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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).

Sensitive and reliable methods are required for the assessment of oxidative DNA damage, which can result from reactive oxygen species that are generated endogenously from cellular metabolism and inflammatory responses, or by exposure to exogenous agents. The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) method is described, that utilises online column-switching valve technology for the simultaneous determination of two DNA adduct biomarkers of oxidative stress, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxodA). To allow for the accurate quantitation of both adducts the corresponding [15N5]-labelled stable isotope internal standards were synthesised and added prior to enzymatic hydrolysis of the DNA samples to 2′-deoxynucleosides. The method required between 10 and 40 µg of hydrolysed DNA on-column for the analysis and the limit of detection for both 8-oxodG and 8-oxodA was 5 fmol. The analysis of calf thymus DNA treated in vitro with methylene blue (ranging from 5 to 200 µM) plus light showed a dose-dependent increase in the levels of both 8-oxodG and 8-oxodA. The level of 8-oxodG was on average 29.4-fold higher than that of 8-oxodA and an excellent linear correlation (r = 0.999) was observed between the two adducts. The influence of different DNA extraction procedures for 8-oxodG and 8-oxodA levels was assessed in DNA extracted from rat livers following dosing with carbon tetrachloride. The levels of 8-oxodG and 8-oxodA were on average 2.9 (p = 0.018) and 1.4 (p = 0.018) times higher, respectively, in DNA samples extracted using an anion-exchange column procedure than in samples extracted using a chaotropic procedure, implying artefactual generation of the two adducts. In conclusion, the online column-switching LC/MS/MS SRM method provides the advantages of increased sample throughput with reduced matrix effects and concomitant ionisation suppression, making the method ideally suited when used in conjunction with chaotropic DNA extraction for the determination of oxidative DNA damage. Copyright © 2008 John Wiley & Sons, Ltd.

The patterns and levels of urinary excreted ribonucleosides which reflect RNA turnover and metabolism in humans offer the potential for early detection of disease and monitoring of therapeutic intervention. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method employing constant neutral loss (CNL) scanning for the loss of the ribose moiety (132 u) was used to detect ribonucleosides in human urine and to evaluate this analytical platform for biomarker research in clinical trials. Ribonucleosides were stable and not influenced by the time spent at room temperature prior to freezing or long-term storage at –80°C. Matrix effects caused variation in the mass spectrometer response which was dependent on the concentration of the analysed urine sample. For the use of urinary ribonucleoside profiling in clinical biomarker studies, adjustment of the urine samples to a common concentration prior to sample preparation is therefore advocated. Changes in the mass spectrometer response should be accounted for by the use of an internal standard added after sample preparation. Diurnal variation exceeded inter-day variation of an individual's ribonucleoside profile, but inter-person differences were predominant and allowed the separation of individuals against each other in a multivariate space. Due to considerable diurnal variation the use of spot urine samples would introduce unnecessary variation and should be replaced by the collection of multiple spot urine samples across the day, where possible. Should such a protocol not be feasible, biological intra-day and inter-day variation must be considered and accounted for in the data interpretation. Copyright © 2011 John Wiley & Sons, Ltd.

This is the pre-peer reviewed version of the following article: Rapid Communications in Mass Spectrometry, 2010, 24 (16), pp. 2329-2340, which has been published in final form at www3.interscience.wiley.com, Doi: 10.1002/rcm.4645

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents a non-invasive biomarker for oxidative stress and may be useful for monitoring chemotherapeutic and chemopreventive interventions associated with cancer-related alterations in oxidative stress. We describe the development and validation of two separate liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) methods for the determination of 8-oxodG and creatinine in both murine and human urine using stable isotope labelled internal standards. Levels of 8-oxodG were normalised to creatinine. The LC/MS/MS methods were applied to two chemoprevention studies utilising tea polyphenols in humans and TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) mice. Patients with benign prostatic hyperplasia received 1 g/day of green tea polyphenols (GTP), 1 g/day of black tea theaflavins (BTT) or no treatment for 4 weeks. TRAMP mice received GTP (0.05% in drinking water) for 4 or 25 weeks. Prostate pathology in TRAMP mice was not affected by GTP. Levels of 8-oxodG were not altered by tea polyphenols in either mice or humans. In TRAMP mice, urinary 8-oxodG levels were elevated with increasing age (p < 0.0001) but not changed by the presence of prostate tumours. In conclusion, the LC/MS/MS SRM methods described here are ideally suited for the accurate determination of 8-oxodG and creatinine in urine samples from both clinical and pre-clinical studies.

