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Background: Idiopathic retroperitoneal fibrosis (RPF) is a rare disease. Asbestos exposure has been proposed as a risk factor for idiopathic RPF., Objective: To investigate the role of asbestos and other occupational agents (such as silica, metals, and organic solvents), as well as environmental agents (such as smoking), and their interactions as potential risk factors for idiopathic RPF., Design: Case-control study., Setting: National referral hospital for idiopathic RPF., Patients: 90 patients with idiopathic RPF and 270 control participants matched for age, sex, and region of residency., Measurements: Occupational history was obtained using structured questionnaires administered by blinded specialists in occupational medicine. Exposure to nonoccupational agents and presence of diseases that were potentially predisposing to idiopathic RPF were assessed through patient interviews and examination of medical records., Results: A history of asbestos exposure was associated with idiopathic RPF (odds ratio [OR], 4.22 [95% CI, 2.14 to 8.33]). Both current smoking (OR, 3.21 [CI, 1.46 to 7.07]) and former smoking (OR, 2.93 [CI, 1.39 to 6.14]) were more prevalent among patients than among those who never smoked. A multiplicative effect was found between tobacco smoke and both occupational asbestos exposure (OR, 12.04 [CI, 4.32 to 38.28]) and extraoccupational asbestos exposure (OR, 8.42 [CI, 2.77 to 30.58])., Limitation: Retrospective, questionnaire-based assessment of occupational exposure., Conclusion: Exposure to asbestos and tobacco smoke resulted in strong risk factors for idiopathic RPF. Coexposure to asbestos and smoke had a multiplicative effect on risk compared with single exposure., Primary Funding Source: None.

Background/aim: In the diagnosis of malignant mesothelioma (MM) there still is a lack of specific and sensitive screening biomarkers: this study examined the discriminatory power of a panel of serum/plasma biomarkers., Patients and Methods: The study involved four groups: (a) individuals previously exposed to asbestos with asbestosis; (b) patients with MM; (c) patients with non-small cell lung cancer; and (d) controls without any evidence of malignancy. The concentrations of mesothelin, chitinase-3-like-1 (YKL-40), vascular endothelial growth factor (VEGF), endothelin-1, interleukin-8 (IL-8) and fibulin-3 in the serum of patients were determined., Results: Patients with MM had significantly higher serum levels of mesothelin (p<0.001), YKL-40 (p<0.001), IL-8 (p<0.001) and VEGF (p<0.01) than controls. The cut-off point for MM was 1.26 nM for mesothelin alone, and 167 pg/ml for YKL-40 alone; the presence of both markers above these cut-off levels improved diagnostic specificity., Conclusion: The addition of YKL-40 may improve the specificity of mesothelin measurements alone for detecting patients with MM.

A standard procedure for exhaled breath condensate (EBC) collection is still lacking. The aim of this study was to compare the concentration of several biomarkers in whole (W-EBC) and fractionated EBC (A-EBC), the latter collected starting from CO2 ≥ 50% increase during exhalation. Forty-five healthy non-smokers or asymptomatic light smokers were enrolled. Total protein concentrations in W-EBC and A-EBC were overlapping (median: 0.7 mg l(-1) in both cases), whereas mitochondrial DNA was higher in A-EBC (0.021 versus 0.011 ng ml(-1)), indicating a concentration rather than a dilution of lining fluid droplets in the last portion of exhaled air. H2O2 (0.13 versus 0.08 µM), 8-isoprostane (4.9 versus 4.4 pg ml(-1)), malondialdehyde (MDA) (4.2 versus 3.2 nM) and 4-hydroxy-2-nonhenal (HNE) (0.78 versus 0.66 nM) were all higher in W-EBC, suggesting a contribution from the upper airways to oxidative stress biomarkers in apparently healthy subjects. NH4(+) was also higher in W-EBC (median: 590 versus 370 µM), with an estimated increase over alveolar and bronchial air by a factor 1.5. pH was marginally, but significantly higher in W-EBC (8.05 versus 8.01). In conclusion, the fractionation of exhaled air may be promising in clinical and occupational medicine.

