Oxidative/nitrosative stress increased the risk of recurrent pregnancy loss-Taiwan Recurrent Pregnancy Loss and Environmental Study (TREPLES).

OBJECTIVE: Oxidative stress biomarkers (OSBs) may be strongly associated with disease progression and recurrent pregnancy loss (RPL). However, the research on associations of most OSBs (e.g., 8-nitroguanine [8-NO2Gua] and 4-hydroxy-2-nonenal-mercapturic acid [HNE-MA]) with RPL is limited. Therefore, we aimed to investigate the effect of OSBs exposure on RPL risk by performing a case-control study. MATERIAL AND METHODS: We use our established dataset, Taiwan Recurrent Pregnancy Loss and Environmental Study (TREPLES), which included 514 Taiwanese reproductive age women (aged 20-50 years; 397 cases and 117 controls) from National Cheng Kung University Hospital. RPL is clinically defined by a history of two or more consecutive miscarriages, where a miscarriage is defined as the termination of pregnancy before 20 weeks of gestation. The urinary levels of several OSBs (e.g., 8-hydroxy-2'-deoxyguanosine [8-OHdG], 8-NO2Gua, 8-isoprostaglandin F2α [8-isoPGF2α], and HNE-MA) and malondialdehyde (MDA) were measured using isotope dilution liquid chromatography-tandem mass spectrometry and thiobarbituric acid reactive substances, respectively. RESULTS: The median levels of 8-NO2Gua (6.15 vs. 3.76 ng/mL) and HNE-MA (30.12 and 21.54 ng/mL) were significantly higher in the RPL group than in the control group. By categorizing the OSBs data into tertiles, after we adjusted for age and urine creatinine levels discovered that the RPL risk associated with 8-NO2Gua and HNE-MA levels in the third tertile were approximately 2 times higher than those in the first tertile (8-NO2Gua, adjusted OR = 3.27, 95 % CI = 1.66-6.43; HNE-MA, adjusted OR = 1.96, 95 % CI = 1.05-3.64; p < 0.05). These findings suggest that the oxidative stress biomarkers of 8-NO2Gua and HNE-MA are risk factors for RPL. CONCLUSION: Our findings indicate that specific OSBs are associated with an increased RPL risk, suggesting that reducing OSB levels can improve RPL risk. Nevertheless, more studies on preventive medicine are required to understand the exposure sources and adverse outcome pathways of OSBs associated with RPL.

Influence of Renal Function on the Single-Dose Pharmacokinetics of Besifovir, a Novel Antiviral Agent for theTreatment of Hepatitis B Virus Infection.

Per the well-known resistance of hepatitis B virus to nucleoside/nucleotide analogs, alternative treatment options with higher resistance barriers have been approved for use in both treatment-naïve and lamivudine-resistant hepatitis B virus infections. This phase I study was conducted in adults with normal and impaired renal function to evaluate the effect of renal impairment on the pharmacokinetics of besifovir, a prodrug of an acyclic nucleotide phosphonate, that is mainly cleared via renal excretion. An open-label, single-dose parallel-group clinical study was conducted in subjects with normal renal function and mild, moderate, and severe renal impairment. Subjects received a single oral dose of besifovir dipivoxil 150 mg, and serial blood and urine samples were collected for up to 72 hours after dosing to assess the pharmacokinetic characteristics of besifovir. The extent of plasma exposure of besifovir, detected as its major and active metabolites, LB80331 and LB80317, respectively, increased with worsening renal function. Compared to the subjects with normal renal function, the mean areas under the concentration-time curves of LB80331 increased by 1.5-, 2.5-, and 4.5-fold in subjects with mild, moderate, and severe impairment, respectively. LB80317 showed a 1.8-, 3.2-, and 6.2-fold increase in subjects with mild, moderate, and severe renal impairment compared to those with normal function. The ratios of LB80331 renal clearance and the average estimated glomerular filtration rate of each renal impairment group with respect to the normal group were similar. The increase in plasma exposure and decrease in renal clearance suggest the need to adjust dosage regimens in patients with moderate to severe renal impairment.

