Urinary 8-oxo-7,8-dihydroguanosine levels are elevated in HCV-infected patients.

HCV patients are usually under substantial oxidative stress because of viral infection. A total of 177 patients with HCV infection and 198 age- and sex-matched healthy controls were enrolled in this study. We evaluated the urinary levels of 8-oxo-7, 8-dihydro-2'deoxyguanosine (8-oxodGuo) and 8-oxo-7, 8-dihydroguanosine (8-oxoGuo) in patients with HCV infection and explored the factors affecting the urinary 8-oxodGuo or 8-oxoGuo levels. Biomarkers of liver function, cancer, and inflammation were determined. Nonparametric correlations were used to evaluate the correlation between 8-oxoGuo or 8-oxodGuo and various laboratory biochemical indicators. Results showed that the levels of urinary 8-oxoGuo both in male and female patients with HCV infection were significantly higher than those in healthy controls (both p < 0.0001), while the urinary 8-oxodGuo levels only in male patients with HCV infection were significantly higher than those in healthy controls (p < 0.01). Urinary 8-oxoGuo was significantly associated with the white blood cell count, C-reactive protein level, and 8-oxodGuo level (p = 0.016, p = 0.003, and p = 0.000, respectively). Urinary 8-oxodGuo was significantly associated with the white blood cell count and 8-oxoGuo level (p = 0.018 and p = 0.000, respectively). A regression equation of urinary 8-oxoGuo or 8-oxodGuo was also established using the biomarkers in plasma. The results suggested that patients with a high C-reactive protein level are likely to have high urinary 8-oxoGuo levels as well, which may be useful for assessing the level of inflammation and oxidative stress in HCV patients.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1961272 .

Effects of a highly controlled carbohydrate-reduced high-protein diet on markers of oxidatively generated nucleic acid modifications and inflammation in weight stable participants with type 2 diabetes; a randomized controlled trial.

Carbohydrate-restricted diets are increasingly recognized as options for dietary management of type 2 diabetes mellitus (T2DM). We investigated the effects of a carbohydrate-reduced high-protein (CRHP) and a conventional diabetes (CD) diet on oxidative stress and inflammation in weight stable individuals with T2DM. We hypothesized that the CRHP diet would improve markers of oxidatively generated RNA and DNA modifications as well as inflammatory parameters. Thirty participants with T2DM were randomized to 6 weeks of CRHP or CD dietary treatment (30/50 energy percentage (E%) carbohydrate, 30/17E% protein, 40/33E% fat), followed by a cross-over to the opposite diet for a subsequent 6-week period. All meals were provided during the study and body weight was controlled. Diurnal urine samples were collected after 4 weeks on each diet and oxidatively generated RNA and DNA modifications were measured as 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), respectively. Fasting concentrations of soluble urokinase plasminogen activator receptor, high-sensitivity C-reactive protein, tumor necrosis factor alpha and interleukin-6 were measured before and after 6 weeks of interventions. Compared with the CD diet, the CRHP diet increased 24-hour urinary excretion of 8-oxoGuo by 9.3% (38.6 ± 12.6 vs. 35.3 ± 11.0 nmol/24 h, p = .03), whereas 8-oxodG did not differ between diets (24.0 ± 9.5 vs. 24.8 ± 11.1 nmol/24 h, p = .17). Changes in plasma inflammatory parameters did not differ between CRHP and CD diets, all p ≥ .2. The clinical implications of increased RNA oxidation following a CRHP diet as well as long-term effects of carbohydrate-restriction on markers of oxidatively generated nucleic acid modifications should be a field of future study.

Targeted and untargeted metabolomics applied to occupational exposure to hyperbaric atmosphere.

