Anna Barbieri

The background of mitochondrial DNA haplogroup J increases the sensitivity of Leber's hereditary optic neuropathy cells to 2,5-hexanedione toxicity.

Lebers hereditary optic neuropathy (LHON) is a maternally inherited blinding disease due to mitochondrial DNA (mtDNA) point mutations in complex I subunit genes, whose incomplete penetrance has been attributed to both genetic and environmental factors. Indeed, the mtDNA background defined as haplogroup J is known to increase the penetrance of the 11778/ND4 and 14484/ND6 mutations. Recently it was also documented that the professional exposure to n-hexane might act as an exogenous trigger for LHON. Therefore, we here investigate the effect of the n-hexane neurotoxic metabolite 2,5-hexanedione (2,5-HD) on cell viability and mitochondrial function of different cell models (cybrids and fibroblasts) carrying the LHON mutations on different mtDNA haplogroups. The viability of control and LHON cybrids and fibroblasts, whose mtDNAs were completely sequenced, was assessed using the MTT assay. Mitochondrial ATP synthesis rate driven by complex I substrates was determined with the luciferine/luciferase method. Incubation with 2,5-HD caused the maximal loss of viability in control and LHON cells. The toxic effect of this compound was similar in control cells irrespective of the mtDNA background. On the contrary, sensitivity to 2,5-HD induced cell death was greatly increased in LHON cells carrying the 11778/ND4 or the 14484/ND6 mutation on haplogroup J, whereas the 11778/ND4 mutation in association with haplogroups U and H significantly improved cell survival. The 11778/ND4 mutation on haplogroup U was also more resistant to inhibition of complex I dependent ATP synthesis by 2,5-HD. In conclusion, this study shows that mtDNA haplogroups modulate the response of LHON cells to 2,5-HD. In particular, haplogroup J makes cells more sensitive to its toxic effect. This is the first evidence that an mtDNA background plays a role by interacting with an environmental factor and that 2,5-HD may be a risk element for visual loss in LHON. This proof of principle has broad implications for other neurodegenerative disorders such as Parkinsons disease.

A rapid and sensitive method for methyl tert-butyl ether analysis in water samples by use of solid phase microextraction and gas chromatography-mass spectrometry

This work describes a rapid and sensitive solid-phase microextraction (SPME) method for the isolation and analysis of methyl tert-butyl ether in water samples. Methyl tert-butyl ether was extracted from aqueous solutions using SPME fibre coated with Divinylbenzene/Carboxen/polydimethylsiloxane (30 microm film thickness) and analysed by GC-MS with a Hewlett Packard 6890/5973 system equipped with a capillary column coated with Vocol (30 m x 0.25 mm, 1.5 microm film thickness). Extraction parameters and chromatographic separation conditions were optimised. The developed method showed good analytical performance in terms of precision (RSD between 2% and 8%) and accuracy (mean recovery from 96% to 104%) with a detection limit of 14 ppt. Finally the method was applied to surface, tap and commercial mineral water samples, as well as snow samples collected along a busy road of Bologna town area. The median concentration of methyl tert-butyl ether in all these samples (0.05-0.4 ppb) was well below the maximum aqueous contamination levels in water adopted in the United States (13 ppb).

Urinary biomarkers and low-level environmental benzene concentration: Assessing occupational and general exposure

The categories of urban workers undergoing prolonged exposure to gasoline due to vehicle emissions, evaporation and traffic fumes are considered occupationally exposed to benzene, even if at low concentrations. The aim of this study was to evaluate the specificity of unmetabolized benzene excreted in urine (UBz) and S-phenylmercapturic acid (SPMA) as biomarkers of exposure to very low levels of benzene, and to study the impact of putative individual confounders like smoking and alcohol habits, co-exposure to other toxicants and body mass index on the exposure assessment. Environmental and biological monitoring of exposure to benzene were performed in 114 Urban Policemen. The mean value of UBz in non-smokers was significantly lower than in either groups of light to moderate smokers and heavy smokers (0.24, 1.82 and 2.82 microg L(-1), respectively). On the contrary, SPMA values did not discriminate exposure resulting from smoking habits. Moreover, the concentration of UBz in non-smokers appears to be correlated with environmental benzene concentration (BenzA) (R(2)=0.13, beta=0.37, p=0.002). On the other hand, no significant correlation was found between SPMA concentration (corrected for creatinine excretion and log transformed, LogSPMA) and LogBenzA (R(2)=0.003, beta=0.05, p=0.6). Our findings reinforce previous research on the use of unmetabolized urinary benzene as a specific and sensitive biomarker of low-level exposure to benzene and confirm that smoking habits strongly influence the excretion of UBz.

