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).

DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).

The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.

An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells.

The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.

Inter-laboratory variation in DNA damage using a standard comet assay protocol.

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

Controlled human wood smoke exposure: oxidative stress, inflammation and microvascular function.

BACKGROUND: Exposure to wood smoke is associated with respiratory symptoms, whereas knowledge on systemic effects is limited. We investigated effects on systemic inflammation, oxidative stress and microvascular function (MVF) after controlled wood smoke exposure. METHODS: In a randomised, double-blinded, cross-over study 20 non-smoking atopic subjects were exposed at rest to 14, 220, or 354 µg/m3 of particles from a well-burning modern wood stove for 3 h in a climate controlled chamber with 2 week intervals. We investigated the level of oxidatively damaged DNA, inflammatory markers and adhesion molecules before and 0, 6 and 20 h after exposure. Six h after exposure we measured MVF non-invasively by digital peripheral artery tonometry following arm ischemia. RESULTS: The MVF score was unaltered after inhalation of clean air (1.58 ± 0.07; mean ± SEM), low (1.51 ± 0.07) or high (1.61 ± 0.09) concentrations of wood smoke particles in atopic subjects, whereas unexposed non-atopic subjects had higher score (1.91 ± 0.09). The level of oxidatively damaged DNA, mRNA of ITGAL, CCL2, TNF, IL6, IL8, HMOX1, and OGG1 and surface marker molecules ICAM1, ITGAL and L-selectin in peripheral blood mononuclear cells were not affected by inhalation of wood smoke particles. CONCLUSIONS: Exposure to wood smoke had no effect on markers of oxidative stress, DNA damage, cell adhesion, cytokines or MVF in atopic subjects.

Expression of adhesion molecules, monocyte interactions and oxidative stress in human endothelial cells exposed to wood smoke and diesel exhaust particulate matter.

Toxicological effects of wood smoke particles are less investigated than traffic-related combustion particles. We investigated the effect of wood smoke particles, generated by smouldering combustion conditions, on human umbilical endothelial cells (HUVECs) co-cultured with or without monocytic THP-1 cells. Standard reference material (SRM) 2975 diesel exhaust particles were used as benchmark particles. Wood smoke particles at 50 µg/ml or 100 µg/ml caused adhesion of THP-1 monocytes onto HUVECs in co-cultures, whereas SRM2975 had no such effect. Both types of particles from 1 µg/ml increased VCAM-1 expression on HUVECs in mono-cultures. However, only the exposure to wood smoke particles was associated with increased expression of TNF and IL8 mRNA in THP-1 cells. We found no effect on the intracellular production of reactive oxygen species by the fluorescent probe DCFH-DA, whereas especially the wood smoke particles caused increased level of DNA strand breaks and oxidised guanines at concentrations with low cytotoxicity. In conclusion, our results indicate that the adherence of monocytes on endothelial cells in wood smoke particle exposed cultures depend on activation of both cell types.

Hazard identification of particulate matter on vasomotor dysfunction and progression of atherosclerosis.

The development and use of nanoparticles have alerted toxicologists and regulators to issues of safety testing. By analogy with ambient air particles, it can be expected that small doses are associated with a small increase in risk of cardiovascular diseases, possibly through oxidative stress and inflammatory pathways. We have assessed the effect of exposure to particulate matter on progression of atherosclerosis and vasomotor function in humans, animals, and ex vivo experimental systems. The type of particles that have been tested in these systems encompass TiO(2), carbon black, fullerene C(60), single-walled carbon nanotubes, ambient air particles, and diesel exhaust particles. Exposure to ambient air particles is associated with accelerated progression of atherosclerosis and vasomotor dysfunction in both healthy and susceptible animal models and humans at risk of developing cardiovascular diseases. The vasomotor dysfunction includes increased vasoconstriction as well as reduced endothelium-dependent vasodilatation; endothelium-independent vasodilatation is often unaffected indicating mainly endothelial dysfunction. Pulmonary exposure to TiO(2), carbon black, and engineered nanoparticles generate vasomotor dysfunction; the effect size is similar to that generated by combustion-derived particles, although the effect could depend on the exposure period and the administered dose, route, and mode. The relative risk associated with exposure to nanoparticles may be small compared to some traditional risk factors for cardiovascular diseases, but superimposed on these and possible exposure to large parts of the population it is a significant public health concern. Overall, assessment of vasomotor dysfunction and progression of atherosclerosis are promising tools for understanding the effects of particulate matter.

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.

Role of oxidative damage in toxicity of particulates.

Particulates are small particles of solid or liquid suspended in liquid or air. In vitro studies show that particles generate reactive oxygen species, deplete endogenous antioxidants, alter mitochondrial function and produce oxidative damage to lipids and DNA. Surface area, reactivity and chemical composition play important roles in the oxidative potential of particulates. Studies in animal models indicate that particles from combustion processes (generated by combustion of wood or diesel oil), silicate, titanium dioxide and nanoparticles (C60 fullerenes and carbon nanotubes) produce elevated levels of lipid peroxidation products and oxidatively damaged DNA. Biomonitoring studies in humans have shown associations between exposure to air pollution and wood smoke particulates and oxidative damage to DNA, deoxynucleotides and lipids measured in leukocytes, plasma, urine and/or exhaled breath. The results indicate that oxidative stress and elevated levels of oxidatively altered biomolecules are important intermediate endpoints that may be useful markers in hazard characterization of particulates.

