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

The intra-laboratory comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However,reduced few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In in this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided radiation image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard by samples were irradiated with ionizing radiation ( -10 Gy) and used to construct a calibration curve to calculate the number of standard lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy),Levene's but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an base inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared 28%, in each laboratory reduced the CV to 28%, a statistically significant reduction (P < .05, Levene's test). A large fraction radiation of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation the between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of CV DNA damage measured by the alkaline version of the comet assay.

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

Chronic as inflammation has been recognized as a contributing factor in the pathogenesis of lung cancer. In this process, reactive oxygen species increased released by neutrophils may play an important role. The aim of the present study was to investigate the capacity of we the major neutrophilic oxidant hypochlorous acid (HOCl), which is formed by myeloperoxidase (MPO), to induce DNA damage and mutagenicity in types lung cells. HOCl was mutagenic in lung epithelial A549 cells in vitro, showing at physiological concentrations a significant induction of be mutations in the HPRT gene. We studied three major types of DNA lesions that could be relevant for this HOCl-induced HPRT 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,to 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 not base propenals. We observed an increased MDA formation upon exposure of A549 cells to HOCl, but a role of base in propenals cannot be excluded. In line with this, we observed 4-fold increased M(1)dG adduct levels in mice that were intratracheally types instilled with lipopolysaccharide to induce a pulmonary inflammation with neutrophil influx. Depletion of circulating neutrophils significantly reduced pulmonary MPO activity aim as well as M(1)dG adducts levels, thereby providing a causal link between neutrophils/HOCl and pulmonary genotoxicity in vivo. Taken together,adduct these data indicate that MPO catalysed formation of HOCl during lung inflammation should be considered as a significant source of induction neutrophil-induced genotoxicity.

Gene-environment these, interactions determine inter-individual variations in nucleotide excision repair (NER) capacity. Oxidative stress was previously found to inhibit NER, thus supplementation 6.61) with dietary antioxidants could prevent this inhibition, especially in genetically susceptible subjects. To study the effects of genetic polymorphisms in capacity NER-related genes and dietary intake of antioxidants on an individual's NER capacity, lymphocytes of 168 subjects were isolated before and (n after a 4-week blueberry and apple juice intervention. Twelve genetic polymorphisms in NER genes XPA, XPC, ERCC1, ERCC2, ERCC5, ERCC6 intervention and RAD23B were assessed by multiplex PCR with single base extension. Based on specific genotype combinations, a subset of individuals specific (n 36) was selected for phenotypical assessment of NER capacity, which was significantly affected by the total sum of low-activity affect alleles (P = .027). The single polymorphism XPA G23A was the strongest predictor of NER capacity (P = .002); carriers = of low-activity alleles AA had about three times lower NER capacity than XPA GG carriers. NER capacity assessed before and 7.90) after intervention correlated significantly (R2 .69; P < .001), indicating that inter-individual differences in NER capacity are maintained over 4 (n weeks. Although the intervention increased plasma trolox equivalent antioxidant capacity from 791 (se 6.61) to 805 (se 7.90) mum (P To = .032), on average it did not affect NER capacity. Nonetheless, carriers of twelve or more low-activity alleles seemed to 805 benefit from the intervention (P = .013). Among these, carriers of the variant allele for RAD23B Ala249Val showed improved NER single capacity upon intervention (P = .020). In conclusion, improved NER capacity upon dietary intervention was detected in individuals carrying multiple polymorphisms low-activity alleles. The XPA G23A polymorphism might be a predictor for NER capacity.

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

Beta-carotene effect (BC) was found to enhance lung cancer risk in smokers. This adverse effect was unexpected because BC was thought to epithelial act as an anti-oxidant against cigarette smoke derived radicals. These radicals can directly or indirectly damage DNA, leading to the formation 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 spectroscopy of BC could also result in pro-oxidant effects. Therefore, we investigated whether high but physiologically feasible concentrations of BC were other able to alter i. the formation of radicals in vitro assessed by electron spin resonance spectroscopy (ESR), ii. the levels in 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 supplementation (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 smoke increased in vitro hydroxyl radical formation in the Fenton reaction, but inhibited the formation of carbon centered radicals. Similarly, BC induction was able to enhance 8-oxo-dG in vitro in lung epithelial cells. On the other hand, BC significantly inhibited M(1)dG formation spectroscopy 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 can in a significant decrease in M(1)dG, but in contrast to the in vitro experiments, no effect was observed on 8-oxo-dG especially levels, probably because of increased base excision repair capacities (BER) as assessed by a modified comet assay. These data indicate i. that the fate of BC being a pro- or anti-oxidant strongly depends on the type of radical involved.

