Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories.

This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).

An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.

The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/10(6) bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose-response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.

Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial.

The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.

DNA damage induced by nitrous oxide: study in medical personnel of operating rooms.

Occupational exposure to anaesthetics such as nitrous oxide (N(2)O) and halogenated hydrocarbons has been suggested to increase risk of genetic damage. However, the dose-dependency of genotoxic effects has not been unequivocally established and their relation to occupational exposure limit (OEL) remain obscure. In this study, the genotoxicity associated with occupational exposure to anaesthetics has been investigated in a group of 55 female nurses and 29 male anaesthesiologists active for at least 5 years in a working environment containing variable concentrations of N(2)O and halogenated hydrocarbons. 83 unexposed health care workers (52 female nurses and 31 male doctors) matched for age, gender, smoking habit and employment duration were included in the control group. Genotoxicity has been assessed using comet test. Concentrations of nitrous oxide, sevoflurane and isoflurane monitored by gas chromatography and mass spectrometry made possible to relate the extent of DNA damage to the level of exposure. Our results for the first time document a positive correlation between the DNA damage and the N(2)O levels in the ambient air. By contrast, no correlation has been observed between genotoxic effects and concentrations of sevoflurane and isoflurane. The extent of genetic injury was especially aggravated among nurses and anaesthesiologists exposed to N(2)O in concentrations exceeding OEL (180 mg/m(3)). We conclude that occupational exposure to N(2)O is associated with increased DNA damage and that the level of exposure plays a critical role in this regard.

Carcinogenic effect of arsenate in C57BL/6J/Han mice and its modulation by different dietary selenium status.

In this study, carcinogenic effects of arsenate in female C57BL/6J/Han mice exposed in drinking water to 50, 200 or 500microgAs/L for 24 months were investigated. All animals were fed low-selenium diet, however half of them were supplemented with sodium selenite in drinking water (200microgSe/L) to ensure the normal dietary level of selenium. Glutathione peroxidase activity in erythrocytes and plasma as well as selenium concentration in plasma after 3, 6, 12 and 18 months in satellite groups showed considerable decrease in animals from non-selenium supplemented groups in comparison to supplemented groups. A clear arsenic concentration-dependent increase in the number of malignant lymphoma associated with increase in the risk of death was observed (hazard ratio=0.91, 1.46, and 2.24, for 50, 200 and 500microgAs/L, respectively). No significant influence of selenium dietary status on arsenic carcinogenicity was shown. A significant association between selenium supplementation status and increased risk of death of the animals from causes other than malignant tumors was found (HR=1.79, p=0.04).

Interleukin-1beta expression in murine J774A.1 macrophages exposed to platinum compounds: the role of p38 and ERK 1/2 mitogen-activated protein kinases.

Although skin and respiratory sensitizing properties of platinum compounds have been proved in humans and mice, little is known about signal transduction pathways leading to cytokine production in the induction phase. It is generally assumed that induction of skin sensitization, but not skin irritation, is associated with a rapid increase in the IL-1beta mRNA expression. In this study, IL-1beta expression and a role of mitogen-activated protein kinases (MAPKs) in this process were investigated in murine macrophages J774A.1 exposed to four platinum compounds. Potassium tetrachloroplatinate (K(2)PtCl(4); TCPP), ammonium tetrachloroplatinate ((NH(4))(2)PtCl(4); TCPA), ammonium hexachloroplatinate ((NH(4))(2)PtCl(6); HCPA) showed a very similar range of cytotoxic concentrations (IC(50) values: 238 microM+/-30; 269 microM+/-39 and 245 microM+/-31, respectively) as assessed in the 24-h MTT reduction test. Cytotoxicity of cis-diammineplatinum dichloride (cisplatin) was considerably higher (IC(50) of 23 microM+/-4). While increased expression of IL-1beta mRNA was observed in the macrophages exposed to each test compound, IL-1beta protein production was detected in cell lysates after treatment with TCPP, TCPA and HCPA for 24h (concentration range of 150-350 microM) as well as for 2h (450-650 microM). The treatment with each compound resulted in the phosphorylation of both p38 MAPK and ERK 1/2 (p44/42). Blocking the activation of p38 MAPK as well as ERK 1/2 with specific inhibitors (SB203580 and U0126, respectively) down-regulated the IL-1beta expression. Interestingly, the skin irritant sodium dodecyl sulfate did not trigger phosphorylation of these kinases, nor induced IL-1beta production. These data suggest that p38 MAPK and ERK 1/2 play an important role in induction of IL-1beta expression in J774A.1 macrophages exposed to test platinum compounds.