PM(2.5) generated by indoor combustion activities can contribute significantly to personal PM exposure. The aims of this study were: (1) to validate a device specifically designed to study the kinetics of particle exhalation and the percentage of airway particle deposition (%DEP) in polluted indoor environments (welding fumes, environmental tobacco smoke - ETS) and (2) to assess the intra- and inter-subject variability of the signal. The device was tested on 14 subjects exposed to welding fumes and 10 subjects exposed to environmental tobacco smoke (ETS), performing repeated measures at different environmental PM concentrations. The intra-subject variability of the signal for particles with diameter 0.3-1.0 μm showed a geometric mean of %CV always below 6%, despite the values of %DEP. In the welding fume study, the increase in airborne 0.5-1.0 μm PM concentrations between the consulting room and production department was explainable in terms of increased density due to the metallic composition of particles. The %DEP of 0.3-1.0 μm ETS particles decreased with airborne PM concentration due to the technical limits of a laser particle counter and the perturbation induced by the physical characteristics of ETS PM. However, also at those extreme conditions, the signal remained repeatable and the individual susceptibility to PM remained substantially unaltered. In conclusion, the versatility and portability of our device, together with the repeatability of the signal, confirmed that the kinetics of exhaled particles and %DEP could be routinely measured in polluted environments and used to define individual susceptibility to airborne particles., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

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.)

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants commonly found as contaminants in the same food sources. Even though their neurotoxic effects are established, the mechanisms of action are not fully understood. In the present study, we have used the mouse hippocampal neuronal cell line HT22 to investigate the mechanisms of neuronal death induced by MeHg, PCB 153, and PCB 126, alone or in combination. All chemicals induced cell death with morphological changes compatible with either apoptosis or necrosis. Mitochondrial functions were impaired as shown by the significant decrease in mitochondrial Ca²+ uptake capacity and ATP levels. MeHg, but not the PCBs, induced loss of mitochondrial membrane potential and release of cytochrome c into the cytosol. Also, pre-treatment with the antioxidant MnTBAP was protective only against cell death induced by MeHg. While caspase activation was absent, the Ca²+-dependent proteases calpains were activated after exposure to MeHg or the selected PCBs. Furthermore, lysosomal disruption was observed in the exposed cells. Accordingly, pre-treatment with the calpain specific inhibitor PD150606 and/or the cathepsin D inhibitor Pepstatin protected against the cytotoxicity of MeHg and PCBs, and the protection was significantly enhanced when the two inhibitors were combined. Simultaneous exposures to lower doses of MeHg and PCBs suggested mostly antagonistic interactions. Taken together, these data indicate that MeHg and PCBs induce caspase-independent cell death via parallel activation of calpains and lysosomal proteases, and that in this model oxidative stress does not play a major role in PCB toxicity.

Interactions among neurotoxicants in in vitro models, where the molecular mechanisms of toxicity are generally studied, represent today an emerging field in the experimental neurotoxicology. In this chapter, we define some general concepts about the optimization of in vitro experiments to assess the dose/concentration-effect/response relationships and to extrapolate the functions describing them. After describing the available models to study interactions (the Bliss independence criterion and the Loewe additivity model), we present a method to practically apply these models to experimental data. Finally, we provide some examples of the theory of interactions among neurotoxicants in in vitro models.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

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.

Introduction: Epidermal growth factor receptor (EGFR) gene intron 1 contains a polymorphic single sequence dinucleotide repeat (CA)n whose length has been found to inversely correlate with transcriptional activity. This study was designed to assess the role of (CA)n polymorphism in predicting the outcome of gefitinib treatment in advanced non-small cell lung cancer (NSCLC)., Methods: Blood and tumor tissue from 58 patients with advanced NSCLC submitted to gefitinib were collected. EGFR intron 1 gene polymorphism, along with EGFR gene mutation, gene copy number and immunohistochemistry expression were determined. Moreover, a panel of lung cancer cell lines characterized for EGFR intron 1 polymorphism was also studied., Results: EGFR intron 1 polymorphism showed a statistically significant correlation with the gefitinib response (response rate 25 versus 0%, for patients with a (CA)16 and with a (CA)else genotype, respectively; p = 0.044). Patients with a (CA)16 genotype had a longer survival compared with those with a (CA)else genotype (11.4 versus 4.8 months, respectively; p = 0.037). In addition, cell lines lacking the (CA)16 allele showed a statistically significant higher IC50 compared with cell lines bearing at least one (CA)16 allele (p = 0.003)., Conclusions: This study supports a potential role of EGFR intron 1 polymorphism in predicting the outcome of gefitinib treatment in advanced NSCLC.