Occupational exposure to hyperbaric atmosphere occurs in workers who carry out their activity in environments where breathing air pressure is at least 10% higher than pressure at sea level, and operations can be divided in Dry or Wet activities. The increased air pressure implies the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), consumption of antioxidants and reduction of antioxidant enzyme activity, causing lipid peroxidation, DNA and RNA damage. The present study was aimed to establish the relation between hyperbaric exposure and metabolic changes due to ROS unbalance, by means of the determination of urinary biomarkers of oxidatively generated damage to DNA and RNA during a controlled diving session. The investigated biomarkers were 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). The experimental session involved six experienced divers subjected to 3 atmospheres absolute for 30 minutes in two different experiments, in both dry and wet conditions. Urine samples were collected at t = 0 (before exposure) and 30 (end of exposure),90, 240, 480 and 720 minutes. The concentration of 8-oxoGua, 8-oxoGuo, and 8-oxodGuo was determined by isotopic dilution high performance liquid chromatography (HPLC-MS/MS). In all subjects there is an increase of the urinary excretion of 8oxo-Guo and 8oxo-dGuo, in both conditions, after 1.5 - 4 hours from the start of the experiment, and that the values tend to return to the baseline after 12 hours. Besides that, also the nucleic magnetic resonance (NMR)-based untargeted metabolomics was employed for the same objective on the same samples, confirming a different metabolic response in the subjects exposed to dry or wet conditions. In particular, the observed hypoxanthine urinary level increases during the underwater hyperbaric exposure, in agreement with the trend observed for 8-oxoGuo and 8-oxodGuo levels. Present results confirmed the relationship between exposure and oxidative stress and depicted a clear temporal trend of the investigated biomarkers. Due to the possible negative consequences of oxidative stress on workers, present research shows a new line in term of risk prevention.

Development of a gas chromatography-mass spectrometry method for breast cancer diagnosis based on nucleoside metabolomes 1-methyl adenosine, 1-methylguanosine and 8-hydroxy-2'-deoxyguanosine.

Metabolomes are small molecule metabolites (<1000 Da) produced by cellular processes. Metabolomes are close counterparts to the genome, transcriptome and proteome. The aim of this study was to develop a method to detect and quantify candidate nucleoside metabolomes 1-methyl adenosine (1-MA), 1-methylguanosine (1-MG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the urine of patients with breast cancer using gas chromatography-mass spectrometry (GC-MS). The method was applied to urine specimens from patients with breast cancer (n = 56) and benign breast tumors (n = 22), as well as from healthy females (n = 20). The relative standard deviations of precision and repeatability analysis were <10%, and recoveries ranged from 88.5 to 105.6%. Limits of detection were 0.014, 0.012, and 0.018 mg/L for 1-MA, 1-MG and 8-OHdG, respectively. The lower limits of quantitation were 0.056, 0.048 and 0.072 mg/L, respectively. There were significant differences in concentrations of candidate metabolomes between patients with cancer and the healthy individuals, especially for those in the early stages of the disease (p < 0.001). No significant differences were observed between the benign and healthy groups. In conclusion, a reliable GC-MS method for the detection and quantification of 1-MA, 1-MG, and 8-OHdG metabolomes in urine has been developed.

8-Hydroxyguanosine as a possible RNA oxidative modification marker in urine from colorectal cancer patients: Evaluation by ultra performance liquid chromatography-tandem mass spectrometry.

Oxidative RNA damage has been found to be associated with a variety of diseases, and 8-hydroxyguanosine (8-OHG) is a typical marker of oxidative modification of RNA. This guanosine modification is an emerging biomarker for disease detection and determination of 8-OHG in human urine is favored because it is noninvasive to patients. However, due to its poor ionization efficiency in mass spectrometry and trace amount in urine, accurate quantification of this modified nucleoside is still challenging. Herein, a rapid, accurate, sensitive and robust method using solid-phase extraction (SPE) combined with isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for detection of this oxidative RNA modification in human urine. The limit of detection can reach 1.5 fmol and the method exhibits good precision on intra-day (1.8-3.3%) and inter-day (0.6-1.2%) analyses. Satisfactory recovery (87.5-107.2%) at three spiked levels was achieved by using HLB cartridge for urine pretreatment. Using this method, we quantified 8-OHG in urine from 65 colorectal cancer (CRC) patients and 76 healthy volunteers. The measured level of urinary 8-OHG for CRC patients and healthy controls is 1.91 ± 0.63 nmol/mmol creatinine and 1.33 ± 0.35 nmol/mmol creatinine, respectively. We found the content of 8-OHG in urine was raised in CRC patients patients, implying this oxidative RNA modification marker could act as a potential noninvasive indicator for early screening of CRC. In addition, this study will make contributions to the investigations of the influences of oxidative stress on the formation and development of CRC.

