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This study was aimed at defining the reference ranges for biomarkers of oxidized guanine in (2'-deoxy)ribonucleotides and nucleic acids from a large Italian sample. We recruited 300 healthy subjects (150 males; mean age 44.1±13.6years; 26% smokers) without any known exposure to occupational oxidizing agents. They were asked to provide a spot urine sample, on which the following markers were determined by liquid chromatography-tandem mass spectrometry: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), 8-oxo-7,8-dihydroguanine (8-oxoGua), and cotinine. The reference ranges, estimated as the 5th-95th percentiles of creatinine-normalized values (pmol/μmol(creat)) were 0.7-4.2, 0.9-4.7, and 5.6-120.7 for 8-oxodGuo, 8-oxoGuo, and 8-oxoGua, respectively. Oxidation biomarkers were correlated with one another (p<0.005) and with urinary creatinine (p<0.0001). Males excreted significantly higher concentrations of 8-oxoGua than females (p<0.0001). 8-OxoGua and 8-oxoGuo showed a positive association with age (p<0.001), also after stratification by gender. Multiple linear regression models including urinary creatinine concentration, age, and smoking habit as independent variables showed a significant effect of age, but not of smoking, on the levels of 8-oxoGuo in males (p<0.0001) and of both 8-oxoGuo and 8-oxoGua in females (p<0.0001). A preliminary assessment in a small group (n=25) of patients affected by advanced non-small-cell lung cancer and receiving platinum-based chemotherapy showed significantly higher values of both 8-oxoGuo and 8-oxodGuo (p<0.0001 for both) compared to the referent population., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Biological monitoring of low level exposure to pollutants is a very challenging analytical activity, and the quality of results is difficult to assess, especially when a certified reference material is unavailable. The aim of this work was to evaluate the reliability of the assays used to measure urinary benzene (Benz-U) and S-phenylmercapturic acid (SPMA), by applying an internal quality control protocol. Urine spot samples from 705 subjects who were either members of the general urban population, gasoline station attendants, or refinery plant workers were assayed for Benz-U and SPMA, using GC/MS and LC/MS/MS, with quantification limits of 15 ng/L and 0.10 μg/L. The median Benz-U concentration was 263 ng/L (60-2789 ng/L, 5th-95th percentile), and the median SPMA concentration was 0.19 μg/L (<0.1-2.5 μg/L, 5th-95th percentile). Linearity of both assays was good, but a less-than-proportional response was found for SPMA concentrations below 1 μg/L. Between-run precision and accuracy for Benz-U concentration determination were assessed using quality controls at 120 ng/L and 1000 ng/L and were 10.3% and 4.8%, and 104.8% and 98.9%, respectively; while the precision and accuracy for SPMA concentration determination at 0.3 μg/L, 2.5 μg/L, and 20 μg/L were 40.3%, 6.2%, and 6.2%, and 48.3%, 96.3%, and 98.8%, respectively. Precision, estimated using duplicates of unknown samples, was 13.4% for Benz-U and 26.5% for SPMA analyses. Control charts for the means of the slope of the linear calibration curve of Benz-U showed good stability of the means over a five-year period. For SPMA, a two-laboratory comparison revealed acceptable agreement between ln-transformed data pairs, with a slope of the linear regression of 0.863 (confidence interval 0.774-0.952), null intercept, and a Pearson's r value of 0.844. Reliable results were obtained for Benz-U analyses over the entire concentration range, and for high and medium SPMA levels. However, the determination of SPMA concentrations at levels close to the limit of quantification was less reliable., (Copyright © 2009 Elsevier B.V. All rights reserved.)

Biological monitoring (BM) and biomarkers are widely applied in occupational toxicology. BM is mainly aimed at (i) defining the existence of an occupational exposure; (ii) quantifying the level of internal dose; (iii) verifying that exposure limits (BEI((R)), BAT, BLV) are respected. As compared to ambient monitoring, BM is more expensive and complex. Several biomarkers are available for the same chemical and the meaning of the marker may depend on the sampling time. Therefore, practical issues, including cost and selection of an adequate sampling strategy, should be dealt with when planning a BM program for specific purposes. In addition, several biological and analytical sources of variability may influence biomarker levels, thus making the interpretation of BM data a difficult task. However, we should recognize that the main aim of BM is not to reduce, but to explain biological variance. The decreasing trend in occupational exposure levels highlighted the specificity problems of traditional biomarkers of exposure and prompted the research to the development of new biomarkers, e.g. unchanged volatile compounds in urine, minor metabolites, DNA and protein adducts. Depending on the scope and context (research or routine) different requirements of biomarkers can be envisaged in terms of validation and acceptable variability.

