Level Of DNA Single-strand Breaks Research Articles

Calibration of the comet assay using ionising radiation

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke’s and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

Neuronal enhancers are hotspots for DNA single-strand break repair.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

Monte Carlo Modeling of DNA Lesions and Chromosomal Aberrations Induced by Mixed Beams of Alpha Particles and X-Rays

Prediction of health risks associated with exposure to mixed beams of high and low LET (linear energy transfer) ionizing radiation is based on the assumption that the biological effect caused by them equals the sum of effects resulting from the action of individual beam components. Experimental studies have been published indicating that the cellular effects in cells exposed to mixed radiations are higher than calculated based on the assumption of additivity. The present work contains a comparative analysis of published results on chromosomal aberrations in human peripheral blood lymphocytes exposed to mixed beams of alpha particles and X-rays with computer simulations using the PARTRAC program based on Monte Carlo methods. PARTRAC was used to calculate the levels of DNA single-strand breaks (SSB) and double-strand breaks (DSB - both complex and simple) and of chromosomal aberrations. The levels and SSB and DSB were found to be additive. A synergistic effect was obtained at the level of chromosomal aberrations, being in good agreement with the experimental results. This result demonstrates that the synergistic action of mixed beams results from processing of SSB and DSB, and not from their initial frequencies. The level of synergy was dependent on the composition of the mixed beam, with highest level at a 50:50 ratio of alpha particles and X-rays.

The effects of 1st and 2nd generation biodiesel exhaust exposure on hematological and biochemical blood indices of Fisher344 male rats – The FuelHealth project

Diesel exhaust emissions (DEE), being one of the main causes of ambient air pollution, exert a detrimental effect on human health and increase morbidity and mortality related to cardiovascular and pulmonary diseases. Therefore, the objective of the present study was to investigate potential adverse effects of exhausts emissions from B7 fuel, the first-generation biofuel containing 7% of fatty acid methyl esters (FAME), and SHB20 fuel, the second-generation biofuel containing 20% FAME/hydrotreated vegetable oil (HVO), after a whole-body exposure with and without diesel particle filter (DPF).The experiment was performed on 95 male Fischer 344 rats, divided into 10 groups (8 experimental, 2 control). Animals were exposed to DEE (diluted with charcoal-filtered room air to 2.1–2.2% (v/v)) for 7 or 28 days (6 h/day, 5 days/week) in an inhalation chamber. DEE originated from Euro 5 engine with or without DPF treatment, run on B7 or SHB20 fuel. Animals in the control groups were exposed to clean air. Our results showed that the majority of haematological and biochemical parameters examined in blood were at a similar level in the exposed and control animals. However, exposure to DEE from the SHB20 fuel caused an increase in the number of red blood cells (RBC) and haemoglobin concentration. Moreover, 7 days exposure to DEE from SHB20 fuel induced genotoxic effects manifested by increased levels of DNA single-strand breaks in peripheral blood lymphocytes. Furthermore, inhalation of both types of DEE induced oxidative stress and caused imbalance of anti-oxidant defence enzymes.In conclusion, exposure to DEE from B7, which was associated with higher exposure to polycyclic aromatic hydrocarbons, resulted in decreased number of T and NK lymphocytes, while DEE from SHB20 induced a higher level of DNA single-strand breaks, oxidative stress and increased red blood cells parameters. Additionally, DPF technology generated increased number of smaller PM and made the DEE more reactive and more harmful, manifested as deregulation of redox balance.

