Diana Poli

Determination of aldehydes in exhaled breath of patients with lung cancer by means of on-fiber-derivatisation SPME–GC/MS

A number of volatile organic compounds (VOCs) have been identified and used in preliminary clinical studies of the early diagnosis of lung cancer. The aim of this study was to evaluate the potential of aldehydes (known biomarkers of oxidative stress) in the diagnosis of patients with non-small cell lung cancer (NSCLC). We used an on-fiber-derivatisation SPME sampling technique coupled with GC/MS analysis to measure straight aldehydes C3-C9 in exhaled breath. Linearity was established over two orders of magnitude (range: 3.3-333.3×10(-12) M); the LOD and LOQ of all the aldehydes were respectively 1×10(-12) M and 3×10(-12) M. Accuracy was within 93% and precision calculated as % RSD was 7.2-15.1%. Aldehyde stability in a Bio-VOC(®) tube stored at +4°C was 10-17 h, but this became >10 days using a specific fiber storage device. Finally, exhaled aldehydes were measured in 38 asymptomatic non-smokers (controls) and 40 NSCLC patients. The levels of all of the aldehydes were increased in the NSCLC patients without any significant effect of smoking habits and little effect of age. The good discriminant power of the aldehyde pattern (90%) was confirmed by multivariate analysis. These results show that straight aldehydes may be promising biomarkers associated with NSCLC, and increase the sensitivity and specificity of previously identified VOC patterns.

Low concentrations of the brominated flame retardants BDE-47 and BDE-99 induce synergistic oxidative stress-mediated neurotoxicity in human neuroblastoma cells.

Polybrominated diphenyl ether (PBDE) flame retardants have become widespread environmental contaminants. The highest body burden has been found in toddlers and infants, due to their exposure through breast milk and house dust, and the current concern for potential adverse health effects of PBDEs relates to their developmental neurotoxicity. The mechanisms underlying the neurotoxicity of PBDEs are largely not understood, though there is evidence that PBDEs may elicit oxidative stress. In this study, two different mathematical models were used to evaluate the interaction between BDE-47 and BDE-99 on viability of neuronal cells. The combined exposure to these compounds induced synergistic effects at concentrations of BDE-47 below its threshold doses, and in a wide range of BDE-99 concentrations below its IC(50). In contrast, at concentrations of BDE-47 near its IC(50) value, and in a wide range of BDE-99 concentrations, antagonistic effects were observed. The interactions observed on cell viability were confirmed by an assessment of induction of oxidative stress. The finding that co-exposure to BDE-47 and BDE-99 could induce synergistic neurotoxic effects, in particular at low doses of BDE-47, is of much toxicological interest, as humans are exposed to mixtures of PBDEs, most notably tetra- and penta-BDE congeners.

Influence of condensation temperature on selected exhaled breath parameters

BackgroundThe 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.MethodsExhaled 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.ResultsThere 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.ConclusionThe 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.

Biological monitoring of low benzene exposure in Italian traffic policemen.

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 1 microg/m(3) (Day 1) and 2 microg/m3 (Day 2). Personal exposure to ambient concentrations of benzene, toluene, ethylbenzene and xylene (BTEX) was assessed by using Radiello((R)) 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] microg/m(3), as assessed by personal sampling. Urinary concentrations of biomarkers in the whole group were 41.8 [34.1-89.8] microg/g creatinine for t,t-MA, 0.67 [0.23-1.32] microg/g creatinine for S-PMA, and 0.16 [0.13-0.26] microg/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 microg/m(3).

Solid-phase microextraction gas chromatographic-mass spectrometric method for the determination of inhalation anesthetics in urine.

Solid-phase microextraction (SPME) has been applied to the headspace sampling of inhalation anesthetics (i.e. nitrous oxide, isoflurane and halothane) in human urine. Analysis was carried out by gas chromatography-mass spectrometry using a capillary column with a divinylbenzene porous polymeric stationary phase. A SPME divinylbenzene-Carboxen-polydimethylsiloxane coated fiber, 2 cm long, was used, and its performances were compared with those of a Carboxen-PDMS in terms of sensitivity, extraction efficiency, extraction time, fiber coating-urine distribution coefficient. For both fibers, linearity was established over four orders of magnitude, limits of detection were below 100 ng/l for nitrous oxide and below 30 ng/l for halogenated. Precision calculated as %RSD was within 3-13% for all intra- and inter-day determinations. The method was applied to the quantitative analysis of anesthetics in the urine of occupationally exposed people (operating room personnel).