Biomonitoring of cyanobacterial blooms in Polish water reservoir and the cytotoxicity and genotoxicity of selected cyanobacterial extracts.

OBJECTIVES: Water pollution with toxic cyanobacterial blooms is a worldwide problem. Cyanobacteria species that mainly produce microcystins predominate in Polish water reservoirs. MATERIALS AND METHODS: In our study, cyanobacterial blooms were monitored during summer of 2004 in the Sulejów reservoir. The concentration of microcystins in water and cyanobacterial cells were determined using liquid chromatography and immunobiotests, while the biological activity of microcystic cyanobacterial extracts was assessed using bacterial tests (SOS Chromotest, UMU test), the comet assay and micronucleus test with human lymphocytes. RESULTS: It was revealed that cyanobacterial bloom was most intensive in mid August and lasted until the end of September. Microcystis aeruginosa and Aphanizomenon flos-aquae dominated in the blooms. The highest concentration of microcystins in cyanobacterial cells was also observed at that time. The concentration of microcystins in water did not exceed 1 microg/l. All cyanobacterial extracts showed weak genotoxicity only for Escherichia coli PQ37. The cyanobacterial extracts prepared at the beginning of September were most toxic to human lymphocytes, the effective microcystin extracts (EC50) concentration was about two or three times lower compared to the other extracts. The level of DNA damage in lymphocytes after short exposure to microcystic extracts (3 and 6 h) was significantly higher than respective levels after longer exposure. The microcystins of cyanobacterial blooms induced a slight increase in micronuclei frequencies in human lymphocytes. CONCLUSION: Phytoplankton biomass and the genotoxicity of massive cyanobacterial blooms should be assessed for eucariotic cells in the Sulejów reservoir to avoid the hazard induced by cyanobacterial blooms.

Genotoxic effects in C57Bl/6J mice chronically exposed to arsenate in drinking water and modulation of the effects by low-selenium diet.

In C57Bl/6J mice chronically exposed to arsenate in drinking water at 50, 200, or 500 microg As/L, genotoxic effects in bone-marrow cells using micronucleus test and in peripheral blood leukocytes using the comet assay were determined after 3, 6 or 12 mo. To assess the modulating role of selenium in development of the effects, the animals were fed a specially prepared low-selenium diet and were supplemented with sodium selenite (200 microg Se/L) in drinking water (supplemented groups) or were without Se supplementation (nonsupplemented groups). Measurements of glutathione peroxidase activity in erythrocytes and plasma as well as selenium concentration in plasma were performed after 3, 6, and 12 mo and showed a marked decrease in values in animals in non-Se supplemented compared to Se-supplemented groups. After 3 mo of arsenic exposure in the Se-supplemented animals the level of DNA fragmentation (without Endo III and Fpg enzymes) did not differ from the control; however, increased oxidative damage of purine and pyrimidine bases was observed. In groups not supplemented with Se, an increase of DNA fragmentation was observed; however, the levels of oxidative DNA damage in these groups did not differ from the control. None of the positive effects observed in the comet assay after 3 mo was related to arsenate concentration. The levels of DNA damage after 6 and 12 mo of exposure to arsenic as well as the frequency of micronuclei after 3, 6, and 12 mo did not differ significantly between exposed and control animals, irrespective of Se supplementation status.

DNA damage in leukocytes of workers occupationally exposed to arsenic in copper smelters.