Background: The effects of changes in cooling temperature on biomarker levels in exhaled breath condensate have been little investigated. The aim of the study was to test the effect of condensation temperature on the parameters of exhaled breath condensate and the levels of selected biomarkers., Methods: Exhaled breath condensate was collected from 24 healthy subjects at temperatures of -10, -5, 0 and +5 C degrees. Selected parameters (condensed volume and conductivity) and biomarkers (hydrogen peroxide, malondialdehyde) were measured., Results: There was a progressive increase in hydrogen peroxide and malondialdehyde concentrations, and condensate conductivity as the cooling temperature increased; total condensate volume increased as the cooling temperature decreased., Conclusion: The cooling temperature of exhaled breath condensate collection influenced selected biomarkers and potential normalizing factors (particularly conductivity) in different ways ex vivo. The temperature of exhaled breath condensate collection should be controlled and reported.

In the field of occupational and/or environmental toxicology, the measurement of specific metabolites in urine may serve to assess exposure to the parent compounds (biological monitoring of exposure). Styrene is one of the chemicals for which biological monitoring programs have been validated and implemented in environmental and occupational medicine. However, inter-individual differences in the urinary excretion exist both for the main end-products (mandelic acid and phenylglyoxylic acid) and for its specific mercapturic acids (phenylhydroxyethylmercapturic acids, PHEMA). This limits to a certain extent the use of these metabolites for an accurate assessment of styrene exposure. In a group of 26 volunteers selected with relevant genotypes, and exposed to styrene vapours (50 mg/m3, 8 h) in an inhalation chamber, we evaluated whether genotyping or phenotyping relevant drug-metabolizing enzymes (CYP2E1, EPHX1, GSTM1, GSTT1 and GSTP1) may help to explain the observed inter-individual variability in the urinary metabolite excretion. Peripheral blood lymphocytes were used for genotyping and as reporter cells for the phenotyping of CYP2E1 and EPHX1. The GSTM1 genotype was clearly the most significant parameter explaining the variance in urinary PHEMA excretion (6-fold lower in GSTM1 null subjects; P < 0.0001) so that systematic GSTM1 genotyping should be recommended routinely for a correct interpretation of PHEMA urinary levels. Variant alleles CYP2E1*6 (7632T>A) and His113EPHX1 were associated with a significant reduction of, respectively, the expression (P = 0.047) and activity (P = 0.022) of the enzyme in peripheral blood lymphocytes. In combination with GSTM1 genotyping, the phenotyping approach also contributed to improve the interpretation of urinary results, as illustrated by the combined effect of CYP2E1 expression and GSTM1 allelic status that explained 77% of the variance in PHEMA excretion and allows the recommendation of mercapturates as specific and reliable biomarkers of exposure to styrene., (Copyright 2002 Lippincott Williams & Wilkins)

This cross-sectional study was aimed at reconstructing the exposure to gasoline in 102 petrol station attendants by environmental and biological monitoring of benzene, toluene, ethylbenzene and xylene (BTEX) and biomonitoring of methyl tert-butyl ether (MTBE). Airborne BTEX were higher for manual refuelers than self-service assistants and were highly correlated with each other. Significant relationships were found between airborne BTX and the corresponding urinary solvents (U-BTX) and beween airborne B and urinary MTBE (U-MTBE). Smokers eliminated higher values of U-B, trans,trans-muconic ( t,t-MA) and S-phenylmercapturic ( S-PMA) acids but not U-MTBE. All these biomarkers were, however, significantly raised during the shift, independently from smoking. Linear regression confirmed that occupational exposure was a main predictor of U-MTBE, U-B and S-PMA values, both the latter confounded by smoking habits. The study supports the usefulness of biomonitoring even at low exposure levels. [ABSTRACT FROM AUTHOR]

