The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level of DNA damage and formamidopyrimidine DNA glycosylase-sensitive sites but the laboratories could detect concentration-dependent relationships in coded samples. Standardization of the results with reference standards decreased the inter-laboratory variation. The ECVAG trail indicates substantial reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation.

Nucleotide-excision repair (NER) is important for the maintenance of genomic integrity and to prevent the onset of carcinogenesis. Oxidative stress was previously found to inhibit NER in vitro, and dietary antioxidants could thus protect DNA not only by reducing levels of oxidative DNA damage, but also by protecting NER against oxidative stress-induced inhibition. To obtain further insight in the relation between oxidative stress and NER activity in vivo, oxidative stress was induced in newborn piglets by means of intramuscular injection of iron (200mg) at day 3 after birth. Indeed, injection of iron significantly increased several markers of oxidative stress, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels in colon DNA and urinary excretion of 8-oxo-7,8-dihydroguanine (8-oxoGua). In parallel, the influence of maternal supplementation with an antioxidant-enriched diet was investigated in their offspring. Supplementation resulted in reduced iron concentrations in the colon (P=0.004) at day 7 and a 40% reduction of 8-oxodG in colon DNA (P=0.044) at day 14 after birth. NER capacity in animals that did not receive antioxidants was significantly reduced to 32% at day 7 compared with the initial NER capacity on day 1 after birth. This reduction in NER capacity was less pronounced in antioxidant-supplemented piglets (69%). Overall, these data indicate that NER can be reduced by oxidative stress in vivo, which can be compensated for by antioxidant supplementation.

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.

Chronic pulmonary inflammation is associated with increased lung cancer risk, but the underlying process remains unknown. Recently, we showed that activated neutrophils inhibit nucleotide excision repair (NER) in pulmonary epithelial cells in vitro via the release of myeloperoxidase (MPO). To evaluate the effect of neutrophils on NER in vivo, mice were intratracheally instilled with lipopolysaccharide (LPS)(20 mug), causing acute lung inflammation and associated neutrophil influx into the airways. Three days post-exposure, phenotypical NER capacity was assessed in lung tissue homogenate. LPS exposure inhibited pulmonary NER by approximately 50%. This finding was corroborated by down-regulation of the NER-associated genes Xpa and Xpf. To further elicit the role of neutrophils and MPO in this process, we utilized MPO-deficient mice as well as mice in which circulating neutrophils were depleted by antibody treatment. LPS-induced inhibition of pulmonary NER was not affected by either Mpo(-/-) or by depletion of circulating neutrophils. This contrasts with our previous in vitro observations, suggesting that inhibition of pulmonary NER following acute dosing with LPS is not fully mediated by neutrophils and/or MPO. In conclusion, these data show that LPS-induced pulmonary inflammation is associated with a reduction of NER function in the mouse lung.

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.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory condition characterized by oxidative stress and the formation of volatile organic compounds (VOCs) secreted via the lungs. We recently developed a methodological approach able to identify profiles of VOCs in breath unique for patient groups. Here we applied this recently developed methodology regarding diagnosis of COPD patients. METHODS: Fifty COPD patients and 29 controls provided their breath and VOCs were analyzed by gas chromatography-mass spectrometry to identify relevant VOCs. An additional 16 COPD patients and 16 controls were sampled in order to validate the model, and 15 steroid naïve COPD patients were sampled to determine whether steroid use affects performance. FINDINGS: 1179 different VOCs were detected, of which 13 were sufficient to correctly classify all 79 subjects. Six of these 13 VOCs classified 92% of the subjects correctly (sensitivity: 98%, specificity: 88%) and correctly classified 29 of 32 subjects (sensitivity: 100%, specificity: 81%) from the independent validation population. Fourteen out of 15 steroid naïve COPD patients were correctly classified thus excluding treatment influences. INTERPRETATION: This is the first study distinguishing COPD subjects from controls solely based on the presence of VOCs in breath. Analysis of VOCs might be highly relevant for diagnosis of COPD.

Chronic inflammation has been recognized as a contributing factor in the pathogenesis of lung cancer. In this process, reactive oxygen species released by neutrophils may play an important role. The aim of the present study was to investigate the capacity of the major neutrophilic oxidant hypochlorous acid (HOCl), which is formed by myeloperoxidase (MPO), to induce DNA damage and mutagenicity in lung cells. HOCl was mutagenic in lung epithelial A549 cells in vitro, showing at physiological concentrations a significant induction of mutations in the HPRT gene. We studied three major types of DNA lesions that could be relevant for this HOCl-induced mutagenicity. Single strand DNA breakage and 8-oxo-7,8-dihydro-2'-deoxyguanosine were not found to be increased following HOCl treatment. On the other hand, HOCl caused a significant increase in the formation of 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M(1)dG), which can be formed by either malondialdehyde (MDA) or base propenals. We observed an increased MDA formation upon exposure of A549 cells to HOCl, but a role of base propenals cannot be excluded. In line with this, we observed 4-fold increased M(1)dG adduct levels in mice that were intratracheally instilled with lipopolysaccharide to induce a pulmonary inflammation with neutrophil influx. Depletion of circulating neutrophils significantly reduced pulmonary MPO activity as well as M(1)dG adducts levels, thereby providing a causal link between neutrophils/HOCl and pulmonary genotoxicity in vivo. Taken together, these data indicate that MPO catalysed formation of HOCl during lung inflammation should be considered as a significant source of neutrophil-induced genotoxicity.