Identification and quantification of isoguanosine in humans and mice.

Isoguanine (2-hydroxyadenine), considered to be a non-natural nucleobase has, however, been shown to occur in the croton bean, butterfly wings and a mollusk. For the first time, to the best of our knowledge, we report the identification of isoguanosine (2-hydroxyadenosine), the ribonucleoside, in humans and mouse. Isoguanosine is identified and quantified in RNA from mouse liver samples and in human urine and cerebrospinal fluid. Isoguanine could not be detected as the 2'-deoxyribonucleoside in mouse liver DNA. It could be speculated that the source of isoguanosine was formation from adenosine during oxidative stress in the body. However, the urinary concentrations of isoguanosine and the levels in the liver found here by using isotope dilution liquid chromatography-tandem mass spectrometry are identical to or exceed those of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine. Guanine is the nucleobase that is oxidized the easiest, so it appears spectacular that the levels of isoguanosine are higher than the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine. It also appears intriguing that it was only possible to detect the ribonucleoside isoguanosine and not the 2'-deoxyribonucleoside. These observations could indicate that the isoguanosine found is not formed by oxidative stress and could have biological functions.

Reduced levels of modified nucleosides in the urine of autistic children. Preliminary studies.

The aim of this study was to investigate and compare the levels of concentration of modified nucleosides in the urine of autistic and healthy children. The compounds have never been analyzed before. The levels of nucleosides in the urine of both groups were determined by validated high performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved with HILIC column and tubercidin was used as the internal standard for the quantification of urinary nucleosides. The within run accuracy and precision ranged from 89 to 106% and from 0.8% to 4.9%, respectively. Lower levels of O-methylguanosine, 7-methylguanosine, 1-methyladenosine, 1-methylguanine, 7-methylguanine and 3-methyladenine in the urine of 22 children with autism, aged 3 to 16 were observed. The differences were not observed in 20 healthy volunteers, in a similar age group. These findings show that modified nucleosides there are metabolic disturbances and nutritional deficiencies in autistic children.

2',3'-cGMP exists in vivo and comprises a 2',3'-cGMP-guanosine pathway.

The discovery in 2009 that 2',3'-cAMP exists in biological systems was rapidly followed by identification of 2',3'-cGMP in cell and tissue extracts. To determine whether 2',3'-cGMP exists in mammals under physiological conditions, we used ultraperformance LC-MS/MS to measure 2',3'-cAMP and 2',3'-cGMP in timed urine collections (via direct bladder cannulation) from 25 anesthetized mice. Urinary excretion rates (means ± SE) of 2',3'-cAMP (15.5 ± 1.8 ng/30 min) and 2',3'-cGMP (17.9 ± 1.9 ng/30 min) were similar. Mice also excreted 2'-AMP (3.6 ± 1.1 ng/20 min) and 3'-AMP (9.5 ± 1.2 ng/min), hydrolysis products of 2',3'-cAMP, and 2'-GMP (4.7 ± 1.7 ng/30 min) and 3'-GMP (12.5 ± 1.8 ng/30 min), hydrolysis products of 2',3'-cGMP. To validate that the chromatographic signals were from these endogenous noncanonical nucleotides, we repeated these experiments in mice (n = 18) lacking 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), an enzyme known to convert 2',3'-cyclic nucleotides to their corresponding 2'-nucleotides. In CNPase-knockout mice, urinary excretions of 2',3'-cAMP, 3'-AMP, 2',3'-cGMP, and 3'-GMP were increased, while urinary excretions of 2'-AMP and 2'-GMP were decreased. Infusions of exogenous 2',3'-cAMP increased urinary excretion of 2',3'-cAMP, 2'-AMP, 3'-AMP, and adenosine, whereas infusions of exogenous 2',3'-cGMP increased excretion of 2',3'-cGMP, 2'-GMP, 3'-GMP, and guanosine. Together, these data suggest the endogenous existence of not only a 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP/3'-AMP → adenosine), which was previously identified, but also a 2',3'-cGMP-guanosine pathway (2',3'-cGMP → 2'-GMP/3'-GMP → guanosine), observed here for the first time. Because it is well known that adenosine and guanosine protect tissues from injury, our data support the concept that both pathways may work together to protect tissues from injury.

