Association of exposure to benzene and smoking with oxidative damage to nucleic acids by means of biological monitoring of general population volunteers.

In this study, the validation of liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) isotopic dilution method for the determination of benzene and nicotine metabolites in urine was carried out. Limit of detection are 0.026 µg/L for S-phenylmercapturic acid (SPMA), 0.55 µg/L for t,t-muconic acid (t,t-MA), and 12.41 µg/L for the cotinine, and the relative combined uncertainty was also calculated. The study involves 446 healthy volunteer residents since at least 10 years in an area of central Italy. SPMA resulted to be strongly correlated with cotinine (p = 0.75), its concentration in smokers (93) being about ten times than in non/ex-smokers (197/156), while the t,t-MA of smokers is about twice the non/ex-smokers value. A cutoff value for the definition of smoker is set at 100 µg/g creat. Oxidative stress was studied in smokers and non- and ex-smokers by means of the determination of the biomarkers 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 8-Oxo-7,8-dihydroguanosine (8-oxoGuo), and 8-Oxo-7,8-dihydroguanine (8-oxoGua): no significant differences were found between smokers and non/ex-smokers, but when subjects are classified according to the cotinine cutoff value, a correlation in smokers' urinary 8-oxodGuo is found with SPMA and cotinine (p = 0.60 and p = 0.57). Results were confirmed by chemometric analysis that also identified the experimental variables most contributing the discrimination as cotinine and t,t-MA.

Oxidative stress biomarkers and otoacoustic emissions in humans exposed to styrene and noise.

OBJECTIVE: Evaluating the correlation between otoacoustic emission levels, styrene exposure, and oxidative stress biomarkers concentration in styrene-exposed subjects, to investigate the role of oxidative stress in outer hair cell damage. DESIGN: Distortion product otoacoustic emissions were measured in the exposed workers and in a control group. Separation between the distortion and reflection otoacoustic components was performed by time-frequency-domain filtering. The urinary concentration of the DNA and RNA oxidation products, namely 8-oxo-7,8-dihydroguanine (oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxodGuo), and 8-oxo-7,8-dihydroguanosine (oxoGuo), were evaluated. STUDY SAMPLE: Nine subjects exposed to styrene in a fiberglass factory, eight control subjects. The two groups were statistically equivalent in mean age. RESULTS: Statistically significant differences were found in the distortion component levels between the exposed and the control group. High levels of the oxidative damage biomarkers were found in the workers exposed to high levels of styrene. Significant negative correlation was found between the otoacoustic emission distortion component levels and the concentration of the oxoGuo biomarker. CONCLUSIONS: Exposure-induced damage of the cochlear amplifier is shown in the mid-frequency range, confirming animal experiments, in which hair cells in the cochlear middle turn were damaged. Hearing damage is consistent with the outer hair cell apoptosis pathway associated with oxidative stress.

Urinary biomarkers of exposure and of oxidative damage in children exposed to low airborne concentrations of benzene.

The aim of this work was to evaluate the oxidative damage to nucleic acids in children (5-11 years) associated with exposure to environmental pollutants and tobacco smoke (ETS). For each subject, urinary sampling was done twice (evening and next morning) to measure by tandem LC-MS-MS such oxidated products of nucleic acids as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and 8-oxo-7,8-dihydroguanine (8-oxoGua). Methyl tert-butyl ether (U-MTBE), benzene (U-Benz), and its metabolites (t,t-muconic and S-phenylmercapturic acids, t,t-MA and S-PMA, respectively) were determined as biomarkers of exposure to air pollution, and cotinine as a biomarker of exposure to ETS. Biomarkers of exposure (S-PMA and U-MTBE) and of DNA oxidation (8-oxodGuo) were dependent on the urbanization and industrialization levels and increased in the evening sample as compared to next morning (p<0.05). In both evening and next morning samples, 8-oxodGuo and 8-oxoGuo correlated with each other (r=0.596 and r=0.537, respectively, p<0.01) and with biomarkers of benzene exposure, particularly S-PMA (r=0.59 and r=0.45 for 8-oxodGuo and r=0.411 and r=0.383 for 8-oxoGuo, p<0.01). No such correlations were observed for U-MTBE and cotinine. Multiple linear regression analyses showed that 8-oxodGuo was positively associated with S-PMA at both sampling times (ß=0.18 and ß=0.14 for evening and next morning sampling, respectively; p<0.02) and weakly with U-MTBE (ß=0.07, p=0.020) only in the evening urines. These results suggest that the selected biomarkers of exposure to benzene, particularly S-PMA, are good tracers of exposure to complex mixtures of oxidative pollutants and that the associated oxidative damage to nucleic acids is detectable even at very low levels of exposure.