Inorganic arsenic (i-As) is a known human carcinogen; however, humans continue to be exposed to i-As in drinking water and in certain occupational settings. In this study, we used the Comet assay to evaluate DNA damage in the somatic cells of workers from three Polish copper smelters who were occupationally exposed to i-As. Blood samples were collected from 72 male workers and 83 unexposed male controls and used for the detection of DNA damage, oxidative DNA damage, and DNA damage after a 3-hr incubation in culture. Urine samples were collected to assess the level of exposure. The mean concentration of arsenic metabolites in urine [the sum of arsenite (AsIII), arsenate (AsV), monomethylarsenate (MMA) and dimethylarsenate (DMA)] and the concentrations of DMA (the main metabolite in urine) were higher in workers than in controls, but the differences were not statistically significant. By contrast, the level of DNA damage, expressed as the median tail moment, was significantly higher in the leukocytes of workers than in the controls. Comet assays conducted with formamidopyrimidine glycosylase (FPG) digestion to detect oxidative DNA damage indicated that oxidative lesions were present in leukocytes from both the exposed and control groups, but the levels of damage were significantly higher among the workers. Incubation of the cells in culture resulted in a significant reduction in the levels of DNA damage, especially among leukocytes from the workers, suggesting that the DNA damage was subject to repair. Our findings indicate that copper smelter workers have increased levels of DNA damage in somatic cells, suggesting a potential health risk for the workers. Although i-As was present in air samples from the smelters and in urine samples from workers, no clear association could be made between i-As exposure and the DNA damage.

The effect of selenium supplementation in the prevention of DNA damage in white blood cells of hemodialyzed patients: a pilot study.

Patients with chronic kidney disease (CKD) have an increased incidence of cancer. It is well known that long periods of hemodialysis (HD) treatment are linked to DNA damage due to oxidative stress. In this study, we examined the effect of selenium (Se) supplementation to CKD patients on HD on the prevention of oxidative DNA damage in white blood cells. Blood samples were drawn from 42 CKD patients on HD (at the beginning of the study and after 1 and 3 months) and from 30 healthy controls. Twenty-two patients were supplemented with 200 µg Se (as Se-rich yeast) per day and 20 with placebo (baker's yeast) for 3 months. Se concentration in plasma and DNA damage in white blood cells expressed as the tail moment, including single-strand breaks (SSB) and oxidative bases lesion in DNA, using formamidopyrimidine glycosylase (FPG), were measured. Se concentration in patients was significantly lower than in healthy subjects (P < 0.0001) and increased significantly after 3 months of Se supplementation (P < 0.0001). Tail moment (SSB) in patients before the study was three times higher than in healthy subjects (P < 0.01). After 3 months of Se supplementation, it decreased significantly (P < 0.01) and was about 16% lower than in healthy subjects. The oxidative bases lesion in DNA (tail moment, FPG) of HD patients at the beginning of the study was significantly higher (P < 0.01) compared with controls, and 3 months after Se supplementation it was 2.6 times lower than in controls (P < 0.01). No changes in tail moment was observed in the placebo group. In conclusion, our study shows that in CKD patients on HD, DNA damage in white blood cells is higher than in healthy controls, and Se supplementation prevents the damage of DNA.

Evaluation of biological effects of nanomaterials. Part I. Cyto- and genotoxicity of nanosilver composites applied in textile technologies.