Modified Nucleosides - Molecular Markers Suitable for Small-volume Cancer?

BACKGROUND: Modified nucleosides (mNS) in urine are shown to be encouraging markers in cancer, mostly in patients presenting with high tumor mass such is breast and lung cancer. To our knowledge, mNS have not been investigated in head and neck squamous cell carcinoma (HNSCC). HNSCC is characterized by early metastasis into locoregional lymph nodes and slow infiltrating growth, but even in the advanced stage exhibits only a relatively low cancer volume. Therefore, reliable distinction between HNSCC and healthy controls by urinary mNS might pose substantial analytical problems and even more as patients with HNSCC mostly have an increased exposure to tobacco smoke and excessive alcohol consumption which affect the renal mNS pattern. MATERIALS AND METHODS: Urinary mNS in samples of 93 therapy-naive patients with HNSCC and 242 healthy controls were quantified by reversed-phase high-performance liquid chromatography. Considering that the circadian rhythm causes diuresis-induced variations in concentration, the mNS-to-creatinine ratio was chosen to compare patients and controls. For sensitivity and specificity in discriminating between patients and controls, the corresponding curve was plotted. Additionally, logistic regression was carried out and a multilayer perceptron neuronal network (NN) was created. RESULTS: Fifteen mNS were detectable in cases and controls; concentrations of 11 were found to be significantly different. The sensitivity and specificity depend on the total volume of the lesion; HNSCC with volume <20 ml was reliably detected, but those with a volume of 20 ml or greater produced amounts of mNS which led to the most accurate detection of HNSCC based on HNSCC-specific mNS patterns. CONCLUSION: Analysis of urinary mNS allows for detection of small-volume HNSCC, with acceptable specificity and sensitivity if the tumor volume exceeds 20 ml.

RNA oxidation and iron levels in patients with diabetes.

AIM: The urinary biomarker for oxidative stress to RNA, 8-oxo-7,8-dihydro-guanosine (8-oxoGuo) is associated with mortality in patients with type 2 diabetes. Iron has also been linked to diabetes. In individuals with untreated hereditary iron overload it has been observed that 8-oxoGuo was higher compared to controls. In the current study, we hypothesized that 8-oxoGuo was associated with diagnosis of diabetes, and that iron confounded this association. METHODS: Participants from a general Danish population were included in the study (n = 3567). UPLC-MS/MS method was used for 8-oxoGuo (nmol/mmol creatinine) measurement in spot urine. Iron biomarkers included total plasma iron, ferritin, transferrin saturation (TS) and transferrin. RESULTS: 8-oxoGuo was 17% higher in diabetes patients (n = 208) compared to non-diabetes controls. Unadjusted logistic regression model showed an odds ratio of diabetes of 1.38 (95%CI:1.21-1.57, P < 0.0001) per unit increase of 8-oxoGuo. When the model was adjusted for possible confounders the odds ratio was 1.09 (95%CI:0.94-1.26, P = 0.24). When additional adjustment was performed including ferritin, TS, or transferrin, respectively, the OR were 1.14 (95%CI:0.97-1.33, P = 0.09), 1.10 (95%CI: 0.95-1.28, P = 0.18), and 1.17 (95%CI:1.01-1.38, P = 0.04). CONCLUSIONS: Our study indicates that 8-oxoGuo is higher in diabetes patients. The lack of association between 8-oxoGuo and diabetes in the adjusted model may be due to the cross-sectional design including post-treatment bias. Our data did not show consistent effect of all iron biomarkers in relation to diabetes. Most likely, the iron biomarkers were affected by inflammation thus not reflecting true iron levels.