Atrial fibrillation becomes a self-perpetuating arrhythmia as a consequence of electrophysiologic and structural remodeling involving the atrium. Oxidative stress may be a link between this rhythm disturbance and electrophysiologic remodeling. The aim of this study was to evaluate whether the heme oxygenase-1 (HO-1) marker of oxidative stress was more expressed in left atrial sites with stronger structural remodeling in patients affected by chronic atrial fibrillation (CAF) and mitral valve disease (MD). Myocardial samples were taken from the left atrial posterior wall (LAPW) and left atrial appendage (LAA) of 24 patients with CAF-MD in addition to 10 autopsy controls. The levels of HO-1 messenger RNA (mRNA) and HO-1 protein in each pathologic LAPW and LAA were quantified using reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Furthermore, light microscopy was used to morphometrically evaluate the differential myocyte and interstitial changes in the same CAF-MD LAPW and LAA samples. In controls, HO-1 protein was quantified using enzyme-linked immunosorbent assay. Unlike controls, patients with CAF-MD had higher levels of HO-1 mRNA and its protein product, expressed as LAPW/LAA ratios, in the LAPW (2.18 +/- 1.18, P < .0001, and 1.55 +/- 0.67, P < .005), and their LAPW also showed greater histologic changes in myocytolytic myocytes (15.1% +/- 3.1% versus 6.9% +/- 3.3%, P < .0001), interstitial fibrosis (8.2% +/- 2.2% versus 2.8% +/- 1.2%, P < .0001), and capillary density (816 +/- 120 number/mm(2) versus 1114 +/- 188 number/mm(2); P < .05). In addition, markers of oxidative stress were immunohistochemically studied with antinitrotyrosine and anti-iNOS antibodies. In patients with CAF-MD, the inducible enzyme HO-1 is more expressed in the left atrial areas that show greater structural remodeling. This finding strongly suggests a pathogenetic relationship between oxidative stress and the degree of histologic change.

Objective: Chromium in exhaled breath condensate (EBC) has recently been proposed as a biomarker of pulmonary exposure. The aim of this study was to measure the Cr levels in the EBC and pulmonary tissue of patients with early, operable non-small cell lung cancer (NSCLC) who had not been occupationally exposed to Cr before and after tumour resection and to correlate Cr in lung tissue with that in EBC., Methods: Cr levels in the EBC and pulmonary tissue of 20 NSCLC patients were measured by means of electrothermal atomic absorption before and after tumour resection. Cr levels were also measured in the urine of 15 of these patients., Results: The pre-surgery EBC Cr levels of the NSCLC patients were not different from those of the controls, but both EBC and urinary Cr levels increased after surgery. There was a significant correlation between Cr levels in EBC and pulmonary tissue (R = 0.55, P = 0.01), but not between these and urinary Cr levels., Conclusion: Cr levels in EBC and urine of NSCLC patients were increased after surgical intervention. Measured Cr EBC levels were by one order of magnitude lower than those observed in moderately exposed workers. This fact, together with the correlation between Cr in EBC and in pulmonary tissue, confirms that EBC is a promising biological fluid to test pulmonary exposure to Cr, giving complementary information to that provided by urinary Cr, not correlated with EBC and tissue.

Intracellular reduction of hexavalent chromium [Cr(VI)] is associated with the production of reactive oxygen species (ROS) and subsequent oxidative damage to different intracellular molecules like DNA, proteins and lipids is believed to contribute to the process of carcinogenesis. Aim of this study was to develop a model to establish a relationship between intracellular and macromolecule-bound chromium and some biomarkers of oxidative stress in two in vitro cell lines. Human lung adenocarcinoma (A549) and human bronchial epithelial (BEAS2B) cells were exposed for 3, 8 and 24 hours to relatively low doses (0.5--1--2 microM) of Cr(VI), i.e., to concentrations similar to what measured and reported by some authors in unexposed subjects and chromate workers. The results show that the differential cytotoxicity of Cr(VI) on the A549 and BEAS2B cell lines may be related both to their different polymorphism of Glutathione S-transferases genes and probably to their unlike permeability to Cr(VI). The glutathione decrease and the induction of HO-1 observed only in BEAS2B cells after Cr(VI) exposure strengthen the idea that glutathione S-transferases activity may accelerate the reduction of Cr(VI) to Cr(III) with the concomitant induction of oxidative stress. In conclusion, the determination of intracellular Cr in cellular models can be considered an important step in comparing in vitro and in vivo models on the basis of target doses and a promising approach to study the effects of pneumotoxic compounds.