Effects of side-stream tobacco smoke and smoke extract on glutathione- and oxidative DNA damage repair-deficient mice and blood cells

Cigarette smoke causes direct oxidative DNA damage as well as indirect damage through inflammation. Epidemiological studies show a strong relationship between secondhand smoke and cancer; however, the mechanisms of secondhand smoke-induced cancer are not well understood. Animal models with either (i) deficient oxidative DNA damage repair, or (ii) a decreased capacity to combat oxidative stress may help determine the pathways important in mitigating damage caused by smoke. In this study, we used mice lacking Ogg1 and Myh, both of which are involved in base excision repair by removing oxidatively damaged DNA bases. Gclm-deficient mice, which have decreased levels of glutathione (GSH), were used to look at the role of smoke-induced oxidative damage. Ex vivo experiments show significantly elevated levels of DNA single-strand breaks and chromosomal aberrations in peripheral blood lymphocytes from Ogg1−/−Myh−/− double knockout mice compared to wild type (WT) mice after 24h of exposure to cigarette smoke extract (CSE). The average γH2AX foci per cell was significantly elevated 3h after exposure to CSE in cells from Ogg1−/−Myh−/− double knockout mice compared to wildtype mice. In vivo we found that all mice had increased markers of DNA damage after exposure to side-stream tobacco smoke (SSTS). Ogg1−/−Myh−/− and Gclm−/− mice had altered levels of peripheral blood glutathione after SSTS exposure whereas wild type mice did not. This may be due to differential regulation of glutathione synthesis in the lung. We also found that Ogg1−/−Myh−/− mice had a decreased lifespan after oral gavage with benzo[a]pyrene compared to wildtype mice and sham-exposed Ogg1−/−Myh−/− mice. Our results are important in investigating the roles of oxidative stress and oxidative DNA damage repair in cigarette smoke-induced cancers and characterizing the role of genetic polymorphisms in smoke-related disease susceptibility.

DNA damage and nucleotide excision repair capacity in healthy individuals

Interindividual differences in DNA repair capacity (DRC) represent an important source of variability in genome integrity and thus influence health risk. In the last decade, DRC measurement has attracted attention as a potential biomarker in cancer prediction. Aim of the present exploratory study was to characterize the variability in DNA damage and DRC on 100 healthy individuals and to identify biological, lifestyle, or genetic factors modulating these parameters. The ultimate goal was to obtain reference data from cancer-free population, which may constitute background for further investigations on cancer patients. The endogenous DNA damage was measured as a level of DNA single-strand breaks and DRC, specific for nucleotide excision repair (NER), was evaluated using modified comet assay, following the challenge of peripheral blood mononuclear cells with benzo[a]pyrene diolepoxide. Additionally, genetic polymorphisms in NER genes (XPA, XPC, XPD, and XPG) were assessed. We have observed a substantial interindividual variability for both examined parameters. DNA damage was significantly affected by gender and alcohol consumption (P = 0.003 and P = 0.012, respectively), whereas DRC was associated with family history of cancer (P = 0.012). The stratification according to common variants in NER genes showed that DNA damage was significantly modulated by the presence of the variant T allele of XPC Ala499Val polymorphism (P = 0.01), while DRC was modulated by the presence of the A allele of XPA G23A polymorphism (P = 0.048). Our results indicate the range of endogenous DNA single-strand breaks and capacity of NER in healthy volunteers as well as the role of potentially relevant confounders. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc.

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O 6-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl- N′-nitro- N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced γH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.

ATR-FTIR spectroscopy detects alterations induced by organotin(IV) carboxylates in MCF-7 cells at sub-cytotoxic/-genotoxic concentrations

The environmental impact of metal complexes such as organotin(IV) compounds is of increasing concern. Genotoxic effects of organotin(IV) compounds (0.01 μg/ml, 0.1 μg/ml or 1.0 μg/ml) were measured using the alkaline single-cell gel electrophoresis (comet) assay to measure DNA single-strand breaks (SSBs) and the cytokinesis-block micronucleus (CBMN) assay to determine micronucleus formation. Biochemical-cell signatures were also ascertained using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. In the comet assay, organotin(IV) carboxylates induced significantly-elevated levels of DNA SSBs. Elevated micronucleus-forming activities were also observed. Following interrogation using ATR-FTIR spectroscopy, infrared spectra in the biomolecular range (900 cm-1 – 1800 cm-1) derived from organotin-treated MCF-7 cells exhibited clear alterations in their biochemical-cell fingerprint compared to control-cell populations following exposures as low as 0.0001 μg/ml. Mono-, di- or tri-organotin(IV) carboxylates (0.1 μg/ml, 1.0 μg/ml or 10.0 μg/ml) were markedly cytotoxic as determined by the clonogenic assay following treatment of MCF-7 cells with ≥ 1.0 μg/ml. Our results demonstrate that ATR-FTIR spectroscopy can be applied to detect molecular alterations induced by organotin(IV) compounds at sub-cytotoxic and sub-genotoxic concentrations. This biophysical approach points to a novel means of assessing risk associated with environmental contaminants.PACS codes: 87.15.-v, 87.17.-d, 87.18.-h