Biomarkers of early genotoxicity and oxidative stress for occupational risk assessment of exposure to styrene in the fibreglass reinforced plastic industry.

This study aimed to identify sensitive and not-invasive biomarkers of early genotoxic/oxidative effect for exposure to styrene in the fibreglass reinforced plastic manufacture. We studied 11 workers of a plastic manufacture using open molding process (A), 16 workers of a manufacture using closed process (B) and 12 controls. We evaluated geno/cytotoxic effects on buccal cells by Buccal Micronucleus Cytome (BMCyt) assay and genotoxic/oxidative effects on lymphocytes by Fpg-comet test. On A workers we also evaluated urinary 8oxoGua, 8oxodGuo and 8oxoGuo to investigate oxidative stress. Personal inhalation exposure to styrene was monitored by passive air sampling and GC/MS. Biological monitoring included urinary metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA). The findings show higher styrene exposure, urinary MA + PGA levels and micronucleus frequency in manufacture A. Higher buccal karyolytic cell frequency vs controls were found in both exposed populations. We found in exposed workers, no induction of direct DNA damage but oxidative DNA damage. Fpg-comet assay and urinary oxidized guanine seem to be sensitive biomarkers of oxidative stress and BMCyt assay a good-not invasive biomarker of cyto-genotoxicity at target organ. The study, although limited by the small number of studied subjects, shows the usefulness of used biomarkers in risk assessment of styrene-exposed workers.

Increased oxidative damage of RNA in liver injury caused by hepatitis B virus (HBV) infection.

To evaluate the urinary levels of 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) in liver injury patients with hepatitis B virus (HBV) infection and to explore the relationship between urinary 8-oxo-dGsn or 8-oxo-Gsn and degree of liver damage. We enrolled 138 liver injury patients with HBV infection and 169 age- and sex-matched healthy controls in this study. A sensitive and accurate isotope-diluted liquid chromatograph mass spectrometer/mass spectrometer (LC-MS/MS) method was used to measure the urinary levels of 8-oxo-Gsn and 8-oxo-dGsn. Simultaneously, pathological analysis of liver biopsy tissues was carried out, and immunohistochemistry was carried out for 8-oxo-Guo, 8-oxo-dGuo and MTH1 protein in some liver injury tissues. We analysed the correlation between the degrees of inflammation and fibrosis and levels of 8-oxo-Gsn and 8-oxo-dGsn. We also analysed the levels of urinary 8-oxo-Gsn and 8-oxo-dGsn with clinical data of HBeAg, HBsAg, and HBV genotype and detected the levels of plasma aspartate aminotransferase, alanine aminotransferase (AST), platelet, alkaline phosphatase, prothrombin time (PT) and HBV DNA, and calculated the aspartate amino transferase-to-platelet ratio index (APRI) score. Nonparametric correlations were used to evaluate the correlation between 8-oxo-Gsn, 8-oxo-dGsn or APRI and various laboratory biochemical indicators. Results showed that the levels of urinary 8-oxo-Gsn and 8-oxo-dGsn in patients with liver injury were significantly higher than those of healthy controls (both p < .001). Urinary 8-oxo-Gsn was significantly associated with AST, APRI and PT (p = .013, p = .026 and p = .049). The receiver operating characteristic curves of 8-oxo-Gsn were 0.696 (0.632-0.759) and 0.731 (0.672-0.790) for inflammatory activity and fibrosis, respectively. Patients with higher levels of urinary 8-oxo-Gsn are more likely to have a high degree of fibrosis and urinary 8-oxo-Gsn may have a great potential in assessing liver fibrosis.