The aim of the present study was to compare hydrogen peroxide (H2O2) levels resulting from oxidative stress in exhaled breath condensate (EBC) and bronchoalveolar lavage (BAL) supernatants of subjects with different diffuse interstitial lung diseases (DILDs). Twenty-one patients who underwent BAL procedure for various DILDs were studied. EBC, which was collected the day before bronchoscopy, was obtained by cooling exhaled breath; BAL was processed for differential cellular count and supernatant was stored. H2O2 in both fluids was measured using a commercial fluorimetric kit with a limit of detection of 0.01 microM. No difference in H2O2 levels in EBC and in BAL was observed [median (range), 0.07 microM (0.01-0.6) and 0.08 microM (0.01-0.8), respectively]. No correlation was observed between BAL and EBC levels. H2O2 in BAL did not correlate with differential cellular count, whereas H2O2 EBC did correlate positively with the percentage of epithelial cells (r = 0.5, p = 0.007) and negatively with the number of macrophages (r = -0.4, p = 0.03). No correlation was observed between H2O2 levels either in EBC or BAL and lung function data. The data show that in DILDs H2O2 can be detected in both EBC and supernatants of BAL and that their relative concentrations are similar but not correlated with each other. The positive correlation between H2O2 levels in EBC and percentage of epithelial cells leads to the speculation that airway epithelia may play a relevant contribution in H2O2 production in the airway lumen.

Exhaled volatile organic compounds (VOCs), mainly aliphatic and aromatic hydrocarbons, have been proposed as a diagnostic test for early lung cancer detection, but the effect of lung cancer surgical re-moval on exhaled VOCs pattern has never been specifically addressed. The aim of this study was to compare VOC levels measured in non small cell lung cancer (NSCLC) patients before surgery (T0), one month (T1) and 3 years (T2) after surgical removal of tumour. In order to better understand the pathophysiological meaning of exhaled aromatic hydrocarbons, the same exhaled biomarkers were also assessed in cancerous and macroscopically unaffected lung tissue samples collected during surgical operation. Exhaled breath was collected in a specially designed Teflon bulb trapping the last 150 ml of a single slow vital capacity. After solid phase micro-extraction, VOCs were analysed in gas chromatography-mass spectrometry. VOC levels were unaffected by surgical removal, except for isoprene, whose concentration was significantly reduced. Three years after surgical operation, some VOCs significantly changed from baseline: in particular, we noted a decrease in isoprene and benzene concentrations, whereas the levels of pentane, toluene and ethylbenzene were increased in comparison with baseline values. Finally, lung tissue analysis showed that all aromatic hydrocarbons, except xylenes, were significantly higher in cancerous than in unaffected tissue. This study showed that surgical operation can influence the concentration of some exhaled VOCs opening a new scenario in the use of exhaled VOCs in lung cancer patients, not only for diagnostic but also for follow up purposes.

Exhaled breath condensate (EBC) is composed mainly by water and also contains non-volatile mediators, which are expired in small droplets of airway fluid. Urea has been proposed as a normalization factor for EBC non-volatile biomarkers. Aim of this study was to assess volatility and diffusivity of urea ex vivo and to measure its EBC concentrations in different clinical conditions. Volatility was assessed quantifying EBC concentrations collected at 4 different temperatures, whereas diffusivity was tested by measuring urea concentrations in both plasma and EBC from uraemic patients on intermittent haemodialysis. Urea was also measured in EBC from patients with chronic airway diseases, i.e., chronic obstructive pulmonary disease, asthma, and cystic fibrosis. The concentration of urea but not its absolute amount in EBC increased with condensation temperature. Haemodialysis influenced EBC and plasma urea concentrations in a similar way. The concentrations of urea in chronic airway diseases did not significantly differ from those of controls. Urea is a non-volatile molecule ex vivo and EBC urea depends on its concentrations in plasma. Urea concentrations in EBC are unaffected by three chronic airway diseases. We suggest that there is no need to normalize non-volatile biomarkers in EBC for urea concentrations to account for inter-individual variability. However, in repeated measurements within the same individual, the use of urea either as a normalizing factor or as covariate variable could be proposed to control intra-individual variability.