Genetic Susceptibility to Benzene Toxicity in Humans

Human metabolism of benzene involves pathways coded for by polymorphic genes. To determine whether the genotype at these loci might influence susceptibility to the adverse effects of benzene exposure, 208 Bulgarian petrochemical workers and controls, whose exposure to benzene was determined by active personal sampling, were studied. The frequency of DNA single-strand breaks (DNA-SSB) was determined by alkaline elution, and genotype analysis was performed for five metabolic loci. Individuals carrying the NAD(P)H:quinone oxidoreductase 1 (NQO1) variant had significantly twofold increased DNA-SSB levels compared to wild-type individuals. The same result was observed for subjects with microsomal epoxide hydrolase (EPHX) genotypes that predict the fast catalytic phenotype. Deletion of the glutathione S‐transferase T1 (GSTT1) gene also showed a consistent quantitative 35–40% rise in DNA-SSB levels. Neither glutathione S-transferase M1 (GSTM1) nor myeloperoxidase (MPO) genetic variants exerted any effect on DNA-SSB levels. Combinations of two genetic polymorphisms showed the same effects on DNA-SSB as expected from the data on single genotypes. The three locus genotype predicted to produce the highest level of toxicity, based on metabolic pathways, produced a significant 5.5-fold higher level of DNA-SSB than did the genotype predicted to yield the least genotoxicity.

Cytotoxicity, Apoptosis and DNA Damage Induced by Alpinia galanga Rhizome Extract

Alpinia galanga, or galangal, has been a popular condiment used in Thai and Asian cuisine for many years. However, relatively little is known of the potential beneficial or adverse health effects of this spice. This study was conducted to analyze the capacity of galangal extract to induce cytotoxicity and DNA damage in six different human cell lines including normal and p53-inactive fibroblasts, normal epithelial and tumour mammary cells and a lung adenocarcinoma cell line. We deliberately focused on treatment with the crude aqueous extract of galangal rhizomes, rather than compounds extracted into an organic solvent, to more closely reflect the mode of dietary consumption of galangal. The cell lines displayed a broad range of cytotoxicity. There was no evidence for preferential cytotoxicity of tumour cells, but there was an indication that p53-active cell lines may be more sensitive than their p53-inactive counterparts. The contribution of apoptosis to total cell killing was only appreciable after exposure to 300 microg/mL of extract. Apoptosis appeared to be independent of p53 expression. Exposure to as little as 100 microg/mL galangal extract generated a significant level of DNA single-strand breaks as judged by the single-cell gel electrophoresis technique (comet assay). The three major UV-absorbing compounds in the aqueous extract were identified by mass spectrometry as 1'-acetoxychavicol acetate and its deacetylated derivatives. However, when tested in A549 human lung adenocarcinoma cells, these compounds were not responsible for the cytotoxicity induced by the complete aqueous extract.

