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Waterpipe (aka hookah) tobacco smokers are exposed to toxicants that can lead to oxidative DNA and RNA damage, a precursor to chronic disease formation. This study assessed toxicant exposure and biomarkers of DNA [8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG)] and RNA [8-oxo-7,8-dihydroguanosine (8-oxoGuo)] oxidative damage during smoking of flavored and non-flavored waterpipe tobacco. Thirty waterpipe smokers completed two counterbalanced 2-h lab waterpipe smoking sessions (flavored vs. non-flavored waterpipe tobacco). Urinary concentrations of 8-oxodG and 8-oxoGuo and expired carbon monoxide (eCO) were measured before and after the smoking sessions. A significant increase in the urinary concentrations of 8-oxodG (from 2.12 ± 0.83 to 2.35 ± 0.91 ng/mg creatinine, p = 0.024) and 8-oxoGuo (from 2.96 ± 0.84 to 3.45 ± 0.76 ng/mg creatinine, p = 0.003) were observed after smoking the non-flavored and flavored waterpipe tobacco, respectively. Our results also showed that the mean ± SD of eCO increased significantly after smoking the flavored (from 1.3 ± 1.1 to 20.3 ± 23.6 ppm, p < 0.001) and non-flavored waterpipe tobacco (from 1.8 ± 1.2 to 24.5 ± 26.1 ppm, p < 0.001). There were no significant differences in the means of 8-oxodG (p = 0.576), 8-oxoGuo (p = 0.108), and eCO (p = 0.170) between the flavored and non-flavored tobacco sessions. Smoking non-flavored and flavored waterpipe tobacco leads to oxidative stress and toxicant exposure. Our findings add to the existing evidence about the adverse effects of waterpipe tobacco smoking (WTS) and the need for strong policies to inform and protect young people from the risks of WTS.

Skin melanisation ranges widely across human populations. Melanin has antioxidant properties and also acts as a filter to solar ultraviolet radiation (UVR) incident upon the skin. In this study we firstly examined whether melanin level might influence baseline levels of systemic oxidative stress, in 65 humans in vivo from the same geographical area ranging from the lightest to darkest skin type (phototype I-VI). This was examined in winter-time (latitude 53.5°N). Remarkably, we found that urinary biomarkers of oxidatively-generated DNA damage (8-oxodG) and RNA damage (8-oxoGuo) were significantly correlated with skin lightness (L*), such that 14–15% of the variation in their baseline levels could be explained by skin colour. Next we exposed 15 humans at the extremes of skin melanisation to a simulated summer-time exposure of solar UVR (95% UVA, 5% UVB; dose standardised to sunburn threshold), following which they provided a sample of every urine void over the next five days. We found that UVR induced a small but significant increase in urinary 8-oxodG and 8-oxoGuo, with differing kinetics between skin types. Thus greater melanisation is associated with protection against systemic oxidative stress, which may reflect melanin's antioxidant properties, and solar UVR exposure also influences systemic oxidative stress levels in humans. These novel findings may have profound implications for human physiology and health., Graphical abstract Image 1, Highlights • Biomarkers of systemic oxidative stress correlate with human skin lightness level. • Skin colour contributes 14–15% variation in baseline urinary 8-oxodG and 8-oxoGuo. • UVR exposure of the skin elevates biomarkers of systemic oxidative stress. • Both oxidatively-damaged DNA (8-oxodG) and RNA (8-oxoGuo) increase post-UVR. • Kinetics of urinary biomarker responses following UVR differ between skin types.

A reliable and fast liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of three oxidized nucleic acid damage products in urine, 8-oxoguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo). We applied this method to assess the effect of various urine workup procedures on the urinary concentrations of the oxidized nucleic acid products. Our results showed that frozen urine samples must be warmed (i.e., to 37 °C) to re-dissolve any precipitates prior to analysis. We showed that common workup procedures, such as thawing at room temperature or dilution with deionized water, are not capable of releasing fully the oxidized nucleic acid products from the precipitates, and result in significant underestimation (up to ~ 100% for 8-oxoGua, ~ 86% for both 8-oxodGuo and 8-oxoGuo).With this method, we further assessed and compared the ability of the three oxidized nucleic acid products, as well as malondialdehyde (MDA, a product of lipid peroxidation), to biomonitor oxidative stress in vivo. We measured a total of 315 urine samples from subjects with burdens of oxidative stress from low to high, including healthy subjects, patients with chronic obstructive pulmonary disease (COPD), and patients on mechanical ventilation (MV). The results showed that both the MV and COPD patients had significantly higher urinary levels of 8-oxoGua, 8-oxodGuo, and 8-oxoGuo (P

Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2′-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2′-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease., Graphical abstract Image 1, Highlights • Oxidatively damaged nucleic acids are implicated in the pathogenesis of disease. • LC-MS/MS, comet assay and ELISA are often used to study oxidatively damaged DNA. • Urinary oxidatively damaged nucleic acids non-invasively reflect oxidative stress. • DNA adductomics will aid understanding the role of ROS damaged DNA in disease.