[Biological monitoring of the PAH exposure in the hardening of metals]. / Monitoraggio biologico dell'esposizione ad IPA nella tempra dei metalli.

The hardening of metals involves the immersion of hot components in mineral oil with production of aerosols containing PAHs, to wich workers can be exposed. The determination of airborne PAHs and urinary 1-hydroxypyrene (1-OHPy) was performed for a group of workers and the latter resulted within the reference values. However, the average 1-OHPy concentration on metal workers (0.07 microg/g creatinine) was statistically different from the average value obtained for a group of employees (0.03 microg/g creatinine), highlighting the risk of exposure to PAHs. Therefore, being these potentially carcinogenic compounds, interventions were prescribed in order to reduce the risk of occupational exposure.

Determination of free and total S-phenylmercapturic acid by HPLC/MS/MS in the biological monitoring of benzene exposure.

Urinary S-phenylmercapturic acid (SPMA) is a biomarker suggested by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene. A possible cause of the miscorrelation between environmental monitoring and biological monitoring for benzene exposure, which many authors complain about, is the existence of a urinary metabolite that turns into SPMA by acid hydrolysis. Forty urine samples were tested to determine which concentration value would correspond to the ACGIH Biological Exposure Index (BEI) of 25 microg g(-1) creatinine if exposure assessment was based on the determination of SPMA after quantitative hydrolysis of its precursor. An aliquot of each sample was hydrolysed with 9 M H2SO4, a second one was brought to pH 2 and a third one was used as it was (free SPMA). SPMA was determined by high-performance liquid chromatography/tandem mass spectrometric technique (HPLC/MS/MS) using an internal standard. The analytical method was validated in the range 0.5-50 microg 1(-1). The average SPMA in pH 2 samples is 45-60% of the total, while free SPMA varies from 1% to 66%. The hydrolysis of pre-SPMA reduces the likelihood of variability in the results by reducing pH differences in urine samples and increasing the amount of measured SPMA. The BEI limit value would be about 50 microg g(-1) creatinine.

[Feasibility study of biological monitoring of chemical agents by means of evaluation of the effects on their in vitro effects on the complement system]. / Studio di fattibilità del monitoraggio biologico di agenti chimici mediante valutazione degli effetti su sistemi multienzimatici del plasma coinvolti nella risposta immune.

Immunological methods for the study of the plasma complement system have been standardized in order to be good and reproducible indicators of some biological effects of the substances under study in in vitro experiments. The substances tested were not capable of interfering within 10 times the possible hypothetical plasma concentration reached in vivo with the function of the different reagents used in the study of complement. Five substances (Skin-ACGIH) have been studied for their effects on the complement system in vitro; four of them could be fully studied (allylic alcohol, cyclohexanone, phenol, dimethylacetamide). After this deep insight we can conclude that: 1. These substances are capable of interfering with the immune response through their complement activating capacity 2. These substances, throughout complement activation, can induce inflammation and reduction of important defensive functions that are complement mediated. 3. The results obtained encourage to study the complement system and especially CH50 in workers exposed to the selected substances in order to verify the possibility to enclose this test in the medical surveillance program.

[Isotopic dilution HPLC/MS/MS urinary trans, trans-muconic quantitative analysis among refinery workers]. / Determinazione quantitativa di acido trans, trans-muconico nelle urine dei lavoratori di una raffineria mediante HPLC/MS/MS con diluizione isotopica.