Oncogenic and tumor suppressor pathways in subchronic aflatoxicosis in rats: Association with serum and urinary aflatoxin exposure biomarkers.

In this study, the changes in oncogenic and tumor suppressor signaling pathways in liver and their association with serum and urinary biomarkers of aflatoxin exposure were evaluated in Wistar rats fed diets containing aflatoxin B1 (AFB1) for 90 days. Rats were divided into four groups (n = 15 per group) and assigned to dietary treatments containing 0 (control), 50 (AFB50), 100 (AFB100) and 200 µg AFB1 kg-1 diet (AFB200). Multiple preneoplastic foci of hepatocytes marked with glutathione-S-transferase-placental form (GST-P) were identified in AFB100 and AFB200 groups. Hepatocellular damage induced by AFB1 resulted in overexpression of cyclin D1 and ß-catenin. The liver expression of retinoblastoma (Rb) and p27Kip1 decreased in AFB100 and AFB200 groups, confirming the favorable conditions for neoplastic progression to hepatocellular carcinoma. All samples from rats fed AFB1-contaminated diets had quantifiable AFB1-lysine in serum or urinary AFM1 and AFB1-N7-guanine, with mean levels of 20.42-50.34 ng mL-1, 5.31-37.68 and 39.15-126.37 ng mg-1 creatinine, respectively. Positive correlations were found between AFB1-lysine, AFM1 or AFB1-N7-guanine and GST-P+, ß-catenin+ and cyclin D1+ hepatocytes, while Rb + cells negatively correlated with those AFB1 exposure biomarkers. The pathways evaluated are critical molecular mechanisms of AFB1-induced hepatocarcinogenesis in rats.

Simultaneous analysis of two urinary biomarkers of oxidative damage to DNA and RNA based on packed-fiber solid phase extraction coupled with high-performance liquid chromatography.

The determination of the concentrations of urinary biomarkers of oxidative damage to DNA and RNA is difficult due to the low content of targets and the complex matrix of urine. A method using polystyrene/polypyrrole (PS/PPY) electronspun nanofibers as the adsorbent was introduced to the routine urinary treatment and determination of 8-OHdG and 8-oxoG for the first time. And 2-aminoethyl diphenylborate (DPBA) solution was creatively used in the loading and rinsing steps in order to promote the retention of the analytes as well as remove impurities. Under optimal conditions, 8-OHdG, 8-oxoG and IS were separated very well and exhibited a good linearity in the range of 0.5-50 ng mL-1, with correlation coefficients of R2 > 0.996. Limits of detection (LOD) were 0.058 ng mL-1 and 0.093 ng mL-1, and limits of quantification (LOQ) were 0.195 ng mL-1 and 0.309 ng mL-1, respectively. The recoveries were 88.8-104.9%. The proposed method was so simple and economical that it had the potential to be applied to batch quantitative analysis of 8-OHdG and 8-oxoG in urine. And it was successfully applied to real urine samples of cancer patients.

Stochastic microsensors for the assessment of DNA damage in cancer.

Three stochastic microsensors based on graphite powder modified with three different oleamides: N-(2-piperidin-1-ylethyl)oleamide, N-(3,4-dihydroxyphenethyl)oleamide and N-(2-morpholinoethyl)oleamide, were designed, characterized, and used to assess DNA damage in cancer by assaying two biomarkers namely 8-nitroguanine and 8-hydroxy-2'-deoxyguanosine. The two biomarkers were determined from urine and whole blood samples. The characterization of the microsensors was done at two pHs 7.40 and 3.00. The best microsensor for the simultaneous determination of biomarkers in whole blood and urine samples was the one based on the graphite paste modified with N-(3,4-dihydroxyphenethyl)oleamide. The results indicated that the proposed microsensors can be reliably used for pattern recognition and quantitative determination of 8-nitroguanine and 8-hydroxy-2'-deoxyguanosine in whole blood and urine, and accordingly, for the assessment of DNA damage in cancer patients.