Exposure to low environmental levels of benzene: evaluation of micronucleus frequencies and S-phenylmercapturic acid excretion in relation to polymorphisms in genes encoding metabolic enzymes.

An integrated approach based on environmental and biological monitoring, including the analysis of biomarkers of exposure [excretion of S-phenylmercapturic acid (S-PMA)], early biological effects [micronucleus (MN) frequency] and susceptibility (genetic polymorphisms), was applied to characterize benzene exposure in a group of 70 traffic policemen and 40 employees of the city of Bologna, Italy. Median personal benzene exposure was 6.55-fold higher for traffic policemen than for controls (P<0.0001). This higher exposure was confirmed by a significant, 2.53-fold higher S-PMA excretion in traffic policemen compared with that observed for indoor workers (P<0.0001). Median MN frequency was also significantly higher in policemen compared with indoor workers (P=0.001), emphasizing the genotoxic effect potentially associated with benzene exposure. With regard to biomarkers of susceptibility, the analysis revealed that high epoxide hydrolase (mEH) (predicted) enzyme activity was significantly correlated with a lower median MN frequency (P=0.003). A gene-gender interaction was observed for the glutathione-S-transferase M1 (GSTM1) genotype. The GSTM1-null genotype was associated with a significantly higher median MN frequency in men, not in women. Statistical analysis did not reveal any association between the presence of the protective allele, pushing the pathway towards benzene detoxification, and MN frequency or S-PMA excretion. Even though there are some limitations in the study, our results indicate that policemen are exposed to higher levels of benzene than individuals spending most of the time indoors. This higher exposure may contribute to DNA damage, suggesting an increase health risk from traffic benzene emission. Finally, a more comprehensive study is warranted in order to better elucidate the involvement of EPHX1 genotypes combination in benzene genotoxicity.

Validation of an HPLC–MS/MS method for the simultaneous determination of phenylmercapturic acid, benzylmercapturic acid and o-methylbenzyl mercapturic acid in urine as biomarkers of exposure to benzene, toluene and xylenes

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated, according to U.S. Food and Drug Administration guidance, for the simultaneous determination of phenylmercapturic acid, benzylmercapturic acid and o-methylbenzyl mercapturic acid in human urine as biomarkers of exposure to benzene, toluene and xylenes (BTX). After solid phase extraction and LC separation, samples were analyzed by a triple-quadrupole mass spectrometer operated in negative ion mode, using isotope-labeled analogs as internal standards (ISs). The method meets all the validation criteria required. The limits of detection of the three analytes, ranging from 0.30 to 0.40microgl(-1), and the high throughput make the method suitable for the routine biological monitoring of co-exposure to BTX both in the occupational and environmental settings. The validated method was applied to assess exposure to BTX in a group of 354 urban traffic wardens.

Lack of correlation between environmental or biological indicators of benzene exposure at parts per billion levels and micronuclei induction

Despite growing concern for possible carcinogenic effects associated with environmental benzene exposure in the general population, few studies exist at parts per billion (ppb) levels. We investigated the existence of a relationship between airborne/biological measurements of benzene exposure (i.e., personal/area sampling and unmodified urinary benzene/trans,trans-muconic acid; t,t-MA) and micronuclei induction (cytochalasin B technique) among exposed chemical laboratory workers (n=47) and traffic wardens (n=15). Although urinary t,t-MA (106.9+/-123.17 microg/L(urine)) correlated (R(2)=0.37) with urinary benzene (0.66+/-0.99 microg/L(urine)), neither biological measurement correlated with environmental benzene exposure (14.04+/-9.71 microg/m(3); 4.39+/-3.03ppb), suggesting that, at ppb level (1ppb=3.2 microg/m(3)), airborne benzene constitutes a fraction of the total intake. Traffic wardens and laboratory workers had comparable numbers of micronuclei (4.70+/-2.63 versus 5.76+/-3.11; n.s.), similar to levels recorded in the general population. With univariate/multivariate analysis, no association was found between micronuclei induction and air/urinary benzene exposure variables. Notably, among the personal characteristics examined (including age, gender, smoking, drinking, etc.), high body mass index correlated with micronuclei induction while, among females, use of hormonal medication was associated with less micronuclei. Thus the present study provides no evidence that ppb levels of environmental benzene exposure appreciably affect micronuclei incidence (against the background of other relevant factors). However, this should not be taken as an argument against efforts aiming to reduce environmental benzene pollution.