Gene-environment interactions determine inter-individual variations in nucleotide excision repair (NER) capacity. Oxidative stress was previously found to inhibit NER, thus supplementation with dietary antioxidants could prevent this inhibition, especially in genetically susceptible subjects. To study the effects of genetic polymorphisms in NER-related genes and dietary intake of antioxidants on an individual's NER capacity, lymphocytes of 168 subjects were isolated before and after a 4-week blueberry and apple juice intervention. Twelve genetic polymorphisms in NER genes XPA, XPC, ERCC1, ERCC2, ERCC5, ERCC6 and RAD23B were assessed by multiplex PCR with single base extension. Based on specific genotype combinations, a subset of individuals (n 36) was selected for phenotypical assessment of NER capacity, which was significantly affected by the total sum of low-activity alleles (P = 0.027). The single polymorphism XPA G23A was the strongest predictor of NER capacity (P = 0.002); carriers of low-activity alleles AA had about three times lower NER capacity than XPA GG carriers. NER capacity assessed before and after intervention correlated significantly (R2 0.69; P < 0.001), indicating that inter-individual differences in NER capacity are maintained over 4 weeks. Although the intervention increased plasma trolox equivalent antioxidant capacity from 791 (se 6.61) to 805 (se 7.90) mum (P = 0.032), on average it did not affect NER capacity. Nonetheless, carriers of twelve or more low-activity alleles seemed to benefit from the intervention (P = 0.013). Among these, carriers of the variant allele for RAD23B Ala249Val showed improved NER capacity upon intervention (P = 0.020). In conclusion, improved NER capacity upon dietary intervention was detected in individuals carrying multiple low-activity alleles. The XPA G23A polymorphism might be a predictor for NER capacity.

Summary Background The correct diagnosis of asthma in young children is often hard to achieve, resulting in undertreatment of asthmatic children and overtreatment in transient wheezers. Objectives To develop a new diagnostic tool that better discriminates between asthma and transient wheezing and that leads to a more accurate diagnosis and hence less undertreatment and overtreatment. A first stage in the development of such a tool is the ability to discriminate between asthmatic children and healthy controls. The integrative analysis of large numbers of volatile organic compounds (VOC) in exhaled breath has the potential to discriminate between various inflammatory conditions of the respiratory tract. Methods Breath samples were obtained and analysed for VOC by gas chromatography-mass spectrometry from asthmatic children (n=63) and healthy controls (n=57). A total of 945 determined compounds were subjected to discriminant analysis to find those that could discriminate diseased from healthy children. A set of samples from both asthmatic and healthy children was selected to construct a model that was subsequently used to predict the asthma or the healthy status of a test group. In this way, the predictive value of the model could be tested. Measurements and main results The discriminant analyses demonstrated that asthma and healthy groups are distinct from one another. A total of eight components discriminated between asthmatic and healthy children with a 92% correct classification, achieving a sensitivity of 89% and a specificity of 95%. Conclusion The results show that a limited number of VOC in exhaled air can well be used to distinguish children with asthma from healthy children.

Beta-carotene (BC) was found to enhance lung cancer risk in smokers. This adverse effect was unexpected because BC was thought to act as an anti-oxidant against cigarette smoke derived radicals. These radicals can directly or indirectly damage DNA, leading to the formation of pro-mutagenic DNA lesions such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 3-(2-deoxy-beta-D-erythro-pentafuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one deoxyguanosine (M(1)dG). Later, it was suggested that high concentrations of BC could also result in pro-oxidant effects. Therefore, we investigated whether high but physiologically feasible concentrations of BC were able to alter i. the formation of radicals in vitro assessed by electron spin resonance spectroscopy (ESR), ii. the levels of 8-oxo-dG and M(1)dG in vitro in lung epithelial cells after incubation with H(2)O(2) and the smoke derived carcinogen benzo[a]pyrene (B[a]P) and iii. the levels of 8-oxo-dG and M(1)dG in vivo in ferrets' lung after chronic exposure to B[a]P. BC increased in vitro hydroxyl radical formation in the Fenton reaction, but inhibited the formation of carbon centered radicals. Similarly, BC was able to enhance 8-oxo-dG in vitro in lung epithelial cells. On the other hand, BC significantly inhibited M(1)dG formation in lung epithelial cells, especially after induction of M(1)dG by H(2)O(2) or B[a]P. Finally, BC supplementation of ferrets also resulted in a significant decrease in M(1)dG, but in contrast to the in vitro experiments, no effect was observed on 8-oxo-dG levels, probably because of increased base excision repair capacities (BER) as assessed by a modified comet assay. These data indicate that the fate of BC being a pro- or anti-oxidant strongly depends on the type of radical involved.