The patterns and levels of urinary excreted ribonucleosides which reflect RNA turnover and metabolism in humans offer the potential for early detection of disease and monitoring of therapeutic intervention. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method employing constant neutral loss (CNL) scanning for the loss of the ribose moiety (132 u) was used to detect ribonucleosides in human urine and to evaluate this analytical platform for biomarker research in clinical trials. Ribonucleosides were stable and not influenced by the time spent at room temperature prior to freezing or long-term storage at -80 °C. Matrix effects caused variation in the mass spectrometer response which was dependent on the concentration of the analysed urine sample. For the use of urinary ribonucleoside profiling in clinical biomarker studies, adjustment of the urine samples to a common concentration prior to sample preparation is therefore advocated. Changes in the mass spectrometer response should be accounted for by the use of an internal standard added after sample preparation. Diurnal variation exceeded inter-day variation of an individual's ribonucleoside profile, but inter-person differences were predominant and allowed the separation of individuals against each other in a multivariate space. Due to considerable diurnal variation the use of spot urine samples would introduce unnecessary variation and should be replaced by the collection of multiple spot urine samples across the day, where possible. Should such a protocol not be feasible, biological intra-day and inter-day variation must be considered and accounted for in the data interpretation.

The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures. The provenance of extracellular 8-oxodG remains a subject for debate. However, previous studies have shown that factors, such as diet and cell death, do not appear to contribute to extracellular 8-oxodG, leaving processes, such as the repair of DNA and/or the 2'-deoxyribonucleotide pool, as the sole source of endogenous 8-oxodG. The method in this chapter describes a non-invasive approach for assessing oxidative stress, via the efficient extraction of urinary 8-oxodG using a validated solid-phase extraction procedure. Subsequent analysis by liquid chromatography-tandem mass spectrometry provides the advantages of sensitivity, internal standardisation, and robust peak identification, and is widely considered to be the "gold standard".

The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures. The provenance of extracellular 8-oxodG remains a subject for debate. However, previous studies have shown that factors, such as diet and cell death, do not appear to contribute to extracellular 8-oxodG, leaving processes, such as the repair of DNA and/or the 2'-deoxyribonucleotide pool, as the sole source of endogenous 8-oxodG. The method in this chapter describes a non-invasive approach for assessing oxidative stress, via the efficient extraction of urinary 8-oxodG using a validated solid-phase extraction procedure. Subsequent analysis by liquid chromatography-tandem mass spectrometry provides the advantages of sensitivity, internal standardisation, and robust peak identification, and is widely considered to be the "gold standard".

Background Epidemiological studies suggest that inhalation of carbonaceous particulate matter from biomass combustion increases susceptibility to bacterial pneumonia. In vitro studies report that phagocytosis of carbon black by alveolar macrophages (AM) impairs killing of Streptococcus pneumoniae. We have previously reported high levels of black carbon in AM from biomass smoke-exposed children and adults. We therefore aimed to use a mouse model to test the hypothesis that high levels of carbon loading of AM in vivo increases susceptibility to pneumococcal pneumonia. Methods Female outbred mice were treated with either intranasal phosphate buffered saline (PBS) or ultrafine carbon black (UF-CB in PBS; 500 μg on day 1 and day 4), and then infected with S. pneumoniae strain D39 on day 5. Survival was assessed over 72 h. The effect of UF-CB on AM carbon loading, airway inflammation, and a urinary marker of pulmonary oxidative stress was assessed in uninfected animals. Results Instillation of UF-CB in mice resulted a pattern of AM carbon loading similar to that of biomass-smoke exposed humans. In uninfected animals, UF-CB treated animals had increased urinary 8-oxodG (P = 0.055), and an increased airway neutrophil differential count (P < 0.01). All PBS-treated mice died within 72 h after infection with S. pneumoniae, whereas morbidity and mortality after infection was reduced in UF-CB treated animals (median survival 48 h vs. 30 h, P < 0.001). At 24 hr post-infection, UF-CB treated mice had lower lung and the blood S. pneumoniae colony forming unit counts, and lower airway levels of keratinocyte-derived chemokine/growth-related oncogene (KC/GRO), and interferon gamma. Conclusion Acute high level loading of AM with ultrafine carbon black particles per se does not increase the susceptibility of mice to pneumococcal infection in vivo.