Occupational/environmental exposure to some metallic elements is a risk factor for the development of lung diseases, including lung cancer. We aimed at investigating the levels of arsenic, beryllium, cadmium, cobalt, chromium, nickel and lead in the lung tissue of patients affected by early stage non small cell lung cancer (NSCLC). A small number of patients without a diagnosis of lung cancer were also included as control group. Lung tissue biopsies were collected from 45 NSCLC patients (both cancerous and unaffected tissues) and 8 control subjects undergoing surgery. Patients were stratified for smoking habits, histopathology and cancer sites. Metallic elements were determined in dry tissue after digestion by means of ICP-MS. Cd, Ni and Pb levels were higher in unaffected than in control tissues (0.52 vs 0.18 microg/g dry, p < 0.05 for Cd; 4.49 vs 1.8 microg/g dry,p < 0.05 for Ni; 0.21 vs 0.06 microg/g dry, p < 0.01 for Pb). The three elements, and particularly Cd, were influenced by smoking habits; Pb levels were higher in squamocellular carcinoma than adenocarcinomas; Ni distributed in the lungs in an inhomogeneous way. This study demonstrates that the unaffected lung tissue is more representative than the cancerous tissue of the pulmonary content of metallic elements. Tobacco smoke is a main factor affecting the concentration levels of Cd, Pb, and to a lesser extent Ni in the lung tissues of NSCLC patients. The role of past environmental-occupational exposures could not be fully elucidated, due to the limited sample size and the retrospective nature of the study.

Background: Routine water monitoring in a haemodialysis centre revealed high trichloroethylene (TCE) concentrations. The aim of this study is to describe the measures adopted after organic contamination of dialysis water in order to avoid the possibility of patient exposure. We also carried out in vitro experiments to evaluate the accumulation of TCE in various devices normally used in a dialysis water treatment system (DWTS)., Methods: In vivo and in vitro blood and water TCE levels were determined by means of solid phase microextraction-gas chromatography/mass spectrometer., Results: High TCE concentrations were found throughout the DWTS; acceptably low levels were obtained only by replacing the activated charcoal, ionic-exchange resins, microfilters and PVC pipes. The adsorption and realising capacities of these devices were tested in vitro, and the elimination curves made it possible to calculate the total percentage of the previously absorbed TCE mass released into the water. Evidence of exposure was confirmed by the relatively high TCE levels in the patient blood samples taken 30 days after the last exposure even if the subjects were asymptomatic. In vivo experiments showed that the blood gain of TCE through the low flux membrane during the course of dialysis was about 77+/-10.4% of the amount carried by dialysis fluid as calculated on the basis of its partition coefficient value (K(b/w) 3.75)., Conclusions: This study shows that, when present in dialysis water, the lipophilic TCE contaminant can accumulate in various devices, thus transforming them into possible sources of exposure. This highlights the importance of periodically monitoring dialysis water for organic substances that have a great affinity to the blood compartment, in order to prevent occasional or chronic patient exposure.

Study Objectives: The aim of this study was to characterize the elemental composition of exhaled breath condensate (EBC) in order to identify new biomarkers of exposure and susceptibility in COPD patients. Serum pneumoproteins were used as lung-specific biomarkers of effect., Design: EBC was obtained from 50 healthy subjects, 30 healthy smokers, 30 asthmatics, and 50 patients with stable COPD, and was collected by cooling exhaled air. Trace elements and toxic metals in the samples were measured by means of inductively coupled plasma-mass spectrometry and electrothermal atomic absorption spectroscopy. The serum pneumoproteins were immunoassayed., Results: The EBC of COPD subjects had higher levels of such toxic elements as lead, cadmium, and aluminum, and lower levels of iron and copper, than that of the nonsmoking control subjects. There were no between-group differences in surfactant protein (SP)-A and SP-B levels. Clara-cell protein and SP-D levels were negatively and positively influenced, respectively, by tobacco smoke., Conclusions: Our results show that toxic metals and transition elements are detectable in the EBC of studied subjects. We propose new biomarkers of exposure as a means of assessing the target tissue dose of carcinogenic and pneumotoxic substances from tobacco smoke or polluted workplaces, and the use of the transition elements involved in redox systems of oxidative stress as disease biomarkers associated with effect or susceptibility. Together with biomarkers of effect, such as serum pneumoproteins, the elemental composition of EBC may be clinically useful in distinguishing similar diseases.