The determination of trans, trans muconic acid (t,t-MA) in human urine, a biomarker suggested by the American Conference of Governmental Industrial Hygienists for occupational benzene exposure, with a limit value of 500 microg/g creatinine, is usually performed by means of gas or liquid chromatographic methods. Almost all the HPLC methods make use of strong ionic exchange cartridges for sample cleaning, reverse phase separation and detection either by UV at lambda = 259 nm or; more recently, by electrospray tandem mass spectrometry: yet, not all of these methods have been validated for quantitative analysis considering also the matrix effect. This paper presents the quantitative analysis of t,t-MA in 94 end-shift urine samples from workers of an oil refinery performed by means of an HPLC/MS/MS analytical method that uses a commercially available deuterium labeled isotope as internal standard, that during the validation has highlited the problem of interferences due to urine ion suppression effect and to the interference from isobaric ions both for the analyte and the internal standared. The following mean values have been obtained: 47.37 microg/g creatinine for non smokers non occupationally exposed to benzene, 97.40 microg/g creatinine for non smokers exposed to benzene, 142.38 microg/g creatinine for smokers non occupationally exposed and 149.08 microg/g creatinine for smokers occupationally exposed to benzene. The results obtained demonstrate that using this analytical method for urinary t,t,MA it is possible to discriminate among groups with different levels of benzene exposure, due to all the possible benzenene sources: environmetal, occupational, due to smoking, and their possible combinations.

[Determination of free and total S-phenylmercapturic acid in the biologic monitoring of exposure to benzene]. / Determinazione dell'acido S-fenilmercapturico libero e totale nel monitoraggio biologico dell'esposizione a benzene.

One of the biomarkers suggested by the ACGIH to assess the professional exposure to benzene is the S-phenylmercapturic acid in the end-shift urine. The existence in the urine of N-acetyl-S(1,2-dihydro-2hydroxypHenyl)-L-Cysteine, a precursor of SPMA that can be turned into it by acid hydrolysis, is a possible cause of miscorrelation between environmental and biological monitoring. The amount of measured SPMA depends on the degree of hydrolysis and therefore it is a function both of the urine PH and of the storage conditions of the sample. 40 urine samples have been collected from workers exposed to benzene, both smokers and not smokers, and for each sample the percentage of SPMA measurable at pH 2 and without pH correction (free SPMA) has been calculated with respect to the SPMA measured after quantitative hydrolysis, with the objectives to determine if a correct assessment of the exposure requires the determination of total SPMA and which concentration value could correspond to the BEI of 25 microg/g of creatinine established by the ACGIH. An aliquot of the urine samples has been treated with 9M H2SO4, a second one is brought to pH 2 and a third one is analyzed as it is. All samples are analyzed by HPLC/MS/MS in negative ions/MRM mode, and quantitative analysis is performed using the internal standard method. The percentage found in samples treated at pH 2 is on average 45% of the total SPMA for smokers and 60% for non smokers, while the free SPMA varies from 1% to 66% due to the urine pH variability and to the lower concentrations detected. The determination of total SPMA allows the standardization of the preanalyticalfactors and the dosage with analytical methods less sensitive than HPLC/MS/MS.

Comparison between external and internal standard calibration in the validation of an analytical method for 1-hydroxypyrene in human urine by high-performance liquid chromatography/tandem mass spectrometry.

1-Hydroxypyrene is a metabolite of pyrene, a member of the class of polycyclic aromatic hydrocarbons (PAHs) whose toxic properties in some cases include carcinogenicity. The determination of 1-hydroxypyrene in human urine is used as a biological indicator for exposure to PAHs, which is related to the combustion of organic materials, like smoking, living in urban environments, and eating grilled or smoked food. The determination of 1-hydroxypyrene by high-performance liquid chromatography (HPLC) with fluorescence detection has very good sensitivity but it is not highly specific: this can reduce accuracy in the quantitative determination of low levels of analyte in a complex matrix like urine. An HPLC method that uses triple quadrupole mass detection has been validated with the objective both to improve the signal-to-noise (S/N) ratio and to achieve the maximum specificity for the analyte in those urine samples that are richer in possible inteferents. The calibration range for 1-hydroxypyrene is from 0.005-0.1 microg/L in the urine of non-smoking healthy volunteers. After solid-phase extraction, samples were analyzed by HPLC/tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode. In order to obtain reliable results quantitative analysis must be performed by means of the internal standard method (we used deuterium-labelled 1-hydroxypyrene): the method accuracy is not less than 85%. The S/N ratio at a concentration of 0.1 microg/L is about 10, and therefore this can be considered the lowest limit of quantitation. The method performance does not change if urine samples are measured using a calibration curve prepared in methanol, thus reducing the time of analysis and costs.