BACKGROUND: Arsenic (As) causes oxidative stress through generation of reactive oxygen species. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a sensitive marker of oxidative DNA damage, has been associated with As exposure in some studies, but not in others, possibly due to population-specific genetic factors. OBJECTIVES: To evaluate the association between As and 8-oxodG in urine in a population with a low urinary monomethylated As (%MMA) and high dimethylated As (%DMA), as well as the genetic impact on (a) 8-oxodG concentrations and (b) the association between As and 8-oxodG. MATERIALS AND METHODS: Women (N=108) in the Argentinean Andes were interviewed and urine was analyzed for arsenic metabolites (ICPMS) and 8-oxodG (LC-MS/MS). Twenty-seven polymorphisms in genes related to oxidative stress and one in As(+III)methyltransferase (AS3MT) were studied. RESULTS: Median concentration of 8-oxodG was 4.7nmol/L (adjusted for specific weight; range 1.6-13, corresponding to 1.7mug/g creatinine, range 0.57-4.8) and of total urinary As metabolites (U-As) 290mug/L (range 94-720; 380mug/g creatinine, range 140-1100). Concentrations of 8-oxodG were positively associated with %MMA (strongest association, p=0.013), and weakly associated with U-As (positively) and %DMA (negatively). These associations were strengthened when taking ethnicity into account, possibly reflecting genetic differences in As metabolism and genes regulating oxidative stress and DNA maintenance. A genetic influence on 8-oxodG concentrations was seen for polymorphisms in apurinic/apyrimidinic endonuclease 1 (APEX1), DNA-methyltransferases 1 and 3b (DNMT1, DNMT3B), thioredoxin reductase 1 (TXNRD1) and 2 (TXNRD2) and glutaredoxin (GLRX). CONCLUSION: Despite high As exposure, the concentrations of 8-oxodG in this population were low compared with other As-exposed populations studied. The strongest association was found for %MMA, stressing that some inconsistencies between As and 8-oxodG partly depend on population variations in As metabolism. We found evidence of genetic impact on 8-oxodG concentrations. (Less)

2,3-dimethoxy-1,4-naphthoquinone (CAS-RN 6959-96-3) (DMNQ) and 2-methyl-1,4-naphthoquinone (CAS-RN 58-27-5) (MNQ:menadione) are effective one electron redox cycling chemicals in vitro. In addition, in vitro MNQ forms a thioether conjugate with glutathione by nucleophilic attack at the third carbon. In contrast, here we demonstrate that in vivo the major metabolic route is directly to the dihydronaphthoquinone for both DMNQ and MNQ followed by conjugation to mono- and di-glucuronides and sulfate. Analysis of urine and bile showed that glutathione conjugation of MNQ was only a very minor route of metabolism. DMNQ was distributed to all tissues including the brain, and MNQ was much less widely distributed. For DMNQ tissue half-life, in particular for the heart, was considerably longer than the plasma half-life. For both DMNQ and MNQ, urine 8-oxo-7,8-dihydro-2'-deoxyguanosine and liver transcriptomic analysis failed to show any evidence of redox stress. Oxidized glutathione (GSSG) in liver increased significantly at the 10 min postdosing time point only. Metabonomic analysis 96 h after DMNQ administration indicated decreased liver glucose and increased lactate and creatine suggesting an impairment of oxidative metabolism. We conclude that in vivo DMNQ and MNQ are primarily two electron reduced to the dihydronaphthoquinones and undergo little one electron redox cycling. For DMNQ, disruption of cellular oxidative metabolism may be a primary mechanism of toxicity rather than redox stress.

In this study, we have used metabolic profiling (metabolomics/metabonomics) via high resolution magic angle spinning (HRMAS) and solution state (1)H NMR spectroscopy to characterize small bowel and colon tissue from the Apc(Min/+) mouse model of early gastrointestinal (GI) tumorigenesis. Multivariate analysis indicated the presence of metabolic differences between the morphologically normal/non-tumor tissue from approximately 10 week-old Apc(Min/+) mice and their wild-type litter mates. The metabolic profile of isolated lamina propria and epithelial cells from the same groups could also be discriminated on the basis of genotype. Accounting for systematic variation in individual metabolite levels across different anatomical regions of the lower GI tract, the metabolic phenotype of Apc(Min/+) lamina propria tissue was defined by significant increases in the phosphocholine/glycerophosphocholine ratio (PC/GPC, +21%) and decreases in GPC (-25%) and the gut-microbial cometabolite dimethylamine (DMA, -40%) relative to wild type. In the whole tissue, elevated lactate (+15%) and myo-inositol (+19%) levels were detected. As the metabolic changes occurred in non-tumor tissue from animals of very low tumor burden (