Biomarkers of internal dose for the assessment of environmental exposure to benzene

The urinary excretion of t,t-muconic acid (t,t-MA), S-phenylmercapturic acid (SPMA) and urinary benzene and the influence of a smoking habit and of exposure to urban traffic on the urinary excretion of these biomarkers were investigated in 137 male adults from the general population. All subjects were not occupationally exposed to benzene and resident in two cities in Puglia (Southern-Italy). Environmental exposure to benzene was measured using passive personal samplers. The biomarkers t,t-MA, SPMA and urinary benzene were determined in urine samples collected from each subject at the end of the environmental sampling. The percentage of cases above the limit of detection was higher for SPMA and urinary benzene in smokers than in non-smokers, and for airborne benzene and urinary benzene in subjects exposed to urban traffic. Airborne benzene was correlated with the time spent in urban traffic during the environmental sampling. Among the biomarkers, urinary benzene was found to be correlated with airborne benzene only in non-smokers, and with the time spent in urban traffic, both in smokers and non-smokers considered together, and in non-smokers only. Finally, multiple regression analysis showed that the urinary excretion of all the biomarkers was dependent on the number of cigarettes smoked per day and, for urinary benzene, also on the time spent in urban traffic. In conclusion, urinary benzene seems to be a more valid biomarker than t,t-MA and SPMA to assess environmental exposure to extremely low concentrations of benzene. Cigarette smoking prevailed over traffic exhaust fumes in determining the internal dose of benzene.

FAILURE OF URINARY trans,trans-MUCONIC ACID AS A BIOMARKER FOR INDOOR ENVIRONMENTAL BENZENE EXPOSURE AT PPB LEVELS

Benzene is a widespread pollutant whose main source in the environment is automotive emission. There is increasing interest in the exposure of the population to this pollutant as benzene is present also in the indoor environment due to cigarette smoke, drinking water, and food. The aim of this study was to evaluate, in an adult nonsmoking population not occupationally exposed to benzene, whether it is possible to detect differences in the urinary concentration of trans,trans-muconic acid (t,t-MA) between low and high environmental exposure to benzene. A study sample of 31 employees working in pharmacies in a large town in Italy with low environmental exposure to benzene (4.8 µg/m³) was compared to a high (8.1 µg/m³) benzene exposure group. Analysis of urinary t,t-MA was carried out by high-performance liquid chromatography (HPLC; photodiode array detector); analysis of environmental benzene samples was by gas chromatography/mass spectroscopy (GC-MS). The statistical analysis revealed no significant differences in urinary levels of t,t-MA of subjects with high (mean concentration: 157.9 µg/gcreatinine) versus low exposure (mean concentration: 114.2 µg/gcreatinine). Data show that it is difficult to correlate urinary t,t-MA with benzene exposure at parts per billion levels.

Lack of sensitivity of urinary trans, trans-muconic acid in determining low-level (ppb) benzene exposure in children

Abstract Benzene is a widespread pollutant of which the main source in the outside environment is automotive traffic. Benzene is also present in cigarette smoke, and small quantities exist in drinking water and food; all of these sources contribute to pollution of indoor environments. Benzene exposure may be studied with biologic indicators. In the present study, the authors evaluated whether differences in urinary concentrations of trans, trans-muconic acid (t,t-MA) were detectable in a sample of 150 children and if the chemical was correlated with environmental exposures to low levels of benzene. The children attended primary schools that had significantly different—but low—environmental benzene levels. Analysis of urinary t,t-MA was achieved with high-performance liquid chromatography (photodiode array detector), and analysis of passive air samplers for benzene was performed with gas chromatography-mass spectrometry. Statistical analysis (Kruskal-Wallis test) indicated that differences in urinary levels of t,t-MA in children from urban and rural areas were not statistically significant (p = .07), nor were there significant differences between children with and without relatives who smoked (p = .69). As has been shown in other studies of children and adults, results of our study evidenced (1) the difficulty of correlating concentrations of urinary biomarkers with environmental exposure to benzene at a parts-per-billion level (i.e., traffic and environmental tobacco smoke) and, consequently, (2) the lack of specificity of t,t-MA as a biological indicator for the study of a populations exposure.

Enflurane as an internal standard in monitoring halogenated volatile anaesthetics by headspace gas chromatography–mass spectrometry

Recently. we proposed the use of a run-only headspace-GC-MS method for the biological monitoring of ppb concentrations of unmodified volatile anaesthetics (isoflurane, sevoflurane and halothane, plus nitrous oxide) in post-shift urine of operating theatre personnel. The adoption of enflurane (a volatile anaesthetic no longer used in clinical practice) as a poper and viable internal standard improves intra-day and inter-day accuracy in halide quantitation, providing a GC-MS reference method useful in the practice of biomonitoring of exposure of operating theatre personnel to modern volatile anaesthetics (isoflurane. sevoflurane, halothane).