Background: Worldwide, smoking is a major public health problem, with exposure to environmental tobacco smoke (ETS) affecting both smokers, and passive smokers, including children. Despite ETS also describing secondhand, and thirdhand smoke (SHS, and THS respectively), the health effects of exposure to passive smoking via these sources are not fully understood, particularly in children. Although cotinine, the primary proximate metabolite of nicotine, has been widely used as a biomarker of ETS exposure, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), provides a uniquely important contribution, both as a biomarker of exposure, and as a specific risk indicator for pulmonary carcinogenesis. Methods: We used LC-MS/MS to study NNK metabolites, cotinine, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (a biomarker of oxidative stress), in the urine of 110 non-smoking adults (age range: 23–62) and 101 children (age range: 9–11), exposed to ETS. Results: In our study of passive smoking adults, and children exposed to ETS, we showed that although the children had a similar urinary level of cotinine compared to the adults, the children had approximately two times higher levels of urinary total NNAL (P = 0.002), and free NNAL (P = 0.01), than adults. The children also had three times lower ability to detoxify NNK than adults (P

WOS: 000319871200001, PubMed ID: 23198723, Aims: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (r(p) 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended., 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" [FOOD-CT-2005-513943]; ECNIS2, a coordination and support action within the European Union FP7 Cooperation Theme 2 Food, Agriculture, Fisheries and Biotechnologies; CISBO; Ingeborg; Leo Dannin Foundation; National Science Council, TaiwanNational Science Council of Taiwan [NSC 97-2314-B-040-015-MY3, NSC 100-2628-B-040-001-MY4]; US NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [P30ES009089]; Instituto Carlos III division of the Government for Clinical Research [PI-10/00802, RD06/0045/0006]; Generalitat ValencianaGeneralitat Valenciana [ACOM/2012/238]; Swedish Council for Working Life and Social ResearchSwedish Research CouncilSwedish Research Council for Health Working Life & Welfare (Forte); TUBITAK (Technical and Scientific Research Council of Turkey)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108Y049]; Grant Agency of the Czech RepublicGrant Agency of the Czech Republic [P503/11/0084]; Sahlgrenska University Hospital, Gothenburg; UK Medical Research Council via a People Exchange Programme Research Leader Fellowship award [G1001808/98136], Some of the authors of this work were 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 No. FOOD-CT-2005-513943), and also ECNIS2, a coordination and support action within the European Union FP7 Cooperation Theme 2 Food, Agriculture, Fisheries and Biotechnologies.; P Moller and S Loft are supported by CISBO and the Ingeborg and Leo Dannin Foundation.; M-R Chao and C-W Hu acknowledge financial support from the National Science Council, Taiwan (Grants NSC 97-2314-B-040-015-MY3 and NSC 100-2628-B-040-001-MY4).; R Santella acknowledges the contribution of Qiao Wang, and support from US NIH P30ES009089.; G Saez and C Cerda acknowledge financial support from the Instituto Carlos III division of the Government for Clinical Research (Grants PI-10/00802 and RD06/0045/0006) and Grant ACOM/2012/238 from Generalitat Valenciana.; K Broberg, C Lindh, and M Hossain acknowledge financial support from the Swedish Council for Working Life and Social Research; H Orhan and N Senduran acknowledge financial support from TUBITAK (Technical and Scientific Research Council of Turkey), grant number 108Y049.; P Rossner, Jr. and RJ Sram acknowledge support from the Grant Agency of the Czech Republic (P503/11/0084).; L Barregard acknowledges financial support from the Sahlgrenska University Hospital, Gothenburg.; MS Cooke acknowledges support from the UK Medical Research Council via a People Exchange Programme Research Leader Fellowship award (G1001808/98136).