Radio-Protective Effect of 2-Deoxy-D-Glucose in Cervical Cancer Patient’s Lymphocytes Exposedin Vitroas Estimated by the Comet Assay

The main objectives of the present study was two fold, namely, first to evaluate the baseline Single- Strand Breaks (SSBs) frequency in Cervical Cancer patient's and secondly, to study the radio-protective effect of 2- deoxy-D-glucose (2-DG) on such cells when exposed to ionising radiation like that of gamma radiation. The reason for proposing such a study is that the DNA of cancer patients has a general tendency to break, i.e. Concept of Common Fragile Sites. This is not yet clearly understood as to whether it is the cause or effect of the pathological processes and necessitates looking into the base-line SSBs in this type of patients. Secondly, the main line of treatment of Cervical Cancer is that of radiotherapy & / or surgery, therefore there is a need to protect the normal cells surrounding the tumour tissue during the course of radiotherapy. This could be achieved by means of a radio- modulator such as 2-DG, which protects normal cells and sensitises the tumour tissue, thus acting as a double-edged sword. Hence the study was designed to evaluate the SSBs by Comet Assay (Single cell Agarose Gel Electrophoresis - SAGE) using a silver staining method, which does not require the usage of fluorescent microscopy and can be analysed with the aid of a simple light microscopy. The results showed that the Cervical Cancer patient's peripheral blood lymphocytes are not entirely fragile and the radiation exposure leads to increasing levels of DNA SSBs, which is reduced by 2-DG showing that the compound can be used as an adjuvant to radiotherapy.

Multibiomarker Responses in Fish from the Moselle River (France)

The response of wild fish to pollutants was studied using two biomakers in chub (Leuciscus cephalus) at five stations in the Moselle River (France) in 1998 and in 1999. The induction of cytochrome P450 1A was quantified by the ethoxyresorufin O-deethylase (EROD) activity in the liver and the level of DNA single-strand breaks was determined in erythrocytes using the comet assay. EROD activity was observed to be up to 10-fold induced in both males and females from the downstream stations in comparison to the fish from the upstream station. Levels of DNA damage did not parallel EROD induction. Chemical analyses did not clearly explain the responses of the studied biomarkers, confirming the great difficulty in relating chemical and biological information in the field. This study confirms the difficulty in assessing the biological effects of mixtures of pollutants and points out the usefulness of a large array of biomarkers.

DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m 3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabelling technique. In lungs after 21 days of exposure to 1500 mg/m 3 the level of 7-SO-guanine was 23.0±11.9 adducts/10 8 normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6±0.2 adducts/10 8 normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood ( r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m 3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4±2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural eliminaton of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei ( r=0.721, P=0.028).

Induction of DNA single-strand breaks by 131I and 99mTc in human mononuclear blood cells in vitro and extrapolation to the in vivo situation.

The radionuclides (131)I and (99m)Tc are frequently used for therapy of benign and malignant thyroid disease ((131)I) and for diagnosis of thyroid and other diseases ((99m)Tc). However, the levels of DNA single-strand breaks (SSBs) induced in cells of patients after administration of (131)I and (99m)Tc are not known. In this study, we measured the number of SSBs per cell induced by (131)I and (99m)Tc in vitro, extrapolated the results to the clinical situation, and assessed their biological relevance by comparing levels of SSBs induced after therapeutic administration of (131)I and (99m)Tc to those induced by endogenous processes or by occupational exposure to genotoxic substances. A linear dose-response relationship between the radioactivity concentrations of (131)I and (99m)Tc and SSBs in human mononuclear blood cells (determined by alkaline elution) was obtained after incubation at 4 and 37 degrees C. At 4 degrees C, where almost no repair of SSBs takes place, (131)I and (99m)Tc induced 81 and 7 SSBs per cell per hour/(MBq/ml), respectively. At 37 degrees C, only 20 and 1.6 SSBs per cell per hour/(MBq/ml) were observed after incubation with (131)I and (99m)Tc. To estimate the induction of SSBs in vivo in cells of patients after administration of 3700 MBq (131)I (oral) or 60 MBq (99m)Tc (i.v.), the rates of induction of SSBs obtained in vitro were extrapolated to the concentrations of (131)I and (99m)Tc measured in blood of patients. The total number of SSBs (mean +/- standard deviation) accumulated after oral administration of 3700 MBq (131)I up to 70 h after administration was calculated as 200 +/- 59 SSBs/cell. After administration of 60 MBq (99m)Tc (i.v.), 0.032 +/- 0.009 SSBs per cell (total SSBs up to 2 h after administration) were cumulated. The induction of SSBs by endogenous processes (estimated 2,000 SSBs per cell per hour) and by occupational exposure to genotoxic substances (125-430 SSBs per cell) has been estimated in earlier studies. In conclusion, the frequency of SSBs induced by thyroid diagnosis with 60 MBq (99m)Tc is approximately 5 orders of magnitude smaller than the frequency of spontaneous SSBs and thus is most probably without biological relevance. Since the frequency of induction of SSBs by therapy with (131)I (3700 MBq) is about 6000-fold higher compared to thyroid diagnosis by (99m)Tc, its biological relevance is more difficult to assess. Nevertheless, the number of SSBs induced by therapy with (131)I is substantially lower than that induced by endogenous processes.