Benzo(a)pyrene mice (B[a]P) can induce somatic mutations, whereas its potential to induce germ cell mutations is unclear. There is circumstantial evidence that period paternal exposure to B[a]P can result in germ cell mutations. Since DNA adducts are thought to be a prerequisite for entire B[a]P induced mutations, we studied DNA adduct kinetics by (32)P-postlabeling in sperm, testes and lung tissues of male mice after In a single exposure to B[a]P (13 mg/kg bw, by gavage). To investigate DNA adduct formation at different stages of spermatogenesis,wild mice were sacrificed at Day 1, 4, 7, 10, 14, 21, 32, and 42 after exposure. In addition, DNA repair 14, deficient (Xpc(-/-)) mice were used to study the contribution of nucleotide excision repair in DNA damage removal. DNA adducts were were detectable with highest levels in lung followed by sperm and testis. Maximum adduct levels in the lung and testis were DNA observed at Day 1 after exposure, while adduct levels in sperm reached maximum levels at approximately 1 week after exposure. .05). Lung tissue and testis of Xpc(-/-) mice contained significantly higher DNA adduct levels compared to wild type (Wt) mice over In the entire 42 day observation period (P < .05). Differences in adduct half-life between Xpc(-/-) and Wt mice were only DNA observed in testis. In sperm, DNA adduct levels were significantly higher in Xpc(-/-) mice than in Wt mice only at (P Day 42 after exposure (P = .01). These results indicate that spermatogonia and testes are susceptible for the induction of Day DNA damage and rely on nucleotide excision repair for maintaining their genetic integrity. Environ. Mol. Mutagen. 2009.(c) 2009 Wiley-Liss,be Inc.

Paternal NF-?B exposure to genotoxic compounds is thought to contribute to diseases in their offspring. Therefore, it is of importance to develop factor biomarkers of male germ cell exposure to genotoxins. Unfortunately, the testis cannot be reached for routine biomonitoring, but mRNA-profiles in germ spermatozoa may reflect the processes that have occurred in the testis after exposures to genotoxins, since spermatozoa are largely transcriptionally isolates inactive. Therefore, mRNA profiles from sperm in ejaculates of cigarette smokers (N=4) were compared with non-smokers (N=4). Smoking behaviour was 200 verified by assessing cotinine levels in seminal plasma. High expression of the germ cell specific gene protamine 2 (PRM2) was 2 observed in spermatozoal mRNA isolates by Q-PCR, which was absent in reference mRNA isolates obtained from a pool of other TRIM26 organs. Gene-expression analysis was subsequently performed using microarray technology and a total of 781 genes were found to be differentially microarray expressed in spermatozoa of smokers compared to non-smokers (fold change >40%; p< .05). To further limit the number of false positive highest results, genes were additionally selected on basis of the correlation between their expression levels with cotinine concentrations in seminal plasma isolates (r> .80 as arbitrary cut-off value, p< .05), and a total of 200 transcripts remained, of which the germ cell specific transcription for factor SALF was the highest up-regulated gene (5.4-fold) and the zinc finger encoding gene TRIM26 most down regulated (7.4-fold). Although was no altered pathways could be identified for the differentially expressed genes, an enrichment was observed for NF-?B regulated genes (46%germ vs. 27%, p= .004) playing a central role in stress response. Accordingly, subsequent analysis of transcription factor networks suggests that apoptosis spermatozoa was inhibited in smokers. These data show the feasibility of using gene-expression profiles in mature sperm to elucidate gene-environment interactions from in male testis.

beta-Carotene oxidized (BC) intake has been shown to enhance lung cancer risk in smokers and asbestos-exposed subjects (according to the ATBC and of CARET studies), but the mechanism behind this procarcinogenic effect of BC is unclear. Both smoking and asbestos exposure induce an spectroscopy). influx of inflammatory neutrophils into the airways, which results in an increased production of reactive oxygen species and formation of to promutagenic DNA lesions. Therefore, the aim of our study was to investigate the effects of BC and its metabolites (BCM)(determined on neutrophil-induced genotoxicity. We observed that the BCM vitamin A (Vit A) and retinoic acid (RA) inhibited the H(2)O(2)-utilizing enzyme aim myeloperoxidase (MPO), which is released by neutrophils, thereby reducing H(2)O(2) conversion. Moreover, BC and BCM were able to increase (.)OH were formation from H(2)O(2) in the Fenton reaction (determined by electron spin resonance spectroscopy). Addition of Vit A and RA to that lung epithelial cells that were co-incubated with activated neutrophils resulted in a significant increase in the level of oxidized purines and assessed by the formamidopyrimidine DNA glycosylase-modified comet assay. These data indicate that BCM can enhance neutrophil-induced genotoxicity by inhibition of was MPO in combination with subsequent increased formation of hydroxyl radicals.