Associations between physical activity, sedentary behavior, and urinary oxidized guanine in colorectal cancer patients: results from the ColoCare Study.

To determine associations between physical activity (PA), sedentary behavior (SB), and oxidative stress in colorectal cancer patients, ColoCare Study participants in Germany wore an accelerometer 6 and/or 12 months after surgery. Spearman partial correlations were used to assess associations between PA and urinary concentrations of oxidized guanine, a validated marker of oxidative stress. There were no significant associations between PA or SB and oxidized guanine in n = 76 measurements (ng/mg creatinine; r = 0.03, p = 0.76 for PA, r = -0.05, p = 0.69 for SB). Novelty Objectively measured PA was not associated with a marker of oxidative stress in colorectal cancer patients.

A new chemiluminescence method for the determination of 8-hydroxyguanine based on l-histidine bound nickel nanoparticles.

A new chemiluminescence aptasensor for sensitive and efficient detection of 8-hydroxyguanine based on the synergistic interaction of Ni NPs@l-histidine@aptamer@MBs has been developed, and it has been applied in the real urine samples of cancer patients.

Colloidal gold clusters formation and chemometrics for direct SERS determination of bioanalytes in complex media.

In this work, we report the sensitive and selective sensing of the purine bases adenine and guanine in urine matrix by using surface-enhanced Raman spectroscopy (SERS) and a colloidal SERS substrate. To identify suitable conditions for quantitative analysis, the pH dependence of spectra of adenine, guanine, urine simulant and their mixtures was studied on gold nanoparticles suspension. Interestingly, although the urine matrix promotes the analytes signal suppression and overlapping bands, it can also cause an improvement in repeatability of the SERS measurements. This effect was associated to the relatively controlled formation of small-sized gold clusters and it was investigated both experimentally and theoretically. Furthermore, a correlation constrained multivariate curve resolution-alternating least squares (MCR-ALS) method was developed to resolve overlapping SERS bands and to quantify physiologically relevant (micromolar) concentrations of the bioanalytes. The performance of the proposed MCR-ALS approach (assessed in terms of figures of merit) was similar to that obtained by using partial least squares regression, but with the additional advantage of retrieving valuable spectral information. Therefore, this method can be used for improving selectivity of colloidal clusters in qualitative and quantitative SERS analysis of complex media, avoiding the need for tedious nanoparticle-surface modification or preliminary chromatographic separation.

Urinary N7-(1-hydroxy-3-buten-2-yl) guanine adducts in humans: temporal stability and association with smoking.

1,3-Butadiene (BD) is a known human carcinogen found in cigarette smoke, automobile exhaust, and urban air. Workers occupationally exposed to BD in the workplace have an increased incidence of leukemia and lymphoma. BD undergoes cytochrome P450-mediated metabolic activation to 3,4-epoxy-1-butene (EB), 1,2,3,4-diepoxybutane (DEB) and 1,2-dihydroxy-3,4-epoxybutane (EBD), which form covalent adducts with DNA. We have previously reported a quantitative nanoLC/ESI+-HRMS3 method for urinary N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts as a mechanism-based biomarker of BD exposure. In the present study, the method was updated to include high throughput 96-well solid phase extraction (SPE) and employed to establish urinary EB-GII biomarker stability and association with smoking. Urinary EB-GII levels were measured bimonthly for 1 year in 19 smokers to determine whether single adduct measurement provides reliable levels of EB-GII in an individual smoker. In addition, association of EB-GII with smoking was studied in 17 individuals participating in a smoking cessation program. EB-GII levels decreased 34% upon smoking cessation, indicating that it is associated with smoking status, but may also originate from sources other than exposure to cigarette smoke.

Development and validation of a rapid LC-MS/MS method for determination of methylated nucleosides and nucleobases in urine.