Chromium is corrosive, cytotoxic, and carcinogenic for humans and can induce acute and chronic lung tissue toxicity. The aim of this study was to investigate Cr levels in exhaled breath condensate (EBC) of workers exposed to Cr(VI) and to assess their relationship with biochemical changes in the airways by analyzing EBC biomarkers of oxidative stress, namely, hydrogen peroxide (H2O2) and malondialdehyde (MDA). EBC samples were collected from 24 chrome-plating workers employed in a chrome-plating plant both before and after the Friday work shift and before the work shift on the following Monday. Cr-EBC levels increased from the beginning (5.3 microg/L) to the end of Friday (6.4 microg/L) but were considerably lower on Monday morning (2.8 microg/L). A similar trend was observed for H2O2-EBC levels (which increased from 0.36 microM to 0.59 microM on Friday and were 0.19 microM on Monday morning) and MDA-EBC levels (which increased from 8.2 nM to 9.7 nM on Friday and were 6.6 nM on Monday). Cr-EBC levels correlated with those of H2O2-EBC (r = 0.54, p < 0.01) and MDA-EBC (r = 0.59, p < 0.01), as well as with urinary Cr levels (r = 0.25, p < 0.05). The results of this study demonstrate that EBC is a suitable matrix that can be used to investigate both Cr levels and biomarkers of free radical production sampling the epithelial-lining fluid of workers exposed to Cr(VI).

Chromium speciation has attracted attention because of the different toxicity of Cr(III), which is considered relatively non-toxic, and Cr(VI), which can cross cell membranes mainly as a chromate anion and has been classified as a class I human carcinogen. The aims of the present study were to measure soluble Cr(VI) levels in environmental samples, to develop a simple method of quantifying Cr(VI) in exhaled breath condensate (EBC), and to follow the kinetics of EBC Cr(VI) in chrome plating workers.Personal air samples were collected from 10 chrome platers; EBC was collected from the same workers immediately after the work shift on Tuesday and before the work shift on the following Wednesday. Environmental and EBC Cr(VI) levels were determined by means of colorimetry and electrothermal absorption atomic spectrometry, respectively.The method of detecting Cr(VI) in environmental air was based on the extraction of the Cr(VI)-diphenylcarbazide (Cr(VI)-DPC) complex in 1-butanol, whereas EBC Cr(VI) was determined using a solvent extraction of Cr(VI) as an ion pair with tetrabutylammonium ion, and subsequent direct determination of the complex (Cr(VI)-DPC) in EBC.Kinetic data showed that airborne Cr(VI) was reduced by 50% in airway lining fluid sampled at the end of exposure and that there was a further 50% reduction after about 15 h. The persistence of Cr(VI) in EBC supports the use of EBC in assessing target tissue levels of Cr(VI).

The aim of the present study was to investigate whether exhaled breath condensate (EBC), a fluid formed by cooling exhaled air, can be used as a suitable matrix to assess target tissue dose and effects of inhaled cobalt and tungsten, using EBC malondialdehyde (MDA) as a biomarker of pulmonary oxidative stress. Thirty-three workers exposed to Co and W in workshops producing either diamond tools or hard-metal mechanical parts participated in this study. Two EBC and urinary samples were collected: one before and one at the end of the work shift. Controls were selected among nonexposed workers. Co, W, and MDA in EBC were analyzed with analytical methods based on mass spectrometric reference techniques. In the EBC from controls, Co was detectable at ultratrace levels, whereas W was undetectable. In exposed workers, EBC Co ranged from a few to several hundred nanomoles per liter. Corresponding W levels ranged from undetectable to several tens of nanomoles per liter. A parallel trend was observed for much higher urinary levels. Both Co and W in biological media were higher at the end of the work shift in comparison with preexposure values. In EBC, MDA levels were increased depending on Co concentration and were enhanced by coexposure to W. Such a correlation between EBC MDA and both Co and W levels was not observed with urinary concentration of either element. These results suggest the potential usefulness of EBC to complete and integrate biomonitoring and health surveillance procedures among workers exposed to mixtures of transition elements and hard metals. Key words: cobalt, exhaled breath condensate, hard metals, lung, malondialdehyde, oxidative stress, tungsten.

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.