We developed a new method for the simultaneous quantitative determination of 8-oxo-7,8-hydro-2′-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and the corresponding non-oxidized forms, 2′-deoxyguanosine (dGuo), guanine (Gua) and guanosine (Guo), in human urine samples by liquid chromatography–tandem mass spectrometry. Differences in the ionization of analytes in different urine batches with variable matrix effects were effectively compensated for by internal standardization with stable isotope-labelled analytes. The method was sensitive enough to allow the determination of background levels of these biomarkers and was applied to characterize the inter- and intraindividual variability of biomarkers in the diurnal profile of concentrations in 24 healthy volunteers. When normalized for creatinine, none of the biomarkers was affected by sampling time, thus ruling out any circadian rhythm for nucleic acid oxidation in urine. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated nucleic acid oxidation associated with exposure to benzene at low levels in 239 workers recruited among traffic policemen, taxi drivers and gasoline pump attendants of the city of Parma (Italy). Biomarkers of exposure, namely urinary t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA), urinary cotinine, and urinary biomarkers of nucleic acid oxidation, namely 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) were determined by liquid chromatography–tandem mass spectrometry. Relevant polymorphisms of NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferases M1-1 (GSTM1), T1-1 (GSTT1), and A1 (GSTA1) were characterized by polymerase chain reaction-based methods in a subgroup of subjects. Biomarkers of nucleic acid oxidation were correlated with each other (r ≥0.32, p <0.0001) and with exposure biomarkers (r ≥0.28, p <0.0001). Multiple linear regression models including age, sex and smoking habits as independent variables demonstrated that benzene exposure is associated with oxidation damage to nucleic acid, particularly to RNA (p <0.0001) and is modulated by the NQO1 polymorphism. The study confirmed a significant modulating effect of GSTM1 (p =0.010), GSTT1 (p =0.023) and GSTA1 (p =0.048) polymorphisms on S-PMA excretion, with a significant interaction between GSTM1 and both GSTT1 and GSTA1 (p =0.006 and p =0.037, respectively). [Copyright &y& Elsevier]

Abstract: This study investigated nucleic acid oxidation associated with styrene exposure, mRNA expression levels of hOGG1 gene and the role of the genetic polymorphism Ser326Cys of human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) in 60 styrene-exposed workers and 50 unexposed clerks. Biomarkers of exposure (styrene in blood, mandelic and phenylglyoxylic acids and 4-vinylphenol in urine) and urinary biomarkers of nucleic acid oxidation, namely 8-oxo-7,8-dihydro-2′-deoxyguanosine (U-8-oxodGuo), 8-oxo-7,8-dihydroguanosine (U-8-oxoGuo) and 8-oxo-7,8-dihydroguanine (U-8-oxoGua) were determined by liquid chromatography–tandem mass spectrometry. The levels of 8-oxodGuo adduct and 2′-deoxyguanosine (dGuo) were measured by HPLC in DNA from white blood cells (WBC). Genomic DNA and RNA from blood samples were used to characterize the Ser326Cys polymorphism and the mRNA expression levels of the hOGG1 gene, respectively, by PCR-based methods. Exposed workers showed lower values of 8-oxodGuo/105 dGuo ratio in WBC-DNA but higher concentrations of U-8-oxoGuo compared to controls (p =0.002 and p =0.008, respectively, t-test for independent samples). In the whole group, all urinary biomarkers of nucleic acid oxidation correlated with both the sum of mandelic and phenylglyoxylic acids (rho >0.33, p <0.0001) and 4-vinylphenol (rho >0.29, p <0.001), whereas 8-oxodGuo/105 dGuo in WBC showed a negative correlation with exposure parameters (rho <−0.24, p <0.02). Subjects bearing the hOGG1 Ser/Ser genotype showed lower values of 8-oxodGuo/105 dGuo in WBC than those with at least one variant Cys allele (0.34±0.16 vs 0.45±0.21, p =0.008). In the subgroup of hOGG1 Ser/Ser subjects, laminators showed lower levels of WBC 8-oxodGuo/105 dGuo ratio and significantly higher concentrations of U-8-oxoGua than controls (p =0.07 and p =0.01, respectively, t-test for independent samples). Interestingly, workers showed higher levels of hOGG1 expression compared to controls (p <0.0005). Styrene exposure seems to be associated with oxidation damage to nucleic acids, particularly to RNA and with an induction of the BER system. [Copyright &y& Elsevier]

Abstract: A comparative evaluation of urinary biomarkers was carried out to characterize benzene exposure in a group of 100 traffic policemen of the city of Parma (Italy). All subjects were monitored once, in two consecutive days characterized by similar climatic conditions but preceded by two windy days. Benzene ambient concentration measured by municipal air monitoring stations was 1μg/m3 (Day 1) and 2μg/m3 (Day 2). Personal exposure to ambient concentrations of benzene, toluene, ethylbenzene and xylene (BTEX) was assessed by using Radiello® passive-diffusive samplers in a subgroup of 24 workers. Benzene metabolites, t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA) were determined by isotopic dilution liquid chromatography–tandem mass spectrometry on spot urine samples collected at the end of the shift. Urinary benzene (U-B) was determined by solid-phase microextraction gas chromatography–mass spectrometry. Airborne benzene concentration expressed as median [and interquartile range] was 6.07 [0.28–9.53]μg/m3, as assessed by personal sampling. Urinary concentrations of biomarkers in the whole group were 41.8 [34.1–89.8]μg/g creatinine for t,t-MA, 0.67 [0.23–1.32]μg/g creatinine for S-PMA, and 0.16 [0.13–0.26]μg/l for U-B. Smokers eliminated significantly higher concentrations of unchanged BTEX and benzene metabolites than non-smokers (p <0.05). When traffic policemen were distinguished into indoor (n =31) and outdoor workers, no significant differences were observed for either airborne benzene or urinary biomarkers. Significantly lower concentrations of S-PMA and U-B were determined in samples collected at Day 1 as compared to Day 2 (p <0.0001 and p =0.003, respectively) suggesting that these biomarkers are enough sensitive and specific to detect changes in airborne benzene concentration even at few μg/m3. [Copyright &y& Elsevier]