Modified nucleosides and nucleobases serve as potential diagnostic and prognostic markers of various diseases, including cancer. These compounds are hydrophilic, but are determined mainly using columns with C18 stationary phase. Moreover, these compounds require purification due to the presence of high amounts of isotopomers in the sample matrix and risk of matrix interferences. The most commonly used method for analyte extraction (i.e. solid-phase extraction on phenylboronic acid sorbent) is not appropriate for all the analytes since compounds with cis-diol groups (e.g. 7-methylguanine) are not absorbed. Thus, the aim of this study was to develop and validate a simple and fast method for the simultaneous determination of the methylated nucleosides and nucleobases (derivatives of adenine and guanine including those with cis-diol groups) in urine. The method was based on hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS). The sample preparation involved only dilution with acetonitrile and centrifugation. The method was validated for selectivity, calibration curve, precision, accuracy, stability, matrix effect, dilution integrity, and carryover, according to the guidelines of the European Medicines Agency (EMA). All the analyzed validation criteria were fulfilled. The method was applied to the urine of rats and humans (adults and children). To our best knowledge, HILIC-MS/MS method established by our team is the first method that does not require the extraction step as well as this method enables simultaneously determination of 1-methylguanine, 2'-O-methylguanosine, 3-methyladenine, and N6-methyl-2'-deoxyadenosine in urine. The method is reliable and can be applied to determine the concentrations of the modified nucleosides and nucleobases in the urine of humans and rats. Because the method is cost-effective, fast, and easy to perform, it may be considered as a routine tool in clinical laboratories.

Prevention by L-carnitine of DNA damage induced by 3-hydroxy-3-methylglutaric and 3-methylglutaric acids and experimental evidence of lipid and DNA damage in patients with 3-hydroxy-3-methylglutaric aciduria.

3-hydroxy-3-methylglutaric aciduria (HMGA) is an inherited disorder of the leucine catabolic pathway in which occurs a deficiency of the 3-hydroxy-3-methylglutaryl-CoA lyase enzyme. Therefore, the organic acids 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA), mainly, accumulate in tissues of affected patients. Lately, much attention has been focused on free radicals as mediators of tissue damage in human diseases, causing lipid peroxidation, protein oxidation and DNA damage. The treatment of this disease is based in a restricted protein ingest and supplementation with l-carnitine (LC), an antioxidant and detoxifying agent. In the present work, we investigated the in vitro oxidative damage to DNA induced by the accumulation of organic acids and oxidative stress parameters in vivo of patients with 3-HMG, as well as the effect of the recommended therapy. The in vitro DNA damage was analyzed by the alkaline comet assay in leukocytes incubated with HMG and MGA (1 mM, 2.5 mM and 5 mM) and co-incubated with LC (90 µM and 150 µM). The in vivo urinary 15-F2t-isoprostane levels and urinary oxidized guanine species were measured by ELISA kits in patient's urine before and after the treatment with LC. HMG and MGA induced a DNA damage index (DI) significantly higher than that of the control group. The DI was significantly reduced in the presence of LC. It was also verified a significant increase of oxidized guanine species and urinary isoprostane levels, biomarker of oxidative DNA damage and lipid peroxidation respectively, in patients before treatment. After the treatment and supplementation with LC, patients presented significantly lower levels of those biomarkers. Analyzing the data together, we can conclude that HMGA patients present oxidative lipid and DNA damage, which is induced by HMG and MGA, and the antioxidant therapy with LC can prevent that kind of injuries.

Leisure-time physical activity and DNA damage among Japanese workers.

BACKGROUND: It remains unclear whether daily physical activity is associated with DNA damage. This cross-sectional study examined the association between leisure-time physical activity and urinary 8-hydroxydeoxyguanosine (8-OH-dG), a biomarker of oxidative DNA damage, or urinary 7-methylguanine (m7Gua), a biomarker of methylating DNA damage. METHODS: Participants included 501 workers (294 men and 207 women), aged 20-65 years, from municipal offices in Japan. Urinary 8-OH-dG and m7Gua were measured using column-switching HPLC. Physical activity was evaluated using a self-reported questionnaire. The associations between leisure-time physical activity and urinary DNA damage markers were assessed by multiple linear regression analysis, with stratification by occupational physical activity. RESULTS: After adjusting for covariates, leisure-time physical activity showed a suggestive inverse correlation with urinary 8-OH-dG levels (P for trend = 0.06), and a significant inverse association with urinary m7Gua levels (P for trend = 0.03). In analysis stratified by occupation, inverse correlations were observed in sedentary workers (walking < 30 min/day at work: P for trend = 0.06 and = 0.03 for urinary 8-OH-dG and m7Gua, respectively), but not in physically active workers (walking ≥ 30 min/day at work). In analysis for each intensity of leisure-time physical activity, light-intensity exercise was associated with lower levels of urinary 8-OH-dG (P for trend = 0.03), whereas moderate-to-high-intensity exercise was associated with lower levels of urinary m7Gua (P for trend = 0.02). CONCLUSIONS: Our results suggest that high levels of leisure-time physical activity are associated with decreased levels of DNA damage in individuals with low physical activity at work.

