Chromium in exhaled breath condensate (EBC), erythrocytes, plasma and urine in the biomonitoring of chrome-plating workers exposed to soluble Cr(VI).

Chromium (Cr) levels measured in exhaled breath condensate (EBC-Cr) and urine (Cr-U) at the beginning and end of working shifts were related to those measured in erythrocytes (Cr-RBC) and plasma in 14 non-smoking male chrome-plating workers exposed to Cr(VI) in soluble aerosol form who did not report any significant current or past respiratory disease. Cr-U mainly correlated with Cr-P (Cr in plasma) at the end of the working shift (r(2) = 0.59, p < 0.01), whereas Cr-RBC correlated with EBC-Cr (r(2) = 0.32, p < 0.05); at the beginning of the shift, the only significant correlation was between Cr-U and Cr-RBC (r(2) = 0.74, p < 0.01). The clearance of Cr(iii) arising from Cr(VI) reduction was rapid, thus making Cr-U and Cr-P ideal biomarkers of the most recent exposure, whereas Cr-RBC may represent the fraction of Cr(VI) that reaches the bloodstream in non-reduced form and therefore depends on the airway inhaled dose represented by EBC-Cr. Cr-RBC clearance is slower and not only involves the free diffusion of Cr(iii) from RBC to plasma, but probably also involves more complicated kinetic phenomena involving other tissues and organs, which may explain the correlation between Cr-RBC and Cr-U and the lack of correlation Cr-RBC and Cr-P at least 36 h after the last exposure. In conclusion, our findings reinforce the idea that measuring Cr in EBC can significantly contribute to traditional biomonitoring by providing specific information at the target organ level and integrating our knowledge of Cr toxicokinetics.

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.

Development and set-up of a portable device to monitor airway exhalation and deposition of particulate matter.

The aim of this study was to assess and monitor airway exhalation and deposition of particulate matter (PM). After standardizing inspiratory/expiratory flow and volumes, a novel device was tested on a group of 20 volunteers and in a field study on workers exposed to cristobalite. Both male and female subjects showed a higher percentage of deposition in the 0.5 microm channel than in the 0.3 microm channel on a laser particle counter, but it was higher in the males because of their higher exhaled lung volumes. The device was tested on a wider range of particles (0.3-0.5-1.0-2.5 microm) in the cristobalite productive division. The device has low intrasubject variability and good reproducibility, with geometric mean of %CV < 5%. Such a measure can be used to assess individual susceptibility to PM, making repeated measures in different environments, and examining the persistence of particles in the airways after a period in polluted environments.

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.

Breath analysis in non small cell lung cancer patients after surgical tumour resection.

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.

Comparison between exhaled and bronchoalveolar lavage levels of hydrogen peroxide in patients with diffuse interstitial lung diseases.

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.

Exposure to low levels of hexavalent chromium: target doses and comparative effects on two human pulmonary cell lines.

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.

Collecting exhaled breath condensate (EBC) with two condensers in series: a promising technique for studying the mechanisms of EBC formation, and the volatility of selected biomarkers.

Exhaled breath condensate (EBC) consists mainly of water, but also contains semivolatile and nonvolatile compounds. The aim of this study was to develop a system in which two condensers are simultaneously used in series to clarify the mechanisms of EBC condensation. Two aliquots of EBC (EBC1 and EBC2) were collected from 20 asymptomatic smokers and 20 healthy young nonsmokers using a specifically designed device having two condensers in series in which total volume, hydrogen peroxide (H(2)O(2)), ammonium (NH(4)(+)), and conductivity before and after lyophilization were measured. Water, NH(4)(+) levels and conductivity before lyophilization were significantly lower in the EBC2 than in the EBC1 of smokers and nonsmokers; the contrary was true for H(2)O(2) levels. Almost all nonvolatile salts were collected in the first condenser, because more than 50% of postlyophilization conductivity was below the detection limit in EBC2. The recovery of volatile molecules and their derivatives (water and NH(4)(+)) was partial in the first condenser, but appreciable amounts of both were measured in the second; however, the condenser immediately in contact with exhaled air was more efficient in terms of water, NH(4)(+) and conductivity before lyophilization. On the contrary, nonvolatile ions (conductivity after lyophilization) were mainly collected in the first condenser. Finally, the behavior of H(2)O(2) cannot be explained on the basis of its chemical and physical properties, and the most probable explanation is that some was byproduced by a radical reaction in the gas phase or during the condensation process in water.