Iron induced RNA-oxidation in the general population and in mouse tissue.

Iron promotes formation of hydroxyl radicals by the Fenton reaction, subsequently leading to potential oxidatively generated damage of nucleic acids. Oxidatively generated damage to RNA, measured as 8-oxo-7,8-dihydroguanosine (8-oxoGuo) in urine, is increased in patients with genetic iron overload, which have led us to test the hypothesis that high iron status, assessed by iron biomarkers and genetic disposition, increases urinary excretion of 8-oxoGuo. In a general Danish population study we used a Mendelian randomization design with HFE genotypes as a proxy for iron status and supplemented with ex vivo experiments in mice muscle tissue exposed to iron(II) sulfate to attempt to clarify this hypothesis. The biomarkers ferritin, transferrin, and transferrin saturation (TS) were associated with 8-oxoGuo (in linear univariable and multivariable regression analyses: P < 0.001). Mendelian randomization indicated a causal pathway between genetically elevated iron biomarkers (assessed by ferritin and TS) and high levels of 8-oxoGuo. The ex vivo experiments showed a monotonically increase in 8-oxoGuo with increased iron concentration (ANOVA: P = 0.0008) that was prevented with iron chelation (P = 0.01). Our results indicate a causal relationship between iron biomarkers and 8-oxoGuo. Furthermore, the ex vivo experiment shows a mechanistic link between iron and 8-oxoGuo formation. Both iron overload and the biomarker 8-oxoGuo have been linked to e.g. diabetes, which merits future studies to investigate if iron induced 8-oxoGuo is involved in disease development.

Randomized Controlled Trial of Mineralocorticoid Receptor Blockade in Children with Chronic Kidney Allograft Nephropathy.

BACKGROUND AND OBJECTIVES: We showed that mineralocorticoid receptor blockade (MRB) prevented acute and chronic cyclosporine nephropathy (CsA-Nx) in the rat. The aim of this translational study was to investigate the effect of long-term eplerenone administration on renal allograft function in children with biopsy-proven chronic allograft nephropathy (CAN). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Renal transplant children <18 years, biopsy-proven CAN, and a GFR>40 ml/min per 1.73 m2 were included. Patients with BK virus active nephritis, recurrence of renal disease, GFR decline in previous 3 months, or treated with calcium antagonists or antifungal drugs were excluded. They were randomized to receive placebo (n=10) or eplerenone 25 mg/d for 24 months (n=13). Visits were scheduled at baseline, 6, 12, and 24 months. At each period, a complete clinical examination was performed and blood and urine samples were taken. Urine creatinine, 8-hydroxylated-guanosine, heat shock protein 72 (HSP72), and kidney injury molecule (KIM-1) levels were also assessed. In kidney biopsy samples, the tubulo-interstitial area affected by fibrosis (TIF) and glomerulosclerosis were measured at baseline and after 24 months. RESULTS: The baseline eGFR was 80±6 in the placebo and 86±6 ml/min per 1.73 m2 in the eplerenone group; at 24 months it was 66±8 and 81±7 ml/min per 1.73 m2, respectively (P=0.33; 95% confidence intervals, -18 to 33 at baseline, and -11 to 40 after 24 months). The albumin-to-creatinine ratio was 110±74 in the placebo, and 265±140 mg/g in the eplerenone group; and after 24 months it was 276±140 and 228±88 mg/g, respectively (P=0.15; 95% confidence intervals, -283 to 593, and -485 to 391, respectively). In addition, the placebo exhibited a greater TIF, glomerulosclerosis, and urinary HSP72 compared with the eplerenone group. CONCLUSIONS: Although this study was underpowered to provide definitive evidence that long-term eplerenone administration attenuates the progression of CAN in pediatric transplant patients, it encourages testing the potential benefit of MRB in this pediatric population.