OBJECTIVES: The aim of this study was to investigate the cyto- and genotoxicity of nanocomposites (NCs) and generation of reactive oxygen species (ROS) as a result of particle-cell interactions. MATERIALS AND METHODS: Titanium dioxide (TiO(2)-Ag) and ion-exchange resin (Res-Ag), both coated with silver (Ag), were examined. The murine macrophage J774A.1 cells were incubated in vitro with NC at different concentrations for 24 h. Cytotoxicity was analyzed by the methylthiazolyldiphenyl-tetrazolium bromide reduction test (MTT reduction test). ROS generation was assessed by incubation of cells with dichlorodihydrofluorescein diacetate (DCF) and flow cytometry. DNA damage was detected by comet assay and included single-strand breaks (SSB), alkali-labile sites (ALS) and oxidative DNA damage after formamidopyrimidine glycosylase (FPG) treatment. The tail moment was used as an indicator of DNA damage. RESULTS: TiO(2)-Ag was not cytotoxic up to 200 µg/ml, whereas IC(50) for Res-Ag was found to be 23 µg/ml. Intracellular ROS levels were elevated after 4 h of exposure to Res-Ag at the concentration of 50 µg/ml. Both types of NC induced fragmentation of DNA strands, but only one of the composites caused damage to purine bases. TiO(2)-Ag induced SSB of DNA at concentrations of 10 and 5 µg/ml. For Res-Ag, a concentration-dependent increase in tail moments was observed. CONCLUSIONS: Silver-coated nanocomposites (both TiO(2)-Ag and Res-Ag) may cause genotoxic effects in murine macrophages J774A.1. Res-Ag increased generation of ROS which suggested that toxicity of Res-Ag in murine macrophages is likely to be mediated through oxidative stress. This paper will support industry and regulators alike in the assessment of hazards and risks and methods for their mitigation at the earliest possible stage in material and product development.

Assessment of the protective effects of selected dietary anticarcinogens against DNA damage and cytogenetic effects induced by benzo[a]pyrene in C57BL/6J mice.

The protective action in C57BL/6J mice from orally administered ellagic acid (EA), benzyl isothiocyanate (BITC), an extract of epigallocatechins (Tegreen®) as well as chlorophyllin (CHL) against benzo[a]pyrene (B[a]P)-induced DNA damage and cytogenetic effects was investigated. In pilot experiment the comet assay indicated protective effects for all compounds, while such activity was confined to EA and CH with respect to B[a]P-DNA adducts and micronuclei. EA and CH were chosen for the main study where the levels of DNA adducts in liver after injection of 30 mg B[a]P/kg b.w. did not differ from those found for animals exposed to B[a]P and treated with the protective substances. In leukocytes no significant protective effect of CHL was detected while a 2-fold increase of adduct concentrations was observed after co-administration of EA. In the comet assay CHL or EA caused a 3-fold decrease of SSB, and a 2-fold decrease of FPG sites in comparison to animals treated with B[a]P. CHL or EA showed a significant protective effect against B[a]P-induced MN in polychromatic erythrocytes in bone marrow. In contrast, flow cytometry measurements in peripheral blood indicated the MN frequency after treatment with CHL or EA almost twice as high as that recorded for B[a]P alone.

Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity.

Genotoxicity of commercial colloidal and laboratory-synthesized silica nanoparticles was tested using the single cell gel electrophoresis or Comet assay. By using a carefully developed protocol and careful characterization of the nanoparticle dispersions, Comet assays were performed on 3T3-L1 fibroblasts with 3, 6, and 24 h incubations and 4 or 40 microg/ml of silica nanoparticles. No significant genotoxicity was observed for the nanoparticles tested under the conditions described, and results were independently validated in two separate laboratories, showing that in vitro toxicity testing can be quantitatively reproducible.

Effects of in vitro exposure to power frequency magnetic fields on UV-induced DNA damage of rat lymphocytes.

The mechanisms of biological effects of 50/60 Hz (power frequency) magnetic fields (MF) are still poorly understood. There are a number of studies indicating that MF affect biochemical processes in which free radicals are involved, such as the biological objects' response to ultraviolet radiation (UVA). Therefore, the present study was aimed to assess the effect of 50 Hz MFs on the oxidative deterioration of DNA in rat lymphocytes irradiated in vitro by UVA. UVA radiation (150 J/m2) was applied for 5 min for all groups and 50 Hz MF (40 microT rms) exposure was applied for some of the groups for 5 or 60 min. The level of DNA damage was assessed using the alkaline comet assay, the fluorescence microscope, and image analysis. It has been found that the 1 h exposure to MF caused an evident increase in all parameters consistent with damaged DNA. This suggest that MF affects the radical pairs generated during the oxidative or enzymatic processes of DNA repair.