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.

Clinical relevance of guanine-derived urinary biomarkers of oxidative stress, determined by LC-MS/MS.

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 < 0.001), but lower MDA levels, compared to healthy controls. Receiver operating characteristic curve analysis revealed that urinary 8-oxoGuo is the most sensitive biomarker for oxidative stress with area under the curve (AUC) of 0.91, followed by 8-oxodGuo (AUC: 0.80) and 8-oxoGua (AUC: 0.76). Interestingly, MDA with AUC of 0.34 failed to discriminate the patients from healthy controls. Emerging evidence suggests a potential clinical utility for the measurement of urinary 8-oxoGuo, and to a lesser extent 8-oxodGuo, which is strongly supported by our findings.

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.

Multiporous molybdenum carbide nanosphere as a new charming electrode material for highly sensitive simultaneous detection of guanine and adenine.

By introduction of Mo metal species (molybdenum-based polyoxometalates) into the Cu-MOF as co-precursor, molybdenum carbide nanosphere (MoxC@C) was prepared via a simple calcining routine and a further etching the metallic Cu process. The obtained MoxC@C showed a unique structure where well-dispersed MoxC nanoparticles (NPs) were encapsulated in porous carbon matrix. As-fabricated novel 3D porous architecture MoxC@C nanosphere exhibited a potent and persistent electro-oxidation behavior followed by well-separated oxidation peaks (peak to peak voltage is about 350 mV) toward adenine (A) and guanine (G) by differential pulse voltammetry (DPV). This excellent electrochemical performance can be attributed to the unique structure and composition of 3D MoxC@C. Furthermore, 3D MoxC@C also revealed high selectivity and sensitivity, good reproducibility, excellent stability and anti-interference ability. The calibration curves for quantitive analysis of G and A were obtained: 0.03-122 µM, and 0.02-122 µM, respectively, the detection limits were 0.0085 µM, 0.008 µM, respectively. The proposed procedure was successfully applied to detect G and A in human urine and serum samples with satisfactory recovery, which manifests its viability application for practical analysis.

The effect of long-term treatment with coenzyme Q10 on nucleic acid modifications by oxidation in children with Down syndrome.

Elevated levels of oxidative nucleic acid modifications have been proposed to be associated with some of the clinical characteristics of Down syndrome. Oral intake of coenzyme Q10 improves oxidative status and shows a tendency toward protective effect on DNA oxidation in certain age groups of children with Down syndrome. Here, we demonstrate that long-term (i.e., 4 years) treatment with coenzyme Q10 (ubiquinone) at the dosage of 4 mg/kg/d does not affect whole body DNA and RNA oxidation.

Dietary non-enzymatic antioxidant capacity and DNA damage in a working population.