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents a non-invasive biomarker for oxidative stress and may be useful for monitoring chemotherapeutic and chemopreventive interventions associated with cancer-related alterations in oxidative stress. We describe the development and validation of two separate liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) methods for the determination of 8-oxodG and creatinine in both murine and human urine using stable isotope labelled internal standards. Levels of 8-oxodG were normalised to creatinine. The LC/MS/MS methods were applied to two chemoprevention studies utilising tea polyphenols in humans and TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) mice. Patients with benign prostatic hyperplasia received 1 g/day of green tea polyphenols (GTP), 1 g/day of black tea theaflavins (BTT) or no treatment for 4 weeks. TRAMP mice received GTP (0.05% in drinking water) for 4 or 25 weeks. Prostate pathology in TRAMP mice was not affected by GTP. Levels of 8-oxodG were not altered by tea polyphenols in either mice or humans. In TRAMP mice, urinary 8-oxodG levels were elevated with increasing age (p0.0001) but not changed by the presence of prostate tumours. In conclusion, the LC/MS/MS SRM methods described here are ideally suited for the accurate determination of 8-oxodG and creatinine in urine samples from both clinical and pre-clinical studies.

BACKGROUND The TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model has frequently been used in preclinical studies with chemotherapeutic/chemopreventive rationales. Here the hypothesis was tested using 1H-NMR-based metabolic profiling that the TRAMP tumor metabolic phenotype resembles that reported for human prostate cancer. METHODS Aqueous extracts or intact tissues of normal prostate from 8- (“young”) or 28-(“old”) week-old C57BL/6J wild-type mice or of prostate tumor from age-matched TRAMP mice were analyzed by 1H-NMR. Results were compared with immunohistochemical findings. Expression of choline kinase was studied at the protein and mRNA levels. RESULTS In young TRAMP mice presenting with zonal hyperplasia, the ratio of glycerophosphocholine (GPC) to phosphocholine (PC) was 22% below that in wild-type mice (P

Sources of analytical variation in high-performance liquid chromatography/mass spectrometry (HPLC/MS), such as changes in retention, mass accuracy or signal intensity, have been investigated to assess their importance as a variable in the metabonomic analysis of human urine. In this study chromatographic retention and mass accuracy were found to be quite reproducible with the most significant source of analytical variation in the data sets obtained being the result of changes in detector response. Depending on the signal intensity threshold used to define the presence of a peak a sample component could be present in some replicate injections and absent in others within the same run. The implementation of a more sophisticated data software analysis package was found to greatly reduce the impact of detector response variability resulting in improved data analysis.

Green tea polyphenols (GTP) have been widely investigated for their potential to prevent prostate cancer. However, results from epidemiological and clinical studies are equivocal. Studies in the TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) mouse suggest that the chemopreventive efficacy of GTP is higher in young animals with early stages of carcinogenesis than in old ones. Here, effects of GTP on prostate carcinogenesis in TRAMP mice were assessed by comparing pathological changes with (1)H-NMR metabolic profiling of plasma and extracts of prostate tissue. Mice received 0.05% GTP in their drinking water for 4 or 25 weeks after weaning. Age-matched wild-type mice were included in the study in order to establish differences in GTP effects between normal and TRAMP mice. Dietary GTP did not markedly alter prostate carcinogenesis as reflected by pathology and prostate tissue metabolic profile. However, a systemic effect of GTP consumption was observed in young mice, regardless of genotype. Plasma lipid signals were decreased in 8 week old mice which received GTP compared to age-matched controls by 19, 61, 27, 34 and 15% (por= 0.05) in the CH(2)CH(2)C[double bond, length as m-dash]C (m 2.00 ppm), CH(2)CH(2)CO (m 1.58 ppm), CH(2) (m 1.26 ppm), CH(3) (m 0.88 ppm) and CH(3) fatty acid resonances (m 0.84 ppm), respectively. GTP consumption did not affect the plasma metabolic profile in 29 week old mice. These results suggest that age rather than disease state determines systemic effects of GTP. More studies are required to investigate factors, such as age or metabolic make-up, inherent to a population or an individual, which may modulate the chemopreventive efficacy of GTP.