alkaline elution studies showed hypoxia was induced, as the oxygen enhancement ratios for DNA strand breaks was 2.4 and 2.6 for the 250 R +/- oxygen and the 500 R +/- oxygen (1 R = 2.58 x 10(-4) C/kg) experiments, respectively. The pattern of adriamycin-induced DNA strand breaks and cross-linking was not affected by hypoxia with 1-h adriamycin exposures between 0.05 and 1.0 microgram/ml. Similarly, 1-h exposures of N-trifluoroacetyladriamycin-14-valerate at 3 or 10 micrograms/mL gave essentially identical alkaline elution profiles in the presence or absence of oxygen. These results do not support the hypothesis that oxygen-derived radicals play a primary role in anthracycline-induced DNA strand breakage.     

Exposure of rat brain to 915 MHz GSM microwaves induces changes in gene expression but not double stranded DNA breaks or effects on chromatin conformation

We investigated whether exposure of rat brain to microwaves (MWs) of global system for mobile communication (GSM) induces DNA breaks, changes in chromatin conformation and in gene expression. An exposure installation was used based on a test mobile phone employing a GSM signal at 915 MHz, all standard modulations included, output power level in pulses 2 W, specific absorption rate (SAR) 0.4 mW/g. Rats were exposed or sham exposed to MWs during 2 h. After exposure, cell suspensions were prepared from brain samples, as well as from spleen and thymus. For analysis of gene expression patterns, total RNA was extracted from cerebellum. Changes in chromatin conformation, which are indicative of stress response and genotoxic effects, were measured by the method of anomalous viscosity time dependencies (AVTD). DNA double strand breaks (DSBs) were analyzed by pulsed-field gel electrophoresis (PFGE). Effects of MW exposure were observed on neither conformation of chromatin nor DNA DSBs. Gene expression profiles were obtained by Affymetrix U34 GeneChips representing 8800 rat genes and analyzed with the Affymetrix Microarray Suite (MAS) 5.0 software. In cerebellum from all exposed animals, 11 genes were upregulated in a range of 1.34-2.74 fold and one gene was downregulated 0.48-fold (P < .0025). The induced genes encode proteins with diverse functions including neurotransmitter regulation, blood-brain barrier (BBB), and melatonin production. The data shows that GSM MWs at 915 MHz did not induce PFGE-detectable DNA double stranded breaks or changes in chromatin conformation, but affected expression of genes in rat brain cells.     

Asp-Ala-His-Lys (DAHK) inhibits copper-induced oxidative DNA double strand breaks and telomere shortening

Both DNA and the telomeric sequence are susceptible to copper-mediated reactive oxygen species (ROS) damage, particularly damage attributed to hydroxyl radicals. In this study, ROS-induced DNA double strand breaks and telomere shortening were produced by exposure to copper and ascorbic acid. Asp-Ala-His-Lys (DAHK), a specific copper chelating tetrapeptide d-analog of the N-terminus of human albumin, attenuated DNA strand breaks in a dose dependent manner. d-DAHK, at a ratio of 4:1 (d-DAHKCu), provided complete protection of isolated DNA from double strand breaks and, at a ratio of 2:1 (d-DAHKCu), completely protected DNA in Raji cells exposed to copper/ascorbate. Southern blots of DNA treated with copper/ascorbate showed severe depletion and shortening of telomeres and Raji cell treated samples showed some conservation of telomere sequences. d-DAHK provided complete telomere length protection at a ratio of 2:1 (d-DAHKCu). The human albumin N-terminus analog, d-DAHK, protects DNA and telomeres against copper-mediated ROS damage and may be a useful therapeutic adjunct in ROS disease processes.     

DNA strand scission by enzymatically reduced mitomycin C: evidence for participation of the hydroxyl radical in the DNA damage

Phage DNA, as well as plasmid and mammalian DNA's, were exposed to a superoxide and hydroxyl radical-generating system containing NADPH-cytochrome P-450 reductase and mitomycin C, both with and without added Fe3+-ADP, in phosphate buffer at pH 7.5. The generation of superoxide (O2-.) and hydroxyl (.OH) radicals in the system was demonstrated by using ESR spectrometry with N-tert -butyl-alpha-phenylnitrone (PBN) as a spin trapping agent. Only the lambda DNA isolated after exposure to the O2-./.OH-generating system containing many lower molecular weight DNA fragments indicating DNA strand breaks. This breakage was completely inhibited by a .OH radical scavenger (sodium benzoate) and by catalase, but only slightly by superoxide dismutase. Thyroid and plasmid DNA's were both cleaved when exposed to the O2-./.OH-generating systems. It is suggested that the mechanism of DNA scission by mitomycin C described here closely resembles that induced by the anthracycline drugs.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.