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane.

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK1, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30-300 micromol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK1 cells (100 micromol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 micromol/L. Flow cytometric analysis showed that DBCP (1-10 micromol/L) exposure for 24 h caused an accumulation of LLCPK1 cells in the G2/M-phase. In HL-60 cells the accumulation in G2/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK1 cells exposed to 100-500 micromol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G1 cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK1 cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G2/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.

Potent Protective Effect of Melatonin on Chromium(VI)-Induced DNA Single-Strand Breaks, Cytotoxicity, and Lipid Peroxidation in Primary Cultures of Rat Hepatocytes

Incubation of primary cultures of rat hepatocytes with K2Cr2O7plus the pineal hormone melatonin resulted in a marked decrease in cellular levels of DNA single-strand breaks caused by K2Cr2O7. Cellular treatment with melatonin also suppressed both dichromate-induced cytotoxicity, as evaluated by the leakage of lactate dehydrogenase, and lipid peroxidation, as monitored by malondialdehyde formation. In addition, treatment with melatonin attenuated the suppression of the levels of vitamins E and C as well as the inhibition of catalase activity attributed to K2Cr2O7. However, melatonin had no influence on cellular level of glutathione and the activity of glutathione reductase, glutathione peroxidase, superoxide dismutase, and alkaline phosphatase suppressed by dichromate. Under the same experimental conditions, cellular uptake and distribution of Cr were not affected by melatonin. Electron spin resonance (ESR) studies showed that melatonin did not affect the formation of Cr(V) complexes in the reaction of K2Cr2O7with reduced glutathione; however, melatonin caused a 25% decrease in the levels of Cr(V)-related hydroxyl radicalsin vitro.These results indicate that melatonin protects cells from Cr(VI)-induced DNA strand breaks, cytotoxicity, and lipid peroxidation, possibly through its ability to increase cellular levels of vitamins E and C as well as catalase activity and/or to directly scavenge toxic hydroxyl radicals in cells.

Protective effect of deferoxamine on chromium (VI)-induced DNA single-strand breaks, cytotoxicity, and lipid peroxidation in primary cultures of rat hepatocytes.

Incubation of primary cultures of rat hepatocytes with K2CR2O7 and deferoxamine (DFO), an iron chelator, resulted in a marked decrease in cellular levels of DNA single-strand breaks caused by K2Cr2O7. Cellular treatment with DFO also suppressed both dichromate-induced cytotoxicity--evaluated by the leakage of lactate dehydrogenase, and lipid peroxidation--as monitored by malondialdehyde formation. In addition, treatment with DFO attenuated the suppression of the levels of vitamin E and C as well as the inhibition of alkaline phosphatase and glutathione peroxidase activity attributed to K2Cr2O7. However, DFO had no influence on the cellular level of glutathione or the activity of glutathione reductase and superoxide dismutase suppressed by dichromate. Under the same experimental conditions, cellular uptake and distribution of chromium were not affected by DFO. These results indicate that DFO protects cells from chromium (VI)-induced DNA strand breaks, cytotoxicity, lipid peroxidation, vitamin E and C depression, and glutathione peroxidase inhibition The role of antioxidants in chromium (VI)-induced cytotoxicity, DNA breaks, and lipid peroxidation is discussed.