Biomarkers of oxidative damage to DNA and repair.

Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation, which is less likely to occur with methods such as the comet assay, which are based on nicking of the DNA strand at modified bases, but offer less specificity. The European Standards Committee on Oxidative DNA Damage has concluded that the true levels of the most widely studied lesion, 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine), in cellular DNA is between 0.5 and 5 lesions per 10(6) dG bases. Base excision repair of oxidative damage to DNA can be assessed by nicking assays based on oligonucleotides with lesions or the comet assay, by mRNA expression levels or, in the case of, e.g., OGG1 (8-oxoguanine DNA glycosylase 1), responsible for repair of 8-oxodG, by genotyping. Products of repair in DNA or the nucleotide pool, such as 8-oxodG, excreted into the urine can be assessed by MS-based methods and generally reflects the rate of damage. Experimental and population-based studies indicate that many environmental factors, including particulate air pollution, cause oxidative damage to DNA, whereas diets rich in fruit and vegetables or antioxidant supplements may reduce the levels and enhance repair. Urinary excretion of 8-oxodG, genotype and expression of OGG1 have been associated with risk of cancer in cohort settings, whereas altered levels of damage, repair or urinary excretion in case-control settings may be a consequence rather than the cause of the disease.

Air pollution, oxidative damage to DNA, and carcinogenesis.

There is growing concern that air pollution exposure increases the risk of lung cancer. The mechanism of action is related to particle-induced oxidative stress and oxidation of DNA. Humans exposed to urban air with vehicle emissions have elevated levels of oxidized guanine bases in blood cells and urine. Animal experimental studies show that pulmonary and gastrointestinal exposure is associated with elevated levels of oxidized guanines in the lung and other organs. Collectively, there is evidence indicating that exposure to traffic-related air pollution particles is associated with oxidative damage to DNA and this might be associated with increased risk of cancer.

Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring.

The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.

Indoor particles affect vascular function in the aged: an air filtration-based intervention study.

RATIONALE: Exposure to particulate matter is associated with risk of cardiovascular events, possibly through endothelial dysfunction, and indoor air may be most important. OBJECTIVES: We investigated effects of controlled exposure to indoor air particles on microvascular function (MVF) as the primary endpoint and biomarkers of inflammation and oxidative stress as secondary endpoints in a healthy elderly population. METHODS: A total of 21 nonsmoking couples participated in a randomized, double-blind, crossover study with two consecutive 48-hour exposures to either particle-filtered or nonfiltered air (2,533-4,058 and 7,718-12,988 particles/cm(3), respectively) in their homes. MEASUREMENTS AND MAIN RESULTS: MVF was assessed noninvasively by measuring digital peripheral artery tone after arm ischemia. Secondary endpoints included hemoglobin, red blood cells, platelet count, coagulation factors, P-selectin, plasma amyloid A, C-reactive protein, fibrinogen, IL-6, tumor necrosis factor-alpha, protein oxidation measured as 2-aminoadipic semialdehyde in plasma, urinary 8-iso-prostaglandin F(2alpha), and blood pressure. Indoor air filtration significantly improved MVF by 8.1% (95% confidence interval, 0.4-16.3%), and the particulate matter (diameter < 2.5 mum) mass of the indoor particles was more important than the total number concentration (10-700 nm) for these effects. MVF was significantly associated with personal exposure to iron, potassium, copper, zinc, arsenic, and lead in the fine fraction. After Bonferroni correction, none of the secondary biomarkers changed significantly. CONCLUSIONS: Reduction of particle exposure by filtration of recirculated indoor air for only 48 hours improved MVF in healthy elderly citizens, suggesting that this may be a feasible way of reducing the risk of cardiovascular disease.

Exposure to ultrafine particles from ambient air and oxidative stress-induced DNA damage.

BACKGROUND: Particulate matter, especially ultrafine particles (UFPs), may cause health effects through generation of oxidative stress, with resulting damage to DNA and other macromolecules. OBJECTIVE: We investigated oxidative damage to DNA and related repair capacity in peripheral blood mononuclear cells (PBMCs) during controlled exposure to urban air particles with assignment of number concentration (NC) to four size modes with average diameters of 12, 23, 57, and 212 nm. DESIGN: Twenty-nine healthy adults participated in a randomized, two-factor cross-over study with or without biking exercise for 180 min and with exposure to particles (NC 6169-15362/cm(3)) or filtered air (NC 91-542/cm(3)) for 24 hr. METHODS: The levels of DNA strand breaks (SBs), oxidized purines as formamidopyrimidine DNA glycolase (FPG) sites, and activity of 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1) in PBMCs were measured by the Comet assay. mRNA levels of OGG1, nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), and heme oxygenase-1 (HO1) were determined by real-time reverse transcriptase-polymerase chain reaction. RESULTS: Exposure to UFPs for 6 and 24 hr significantly increased the levels of SBs and FPG sites, with a further insignificant increase after physical exercise. The OGG1 activity and expression of OGG1, NUDT1, and HO1 were unaltered. There was a significant dose-response relationship between NC and DNA damage, with the 57-nm mode as the major contributor to effects. Concomitant exposure to ozone, nitrogen oxides, and carbon monoxide had no influence. CONCLUSION: Our results indicate that UFPs, especially the 57-nm soot fraction from vehicle emissions, causes systemic oxidative stress with damage to DNA and no apparent compensatory up-regulation of DNA repair within 24 hr.