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Rationale: Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood. Objectives: We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility. Methods: We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility. Measurements and Main Results: We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress–induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin. Conclusions: Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.

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. This work was supported by ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (contract no. 513943).

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve the problems associated with the measurement of background levels of oxidative DNA damage (in particular 8-oxo-7,8-dihydroguanine, or 8-oxoGua) in human cells. A tendency for DNA oxidation to occur during sample preparation prior to chromatography has been recognized as the source of a very substantial artefact. To assess the success of attempts to eliminate the artefact, ESCODD has distributed to its members standard samples of pig liver and HeLa cells for analysis. Estimates of 8-oxoGua in pig liver, using chromatographic techniques, ranged from 2.23 to 441 per 10(6) guanines, with a median of 10.47 per 10(6) guanines. Chromatographic analysis of HeLa cell DNA gave a range of 1.84 to 214 per 10(6) guanines with a median of 5.23 per 10(6) guanines. HeLa cell DNA was also analysed by an enzymic approach, in which whole cell DNA was treated with formamidopyrimidine DNA glycosylase, which nicks DNA at sites of 8-oxoGua, and the breaks measured with the comet assay, alkaline unwinding or alkaline elution. Values with these methods ranged from 0.06 to 4.988-oxoGua per 10(6) guanines, with a median of 0.79 per 10(6) guanines. Although there are clearly still serious discrepancies between methods and laboratories, the lowest estimates by chromatography (arguably those in which the artefact was best controlled) are only 2.5 times higher than the median value obtained with the enzymic approach.

Many recent dioxin contamination events have been traced back to poisoned animal feed or feed ingredients. Therefore, enforcement authorities placed limits on the levels of dioxins in food and feed or implemented strict monitoring and control programs. The levels in force are generally expressed as TEQ values, which inherently accepts the underlying hypothesis that the effects of dioxin-like chemicals are additive. TEQ determination involves either chemo-analysis, with high-resolution gas chromatography in combination with high-resolution mass spectroscopy, or bio-analysis. Bio-analytical methods, more particularly the reporter gene expression method CALUX, are advantageous due to their high throughput rate and low cost. Moreover, the CALUX methodology detects the overall dioxin-like toxicity, rather than the limited number of compounds investigated by chemo-analysis. Bioanalytical methods such as CALUX also differ from chemo-analysis in that the contribution of antagonistic as well as synergistic effects, which violate the additivity principle, can be detected. The application of bio-analytical methods can facilitate a broader assessment of public health risks by intensifying the current monitoring programs in terms of both sample numbers and types. Bio-analysis provides information on the total dioxin-like activity of the samples under study (hazard assessment); however, chemo-analysis is still needed to identify the predominant contaminants (congener identification) for risk management.

Of the DNA-derived biomarkers of oxidative stress, urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is the most frequently measured. However, there is significant discrepancy between chromatographic and immunoassay approaches, and intratechnique agreement among all available chromatography-based assays and ELISAs is yet to be established. This is a significant obstacle to their use in large molecular epidemiological studies. To evaluate the accuracy of intra/intertechnique and interlaboratory measurements, samples of phosphate buffered saline and urine, spiked with different concentrations of 8-oxoG, together with a series of urine samples from healthy individuals were distributed to ESCULA members. All laboratories received identical samples, including 2 negative controls that contained no added 8-oxodG. Data were returned from 17 laboratories, representing 20 methods, broadly classified as mass spectrometric (MS), electrochemical detection (EC), or enzyme-linked immunosorbant assay (ELISA). Overall, there was good within-technique agreement, with the majority of laboratories' results lying within 1 sd of their consensus mean. However, ELISA showed more within-technique variation than did the chromatographic techniques and, for the urine samples, reported higher values. Bland-Altman plots revealed good agreement between MS and EC methods but concentration-dependent deviation for ELISA. All methods ranked urine samples according to concentration similarly. Creatinine levels are routinely used as a correction factor for urine concentration, and therefore we also conducted an interlaboratory comparison of methods for urinary creatinine determination, in which the vast majority of values lay within 1 sd of the consensus value, irrespective of the analysis procedure. This study reveals greater consensus than previously expected, although concern remains over ELISA.-ESCULA [European Standards Committee on Urinary (DNA) Lesion Analysis], Evans, M. D., Olinski, R., Loft, S., Cooke, M. S. Toward consensus in the analysis of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine as a noninvasive biomarker of oxidative stress. Some of the authors of this paper are partners in, and this work was partly supported by, ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a Network of Excellence operating within the European Union 6th Framework Program, Priority 5: Food Quality and Safety (contract 513943)

Background: Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. The noninvasive assessment of such damage, i.e., in urine, and application to large-scale human studies are vital to understanding this role and devising intervention strategies. Methods: We have reviewed the literature to establish the status quo with regard to the methods and meaning of measuring DNA oxidation products in urine. Results: Most of the literature focus upon 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), and whereas a large number of these reports concern clinical conditions, there remains (a) lack of consensus between methods, (b) possible contribution from diet and/or cell death, (c) no definitive DNA repair source of urinary 2′-deoxyribonucleoside lesions, and (d) no reference ranges for healthy or diseased individuals. Conclusions: The origin of 8-oxodG is not identified; however, recent cell culture studies suggest that the action of Nudix hydrolase(s) on oxidative modification of the nucleotide pool is a likely candidate for the 8-oxodG found in urine and, potentially, of other oxidized 2′-deoxyribonucleoside lesions. Literature reports suggest that diet and cell death have minimal, if any, influence upon urinary levels of 8-oxodG and 8-oxo-7,8-dihydroguanine, although this should be assessed on a lesion-by-lesion basis. Broadly speaking, there is consensus between chromatographic techniques; however, ELISA approaches continue to overestimate 8-oxodG levels and is not sufficiently specific for accurate quantification. With increasing numbers of lesions being studied, it is vital that these fundamental issues are addressed. We report the formation of the European Standards Committee on Urinary (DNA) Lesion Analysis whose primary goal is to achieve consensus between methods and establish reference ranges in health and disease. (Cancer Epidemiol Biomarkers Prev 2008;17(1):3–14)

n engl j med 356;22 www.nejm.org may 31, 2007 2329 To the Editor: In the Case Record of a patient with respiratory insufficiency, presented by Boyer et al. (Jan. 25 issue),1 the discussants do not mention a condition known to be associated with idiopathic pulmonary hemosiderosis and to have implications for its management: celiac disease. The association is referred to as the Lane–Hamilton syndrome.2 It is important to recognize this condition, since treatment of celiac disease with a gluten-free diet could lead to the remission of idiopathic pulmonary hemosiderosis.3 Although both celiac disease and idiopathic pulmonary hemosiderosis are believed to be immunologically mediated, the pathogenetic link between them is not clear. A recent systematic review identified 20 patients with the Lane–Hamilton syndrome.4 Of these, 16 had been prescribed a gluten-free diet, with improvement documented in 12. Patients with idiopathic pulmonary hemosiderosis should be screened for celiac disease, even in the absence of gastrointestinal symptoms, by means of serologic testing for antiendomysial antibodies. If the test is positive, an intestinal biopsy should be carried out to confirm the disease, because a gluten-free diet is very effective for the regression of both celiac disease and idiopathic pulmonary hemosiderosis.