DNA damage induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5 H]-furanone (MX) in HL-60 cells and purified DNA in vitro

Chlorinated tap water often contains 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5 H]-furanone (MX), which is a potent directly acting bacterial mutagen. We have investigated the induction of DNA damage by MX in a promyelocytic human leukaemia cell line (HL-60 cells). Exposure of HL-60 cells to 100–300 μM MX resulted in increased levels of DNA single-strand breaks and/or alkali-labile sites (SSBs) as detected by alkaline filter elution. When adding inhibitors of DNA break repair (AraC plus hydroxyurea), increased levels of DNA SSBs were observed at very low concentrations (1–3 μM) of MX, as observed by both alkaline filter elution and the single-cell gel electrophoresis assay. Increased DNA SSBs could also be observed if DNA repair inhibitors were added immediately after exposure to 10 μM MX, indicating that low concentrations of MX cause a relatively stable modification of DNA that may be recognized and incised by DNA repair enzyme activities. Further studies with DNA break repair inhibitors indicated that HL-60 cells exposed to 10 μM MX for 1 h repaired 50% of their initial DNA damage during a 2-h period and the repair appeared to be complete at 22 h. Analysis of MX-treated DNA by sequencing methods indicated that MX preferentially reacts with guanines in DNA.

Basal DNA damage in individual human lymphocytes with age

A role for DNA damage is central to many theories of aging, but attempts to show an increase in DNA damage with age have yielded contradictory results. However, previous experiments have been of limited sensitivity, only able to examine induced (not basal) damage or pooled (not individual) cells. In this report, we apply a novel technique (Singh et al., 1988) to directly measure basal levels of DNA single-strand breaks and alkali-labile sites in individual human peripheral blood lymphocytes (PBL) obtained from young (less than 60 years) and old (more than 60 years) male donors. This approach shows that while average changes with age are small, changes in certain individuals and in certain cells may be large: the mean increase in damage was only 12%, but the increase in a subpopulation of highly damaged lymphocytes was 5-fold. However, most of this increase was contributed by just 3 of 17 older subjects. Further characterization of these individuals may shed light on the relationship between DNA damage and aging.

Induction and repair of DNA single-strand breaks in EM9 mutant cho cells treated with hydrogen peroxide

In this study we investigated the induction and rejoining of DNA single-strand breaks (SSBs) produced by H 2O 2 in the repair-deficient EM9 mutant Chinese hamster ovary (CHO) cell line. The effect of the poly(ADP-ribose)-transferase inhibitor 3-aminobenzamide (3-ABA) on SSB-rejoining and on cell killing was also evaluated. Results were compared with those obtained previously with the parent cell line (AA8). Cells were treated with H 2O 2 on ice for 1 h, after which they were either harvested or allowed to repair their damage at 37°C either in the presence or absence of 3-ABA (5 mM). The cells were then assayed either for survival using a colony-forming assay or for their level of DNA SSBs using alkaline elution. EM9 cells were somewhat more sensitive than AA8 cells to the cytotoxic effects of H 2O 2. However, because the repair mutant showed slightly lower levels of DNA SSBs than did its parental cell line, this sensitivity could not be explained on the basis of alterations in initial damage. The rejoining of the H 2O 2-induced DNA SSBs followed exponential kinetics in both cell lines; however, EM9 cells rejoined these breaks at a slower rate ( t 1 2 of 10 min) than did AA8 cells ( t 1 2 of 5 min). The increased sensitivity of the EM9 cells therefore appears to correlate with a reduced ability to remove these lesions from their DNA. As previously demonstrated for the AA8 cells, 3-ABA treatment resulted in both a retardation of the removal of H 2O 2-induced DNA SSBs and potentiation of cytotoxicity in the EM9 cells. However, the degree of these effects were similar for both AA8 and EM9 cells. These data provide further evidence that the cytotoxic effects of low concentrations of H 2O 2 are mediated by damage to DNA, and suggest that the rate at which DNA SSBs are rejoined is important for cell survival.