The effect of smoking on the urinary excretion of 8-oxodG and 8-oxoGuo in patients with type 2 diabetes.

Over the past decades, attention has been paid to understanding the impact of oxidative stress and related modifications of DNA and RNA on various human health risks. A recent meta-analysis comprising 1915 smokers and 3462 non-smokers found a significantly higher level of DNA oxidation measured as urinary 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) excretion in smokers compared with non-smokers in a healthy population. We aimed to investigate if an increased urinary excretion of 8-oxodG in smokers versus never smokers and former smokers could be verified in a population with type 2 diabetes. Additionally, we measured RNA oxidation levels through urinary excretion of 8-oxo-7, 8-dihydroguanosine (8-oxoGuo). Our study included urinary samples from 2721 type 2 diabetic patients, analyzed using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Logistic regression was used to examine the relationship between daily smokers (n = 462) versus former (n = 1341) and never smokers (n = 918) regarding the RNA and DNA oxidation, respectively. We did not find any significant effect of smoking on urinary excretion of 8-oxodG or 8-oxoGuo in our study. Due to a sparse study area, it is still too early to draw any conclusions on smoking and RNA-oxidation. Regarding DNA oxidation, our study suggests that the effect of smoking seen in healthy populations might be attenuated in patients with type 2 diabetes.

A fully validated bioanalytical method using an UHPLC-MS/MS system for quantification of DNA and RNA oxidative stress biomarkers.

A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.

Smoking modify the effects of polycyclic aromatic hydrocarbons exposure on oxidative damage to DNA in coke oven workers.

PURPOSE: Coke oven emissions containing polycyclic aromatic hydrocarbons (PAHs) are predominant toxic constituents of particulate air pollution that have been linked to increased risk of lung cancer. Numerous epidemiological studies have suggested that oxidative DNA damage may play a pivotal role in the carcinogenic mechanism of lung cancer. Little is known about the effect of interaction between PAHs exposure and lifestyle on DNA oxidative damage. METHODS: The study population is composed by coke oven workers (365) and water treatment workers (144), and their urinary levels of four PAH metabolites and 8-hydroxydeoxyguanosine (8-OHdG) were determined. Airborne samples of exposed sites (4) and control sites (3) were collected, and eight carcinogenic PAHs were detected by high-performance liquid chromatography. RESULTS: The median values of the sum of eight carcinogenic PAHs and BaP in exposed sites were significantly higher than control sites (P < 0.01). The study found that the urinary PAH metabolites were significantly elevated in coke oven workers (P < 0.01). Multivariate logistic regression analysis revealed that the risk of high levels of urinary 8-OHdG will increase with increasing age, cigarette consumption, and levels of urinary 1-hydroxypyrene, and P for trend were all <0.05. Smoking can significantly modify the effects of urinary 1-hydroxypyrene on high concentrations urinary 8-OHdG, during co-exposure to both light or heavy smoking and high 1-hydroxypyrene levels (OR 4.28, 95% CI 1.32-13.86 and OR 5.05, 95% CI 1.63-15.67, respectively). CONCLUSIONS: Our findings quantitatively demonstrate that workers exposed to coke oven fumes and smoking will cause more serious DNA oxidative damage.

Impact of chemotherapy on medium-term physical function and activity of older breast cancer survivors, and associated biomarkers.

