Search

Abstract: We present the application of a novel isotope dilution method, named Alternate Isotope-Coded Derivatization Assay (AIDA), to the quantitative analysis of hydrazone derivatives of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in exhaled breath condensate (EBC) samples using liquid chromatography–tandem mass spectrometry. AIDA is based on the alternate derivatization of the analyte(s) with reagents that are available in two pure isotopic forms, respectively “light” and “heavy”, by using light-derivatized standards for the quantification of the heavy-derivatized analytes, and vice versa. To this purpose, 2,4-dinitro-3,5,6-trideuterophenylhydrazine (d 3 -DNPH) has been synthesized and used as “heavy” reagent in combination with commercial “light” DNPH. Using the AIDA method, any unknown concentration of the analyte in the matrix can be calculated without the need of a calibration curve. An external calibration method has been also investigated for comparative purpose. The stability of DNPH and d 3 -DNPH derivatives was verified by excluding any exchange of hydrazones with each other. In the range of concentrations of biological interest, e.g., 2–40nM for MDA and 0.5–10nM for 4-HNE, the derivatization reactions of MDA and 4-HNE with DNPH and d 3 -DNPH showed overlapping kinetics and comparable yields. The MS response of both DNPH and d 3 -DNPH hydrazones was similar. The precision of AIDA, calculated as %RSD, was within 3.2–8% for MDA and 4.5–11% for 4-HNE. Accuracy was tested by analyzing a spiked EBC pool sample and acceptable results (accuracy within 98–108% for MDA and 93–114% for 4-HNE) were obtained by AIDA after subtraction of the blank, which was not negligible. The results of quantitative analysis of MDA and 4-HNE in EBC samples obtained by AIDA assay with four analyses per sample were in good agreement with those obtained by external calibration method on the same samples. [Copyright &y& Elsevier]

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: 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]

Two workers were accidentally exposed to unusually high styrene concentrations (>1000 ppm) for about 30 min. In addition to the main styrene metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA), other minor metabolites, including specific mercapturic acids, (R,R)- and (S,R)-N-acetyl-S-(1-phenyl-2-hydroxyethyl)-l-cysteine [(R,R)-M1 and (S,R)-M1] and (R,R)- and (S,R)-N-acetyl-S-(2-phenyl-2-hydroxyethyl)-l-cysteine [(R,R)-M2 and (S,R)-M2], 4-vinylphenol-glucuronide and -sulfate, and phenylglycine, were determined by Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI-MS/MS) in urine samples collected 12, 24, 36, 48, 75 and 99 h after the episode. The genotypes of microsomal epoxide hydrolase, glutathione-S-transferases M1-1 (GSTM1), T1-1 (GSTT1) and P1-1 (GSTP1) were characterized by PCR-based methods. The two subjects showed similar peak levels of MA and PGA, as well as 4-vinylphenol conjugates, whereas mercapturic acids were five times higher in the subject bearing the GSTM1pos than in the GSTM1null subject. Also, relative proportions of diasteroisomers of mercapturic acids were influenced by the GSTM1 polymorphism. [Copyright &y& Elsevier]

Abstract: A sensitive and selective liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the determination of several water-soluble vitamins, namely vitamins B1, B2, B6 (pyridoxine, pyridoxal, and pyridoxamine), and PP (nicotinamide and nicotinic acid), pantothenic acid, and folic acid was developed and validated. The analytes were characterized by means of their electrospray (ESI) and atmospheric pressure chemical ionization (APCI) mass spectra. In general, the positive ion spectra were 100- to 1000-fold more intense than the corresponding negative ion ones. Chromatography of water-soluble vitamins was obtained by using a reversed-phase C16 Amide (15cm, 5μm) column and a mobile phase made of ammonium formate buffer (20mM, pH 3.75)/methanol under gradient elution conditions. Linearity of the MS response was observed over three to four orders for both ESI and APCI, and limits of detection were in the low μg/l range for both the ionization techniques. In particular, the sensitivity of ESI was about two- to five-fold higher for all vitamins except PP vitamers, for which APCI produced a better response. Precision calculated at two concentration levels (0.05 and 1.0mg/l) was within 0.2–7.4% for all intra- and inter-day determinations and for all analytes. The LC–ESI-MS/MS method was applied to the quantitative analysis of the natural content of vitamins in typical Italian pasta samples, as well as in fortified pasta samples produced for the US market. [Copyright &y& Elsevier]