OBJECTIVE: The aim of this study was to investigate the potential links between dietary non-enzymatic antioxidant capacity (NEAC) in overall diet and separately from foods and beverages and markers of DNA damage. METHODS: The participants were 513 employees, 20 to 65 y of age. Urinary levels of 8-hydroxydeoxyguanosine (8-OHdG) and 7-methylguanine (m7 Gua) were measured using column-switching high-performance liquid chromatography. Dietary NEAC was determined from databases of NEAC measurements obtained by different assays: ferric reducing-antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and total radical-trapping antioxidant parameter (TRAP). Dietary NEAC for each participant was calculated by multiplying the estimated NEAC values with the consumed amount and summing up those values, which was ascertained by a validated brief self-administered diet history questionnaire. Multiple-regression analyses were performed to assess the associations between dietary NEAC and 8-OHdG and m7 Gua, with adjustment for potential confounders. RESULTS: No statistically significant associations were found between overall dietary NEAC or NEAC from either foods or beverages and urinary 8-OHdG levels, after adjustment for potential confounders (overall: FRAP, Ptrend = 0.40; ORAC, P = 0.27; TRAP, P = 0.45). Likewise, no association was found between overall dietary NEAC and m7 Gua levels (FRAP, Ptrend = 0.30; ORAC, P = 0.65; TRAP, P = 0.41). However, we did identify significant inverse association between NEAC from foods, as estimated by TRAP, and m7 Gua levels (Ptrend = 0.049). CONCLUSION: Overall, dietary NEAC was not associated with 8-OHdG or m7 Gua levels. In contrast, dietary NEAC from foods but not beverages may be inversely associated with DNA damage caused by methylation.

Feasibility of using urinary N7-(2-carbamoyl-2-hydroxyethyl) Guanine as a biomarker for acrylamide exposed workers.

Acrylamide (AA), a probable human carcinogen, is a widely-used industrial chemical but is also present in tobacco smoke and carbohydrate-rich foods processed at high temperatures. AA is metabolized to glycidamide (GA) to cause the formation of DNA adducts. N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GAG), the most abundant DNA adduct induced by GA, was recently detected in urine of smokers and non-smokers. In this study, we assessed the variability of AA exposure and biomarkers of AA exposure in urine samples repeatedly collected from AA-exposed workers and explored the half-life of N7-GAG. A total of 8 AA-exposed workers and 36 non-exposed workers were recruited. Pre-shift and post-shift urine samples were collected from the exposed group in parallel with personal sampling for eight consecutive days and from the control group on day 1 of the study. Urinary N7-GAG and the mercapturic acids of AA and GA, namely N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-(R,S)-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) were analyzed using on-line solid phase extraction-liquid chromatography-electrospray ionization/tandem mass spectrometry methods. We found that N7-GAG levels in urine were significantly higher in exposed workers than in controls and that N7-GAG level correlated positively with AAMA and GAMA levels. Results from this study showed that AAMA and GAMA possibly remain the more preferred biomarkers of AA exposure and that N7-GAG levels could be elevated by occupational exposures to AA and serve as a biomarker of AA-induced genotoxicity for epidemiological studies.

Experimental evidence of oxidative stress in patients with l-2-hydroxyglutaric aciduria and that l-carnitine attenuates in vitro DNA damage caused by d-2-hydroxyglutaric and l-2-hydroxyglutaric acids.

d-2-hydroxyglutaric (D-2-HGA) and l-2-hydroxyglutaric (L-2-HGA) acidurias are rare neurometabolic disorders biochemically characterized by increased levels of d-2-hydroxyglutaric acid (D-2-HG) and l-2-hydroxyglutaric acid (L-2-HG) respectively, in biological fluids and tissues. These diseases are caused by mutations in the specific enzymes involved in the metabolic pathways of these organic acids. In the present work, we first investigated whether D-2-HG and L-2-HGA could provoke DNA oxidative damage in blood leukocytes and whether l-carnitine (LC) could prevent the in vitro DNA damage induced by these organic acids. It was verified that 50µM of D-2-HG and 30µM of L-2-HG significantly induced DNA damage that was prevented by 30 and 150µM of LC. We also evaluated oxidative stress parameters in urine of L-2-HGA patients and observed a significant increase of oxidized guanine species and di-tyrosine, biomarkers of oxidative DNA and protein damage, respectively. In contrast, no significant changes of urinary isoprostanes and reactive nitrogen species levels were observed in these patients. Taken together, our data indicate the involvement of oxidative damage, especially on DNA, in patients affected by these diseases and the protective effect of LC.