Chewing betel quid is a common habit in Taiwan and associated with the risk of oral cancer. Betel quid contains arecoline and arecaidine, which may serve as the exposure biomarkers of a chewing habit. We developed a liquid chromatography-tandem mass spectrometry method for the quantitative analysis of blood arecoline and arecaidine. Because 8-hydroxydeoxyguanosine (8-OH-dG) level in urine is only one early health marker of carcinogenesis, we also examined its association with chewing habit. We found a significant positive correlation between the quantities of betel quid used before the day of drawing blood and arecoline [(Spearman correlation coefficient (r) = 0.81; p value < 0.01) or arecaidine levels (r = 0.86; p value < 0.01)]. Habitual use quantity (quids/day) showed moderate correlation with both arecoline (r = 0.52; p value < 0.05) and arecaidine concentrations (r = 0.51; p value < 0.05). However, there were no significant correlations between total chewing years and concentrations of arecoline and arecaidine in serum or 8-OH-dG in urine. In conclusion, serum arecoline and arecaidine levels are measurable and good indicators for recent betel quid use.

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)

We conducted a repeated-measures cohort study of coke oven workers to evaluate the relationships between the traditional exposure biomarker, urinary 1-hydroxypyrene (1-OHP), and a series of biomarkers, including urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), N7-methylguanine (N7-MeG), acute toxicity, and mutagenicity. A total of eight spot urine samples were collected from each high-exposed (at topside oven area) and low-exposed workers (at side oven area) during the whole working cycle, which consisted of 6 consecutive days of working followed by 2 days off. Our results showed that the high-exposed workers had significantly higher urinary levels of 1-OHP, 8-oxodG, and N7-MeG compared with the low-exposed workers. Acute toxicity and mutagenicity of urine were also found to be markedly increased in the high-exposed workers, as determined by Microtox assay and Ames test, respectively. Multivariate regressions analysis revealed that the urinary 8-oxodG, N7-MeG, or acute toxicity was significantly correlated with 1-OHP concentrations. Overall, the present study showed that exposure to coke oven emissions increased oxidatively damaged DNA products and mutagenicity of urine, and for the very first time, such exposure was also found to increase DNA methylation and urinary acute toxicity. The potential source of methylating agents in coke oven emissions warrants further investigation. Additionally, with repeated measurements, the pattern of time course for urinary 1-OHP was found to be different from those of 8-oxodG and N7-MeG, as well as acute toxicity and mutagenicity. This finding implies that the single measurement that was often conducted in occupational healthy investigations should be used with certain precautions, because single measurement may fail to provide the proper information of interest. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3381–9)

Background: Quantification of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) in urine or blood is used to assess and monitor oxidative stress in patients. We describe the use of on-line solid-phase extraction (SPE) and isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) for automated measurement of urinary 8-oxodGuo. Methods: Automated purification of urine was accomplished with a switching valve and an Inertsil ODS-3 column. After the addition of 15N5-labeled 8-oxodGuo as an internal standard, urine samples were analyzed within 10 min without sample purification. This method was applied to measure urinary 8-oxodGuo in a group of healthy persons (32 regular smokers and 35 nonsmokers). Urinary cotinine was also assayed by an isotope-dilution LC-MS/MS method. Results: The lower limit of detection was 5.7 ng/L on column (2.0 fmol). Inter- and intraday imprecision (CV) was Conclusions: This on-line SPE LC-MS/MS method is sufficiently sensitive, precise, and rapid to provide high-throughput direct analysis of urinary 8-oxodGuo without compromising quality and validation criteria. This method could be applicable for use in daily clinical practice for assessing oxidative stress in patients.

Previous studies demonstrated the presence of unknown direct-acting ethylating agents arising from cigarette smoke. We hypothesized that such agents would also lead to ethylation of guanine in DNA followed by depurination/repair and excretion of N7-ethylguanine (N7-EtG) in urine. In this study, a highly specific and sensitive liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was firstly developed for measuring urinary N7-EtG. With the use of an isotope internal standard (15N5-N7-EtG) and on-line enrichment techniques, the detection limit of this method was estimated as 0.59 pg/ml (0.33 pmol) on-column. This method was then applied to measure urinary samples obtained from 35 non-smokers and 32 smokers with dietary control. The results showed that the mean urinary levels of N7-EtG were 85.5+/-105 and 28.1+/-19.4 pg/mg creatinine for smokers and non-smokers, respectively. Smokers had about three times higher level of N7-EtG than non-smokers (P