OBJECTIVE: Chemotherapy is less often prescribed in older individuals due to concerns about post-treatment morbidity and quality of life. We evaluated the physical performance of breast cancer survivors treated with and without adjuvant chemotherapy. MATERIALS AND METHODS: We conducted a case-control study in 56 estrogen receptor positive breast cancer survivors (BCS) on adjuvant aromatase inhibitors 1-2years after definitive surgery. Cases had received adjuvant chemotherapy (n=27; age 70.5±3.6years) versus age-matched controls who had not (n=29; age 70.0±4.3years). Measures of grip strength, physical activity and performance, walking speed, fatigue, and self-reported physical function were collected. Biological correlates of inflammation, frailty and markers of DNA and RNA oxidation were compared. RESULTS: Grip strength (controls: 21±7.4 vs. CASES: 29.7±5.0kg, p=0.20), physical activity (5403±3204 vs. 6801±9320steps/day, p=0.45), physical performance (short physical performance battery score: 10.1±1.8 vs. 10.4±1.1, p=0.52) and long-distance walking speed (1.2±0.21 vs. 1.3±0.41m/s, p=0.17) were similar between the two groups. Self-reported physical function was marginally lower in cases than controls (controls: 72±24 vs. CASES: 57±34AU, p=0.07). Fatigue disruptiveness was not different between groups (controls: 11.1±13.0 vs. CASES: 15.7±16.2AU, p=0.24). Similarly, the inflammation, oxidation, and frailty markers did not present a significant difference between groups, except for vitamin D levels (p=0.04). CONCLUSION: Older women who received chemotherapy reported having slightly lower physical function, but a similar physical performance compared to women who did not. These data suggest that older BCS treated with chemotherapy recover to an extent similar to survivors who only received hormonal therapy.

Toward Noninvasive Diagnosis of IgA Nephropathy: A Pilot Urinary Metabolomic and Proteomic Study.

IgA nephropathy is diagnosed by renal biopsy, an invasive procedure with a risk of significant complications. Noninvasive approaches are needed for possible diagnostic purposes and especially for monitoring disease activity or responses to treatment. In this pilot project, we assessed the utility of urine samples as source of biomarkers of IgA nephropathy. We used spot urine specimens from 19 healthy controls, 11 patients with IgA nephropathy, and 8 renal-disease controls collected on day of renal biopsy. Urine samples were analyzed using untargeted metabolomic and targeted proteomic analyses by several experimental techniques: liquid chromatography coupled with mass spectrometry, immunomagnetic isolation of target proteins coupled with quantitation by mass spectrometry, and protein arrays. No single individual biomarker completely differentiated the three groups. Therefore, we tested the utility of several markers combined in a panel. Discriminant analysis revealed that combination of seven markers, three metabolites (dodecanal, 8-hydroxyguanosine, and leukotriene C4), three proteins (α1-antitrypsin, IgA-uromodulin complex, and galactose-deficient IgA1), and heparan sulfate, differentiated patients with IgA nephropathy from patients with other renal diseases and healthy controls. Future studies are needed to validate these preliminary findings and to determine the power of these urinary markers for assessment of responses to therapy.

Concurrent quantification of multiple biomarkers indicative of oxidative stress status using liquid chromatography-tandem mass spectrometry.

8-Hydroxy-2-deoxyguanosine (8-OHdG), 8-nitroguanine (8-NO2Gua), 8-iso-prostaglandin F2α (8-IsoPGF2α), and N-acetyl-S-(tetrahydro-5-hydroxy-2-pentyl-3-furanyl)-L-cysteine (HNE-MA) are well-studied and representative biomarkers for oxidative DNA damage, inflammation, and lipid peroxidation; all of which have been associated with increases in risks of various diseases and cancers. A rapid and highly sensitive isotope-dilution liquid-chromatography tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously quantify the aforementioned biomarkers in urine. Upon validation, this method shows excellent feasibility, sensitivity (0.008-0.03 ng/mL) and satisfactory recoveries (88.7-95.4%); the calibration curves displayed excellent linearity with coefficients of determination (R(2)) greater than 0.998. Additionally, low variations were observed in the relative standard deviation for intra- and inter-day measurements for the four analytes. The relative matrix effects for all four analytes ranged from 2.04 to 3.27%, which signaled that interferences from endogenous levels of the analytes were deemed statistically insignificant. This study successfully developed an analytical method capable to simultaneously quantify urinary 8-OHdG, 8-NO2Gua, 8-IsoPGF2α, and HNE-MA. This analytical protocol can be applied towards conducting epidemiological studies to reveal the mechanisms related to disease development, and thus evaluate the associated risks of diseases.