Styrene 7,8-oxide induces caspase activation and regular DNA fragmentation in neuronal cells

Neurobehavioral changes have been described in workers occupationally exposed to styrene vapors. Alterations of neurotransmitters and loss of neurons have been observed in brains of styrene-exposed rats. However, the mechanisms of neuronal damage are not yet clearly understood. We have characterized the cellular alterations induced by the main reactive intermediate of styrene metabolism, styrene 7,8-oxide (SO) in the human neuroblastoma SK-N-MC cell line and primary culture of rat cerebellar granule cells (CGC). SK-N-MC cells exposed to SO (0.3-1 mM) displayed apoptotic morphology, together with chromatin condensation and DNA cleavage into high molecular weight fragments of regular size. These features were accompanied by the activation of class II caspases, as detected with the DEVD assay, by following the cleavage of the caspase-substrate poly (ADP-ribose) polymerase (PARP) and by detection of the active fragment of caspase-3. Pre-incubation of the cells with the caspase inhibitor z-VAD-fmk reduced the cellular damage induced by SO, suggesting that caspases play an important role in SO toxicity. Increased proteolysis by class II caspases was detected also in primary culture of CGC exposed to SO. In addition, the presence of the 150-kDa cleavage product of alpha-fodrin suggests a possible activation of calpains in SK-N-MC cells. Moreover, SO did not affect the level of expression of the p53 protein, even though it is known to cause DNA damage. The identified intracellular pathways affected by SO exposure provides end-points that can be used in future studies for the evaluation of the neurotoxic effect of styrene in vivo.

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography-mass spectrometry : Comparison with solid phase extraction and liquid/liquid extraction

A simple and reliable solid phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85mum PA exposed at 100 degrees C for 40min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2ng/g, serum: 0.2ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.

Neurobehavioral assessment of rats exposed to low doses of PCB126 and methyl mercury during development

Epidemiological and laboratory studies have suggested that polychlorinated biphenyls (PCBs) and methyl mercury (MeHg) may have additive or synergistic effects on CNS function. Aim of this study was to characterize the effects of exposure to low levels of MeHg (0.5mg/kgday in drinking water) and PCB126 (100ng/kgday in food), alone and in combination, on neurobehavioral development in Wistar rats. Dams were treated from gestational day 7 to post-natal day (PND) 21. Animals were tested for developmental landmarks and reflexes (PND1-21), attention deficits (PND40), locomotor activity (PND30, 110), spatial learning (PND75), coordination and balance (PND90), object discrimination (PND80), anxiety (PND100), and conditioned learning (PND110). Parameters related to pregnancy, sex ratio at birth, and physical development (at weaning) did not differ among groups, though PCB126 decreased number of pups at birth. A slight delay in negative geotaxis was found in female rats in all treatment groups. No significant effects were seen in attention, coordination and balance, object discrimination, and spatial and conditioned learning. Increased motor activity was present in PCB126-treated male and in MeHg+PCB-treated female rats in the elevated plus maze test, and in PCB126-treated male rats in the open field test (PND110). The results do not support the hypothesis that co-exposure to MeHg and PCB126 results in additive or synergistic effects. This finding is in agreement with more recent in vitro and in vivo studies.

Biomarkers of exposure to aromatic hydrocarbons and methyl tert-butyl ether in petrol station workers

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.

Exhaled breath analysis in suspected cases of non-small-cell lung cancer: a cross-sectional study

Lung cancer is one of the most frequently diagnosed cancers worldwide and is still the leading cause of cancer-related deaths. There is a considerable interest in finding diagnostic methods in the diseases earliest stages. A complementary approach to imaging techniques could be provided by exhaled breath gas phase and exhaled breath condensate (EBC) analysis. The aim of this study was to quantify various biomarkers in the exhaled breath gas phase and EBC in suspected cases of non-small-cell lung cancer (NSCLC). The study involved 138 subjects with suspected lung cancer, 71 of whom had a subsequent diagnosis of NSCLC. The diagnostic power of a combination of hydrogen peroxide (H₂O₂)-EBC, and exhaled pentane, 2-methyl pentane, hexane, ethyl benzene, heptanal, trans-2-nonenal in distinguishing NSCLC and non-NSCLC subjects was poor-to-fair (area under the curve (AUC) = 0.68), similar to that of smoking history alone (expressed as pack-years, AUC = 0.70); a further improvement was observed when smoking history was combined with exhaled compounds (AUC = 0.80). The diagnostic power was increased in those patients with little or no past smoke exposure (AUC = 0.92) or where past smoke exposure was up to 30 pack-years (AUC = 0.85). Exhaled substances had a good accuracy in discriminating suspected cancerous cases only in those subjects with a modest smoking history (≤ 30 pack-years), but the inclusion of other exhaled biomarkers may increase the overall accuracy, regardless of tobacco smoke.