Abstract: Recent advances on biomarker research are discussed, primarily relying on experience gained with technologies based on liquid chromatography–tandem mass spectrometry (LC–MS–MS). Determination of urinary metabolites of industrial chemicals (n-hexane, benzene, toluene, and styrene) in samples from occupationally exposed workers and controls was performed by LC–MS–MS with either electrospray (ESI) or atmospheric pressure chemical ionization (APCI), as appropriate. Both phase I and II metabolites (glucuronides, sulfates, and mercapturic acids) can be detected with little or no sample manipulation, thus allowing the identification of a number of artifacts and “new” metabolites. However, experimental evidence indicates the need for properly addressing the matrix effect, which is always associated with the analysis of biological samples. Both efficient sample preparation and the use of isotopically labeled internal standards seem to be necessary to develop validated quantitative methods. [Copyright &y& Elsevier]

Abstract: Biological monitoring and biomarkers are used in occupational toxicology for a more accurate risk assessment of occupationally exposed people. Appropriate and validated biomarkers of internal dose, like urinary metabolites, besides to be positively correlated with external exposure, have a predictive value to the risk of adverse effects. The application of liquid chromatography–mass spectrometry (LC–MS) in occupational and environmental toxicology, although relatively recent, has been demonstrated valid in the determination of traditional biomarkers of exposure, as well as in metabolism studies aimed at investigating minor metabolic routes and new more specific biomarkers. This review presents selected applications of LC–MS to the study of the metabolism of industrial chemicals, like n-hexane, benzene and other aromatic hydrocarbons, styrene and other monomers employed in plastic industry, as well as to other chemicals used in working environments, like pesticides used by farmers, and antineoplastic agents prepared by hospital personnel. Analytical and pre-analytical factors, which affect quantitative determination of urinary metabolites, i.e. sample preparation, matrix effect, ion suppression, use of internal standards, and calibration, are emphasized. [Copyright &y& Elsevier]

Abstract: The aims of the study were to evaluate the feasibility of urinary trans, trans-muconic acid (u-t,t-MA) and urinary S-phenylmercapturic acid (u-SPMA) as markers of exposure to urban benzene pollution for biomonitoring studies performed on children and to investigate the impact that creatinine correction may have on the classification of children exposure status. U-t,t-MA, u-SPMA, u-cotinine, and u-creatinine levels were measured in urine samples of 396 Italian children (5–11years) living in three areas with different degrees of urbanisation (very, fairly and non-urban). The median u-SPMA levels significantly increased with increased urbanisation: non-urban (0.19μg/L; 0.22μg/g creatinine)

Abstract: Benzene is a widespread air pollutant and a well-known human carcinogen. Evidence is needed regarding benzene intake in the pediatric age group. We investigated the use of urinary (u) trans,trans-muconic acid (t,t-MA), S-phenylmercapturic acid (SPMA), and unmodified benzene (UB) for assessing exposure to low concentrations of environmental benzene and the role of living environment on benzene exposure in childhood. u-t,t-MA, u-SPMA, u-UB and u-cotinine were measured in urine samples of 243 Italian children (5–11years) recruited in a cross-sectional study. Analytical results were compared with data obtained from questionnaires about participants'' main potential exposure factors. u-UB, u-t,t-MA and u-SPMA concentrations were about 1.5-fold higher in children living in urban areas than in those in the rural group. Univariate analyses showed that u-UB was the only biomarker able to discriminate secondhand smoke (SHS) exposure in urban and rural children (medians=411.50 and 210.50ng/L, respectively); these results were confirmed by the strong correlation between u-UB and u-cotinine in the SHS-exposed group and by multivariate analyses. A regression model on u-SPMA showed that the metabolite is related to residence area (p <0.001), SHS exposure (p =0.048) and gender (p =0.027). u-UB is the best marker of benzene exposure in children in the present study, and it can be used as a good carcinogen-derived biomarker of exposure to passive smoking, especially related to benzene, when urine sample is collected at the end of the day. In addition, it is important to highlight that SHS resulted the most important contributor to benzene exposure, underlining the need for an information campaign against passive smoking exposure. [Copyright &y& Elsevier]