Heme oxygenase-1 expression in the left atrial myocardium of patients with chronic atrial fibrillation related to mitral valve disease: its regional relationship with structural remodeling.

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.

Polybrominated diphenyl ether (PBDE) flame retardants: environmental contamination, human body burden and potential adverse health effects.

Polybrominated diphenyl ethers (PBDEs) are an important class of flame retardants, widely used in a variety of consumer products. In the past several years, PBDEs have become widespread environmental pollutants, and have been detected in water, soil, air, animals and human tissues. Exposure occurs in particular through the diet and the indoor environment. Infants and toddlers have the highest body burden, due to exposure via maternal milk and through house dust. Tetra-, penta- and hexa-BDEs are the congeners most commonly found in humans. Recent concerns on possible adverse health effects of PBDEs are focusing on their potential endocrine disrupting effects and on developmental neurotoxicity.

Chromium in exhaled breath condensate and pulmonary tissue of non-small cell lung cancer patients.

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.

Determination of hexavalent chromium in exhaled breath condensate and environmental air among chrome plating workers.

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).

Clusterin decreases oxidative stress in lung fibroblasts exposed to cigarette smoke.

RATIONALE: Cigarette smoke causes injury to lung fibroblasts, partly by means of oxidative stress, and oxidative stress can lead to various lung diseases, such as chronic obstructive pulmonary disease. Clusterin is a widely distributed protein with many functions, including cellular protection in response to oxidative stress. OBJECTIVES: To determine whether clusterin is involved in the defense of the lung against cigarette smoke, we investigated the effects of cigarette smoke extract on clusterin expression and its protective effect, if any, against oxidative stress. METHODS: Fibroblasts were coincubated with conditioned medium and cigarette smoke extract, and bronchial biopsy specimens obtained from nonsmokers, smokers, and ex-smokers were analyzed by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: At concentrations of 2.5 and 5.0%, cigarette smoke extract induced oxidative stress. It also markedly increased the expression of two clusterin isoforms (60 and 76-80 kD) and the 76-80-kD isoform was secreted in the incubation medium. Coincubation of fibroblasts with conditioned medium significantly decreased the cellular oxidation caused by the cigarette smoke extract. Immunohistochemical analysis of clusterin on bronchial biopsy specimens obtained from smokers and ex-smokers showed localization of clusterin mainly in the submucosa. CONCLUSIONS: We conclude that clusterin may have a protective effect against cigarette smoke-induced oxidative stress in lung fibroblasts.

The effect of inhaled chromium on different exhaled breath condensate biomarkers among chrome-plating workers.

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).

Influence of condensation temperature on selected exhaled breath parameters.

BACKGROUND: The 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. METHODS: Exhaled 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. RESULTS: There 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. CONCLUSION: The 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.

Models of neurotoxicity: extrapolation of benchmark doses in vitro.

In risk assessment, no observed exposure level (NOAEL) and benchmark dose (BMD) are usually derived either from epidemiological studies in humans or from animal experiments. In many in vitro studies, concentration-effect/response curves have been analyzed using different mathematical models finalized to the identification of EC50. In the present article, we propose a model to fit dose-response curves in vitro. The BMD approach has been used to compare the cell viability (MIT assay) of different rat (C6 and PC12, glial and neuronal, respectively) and human cell lines (D384 and SK-N-MC, glial and neuronal, respectively) after 24-hour exposure to the following neurotoxic substances: manganese chloride (MnCl2), methyl-mercury (Me-Hg), and the enantiomers of styrene oxide (SO). For all rat and human cell lines, the potency of the examined compounds was: MnCl2 < S-SO < R-SO < Me-Hg. A preliminary comparison with in vivo toxicity data for these substances gave rise to consistent results. Whereas a reasonable agreement between in vitro and in vivo data has been found for Mn and styrene oxide, a wide scatter of LOAEL has been reported for Me-Hg and these appear to be either much higher or lower than the BMD for the MIT assay we observed in vitro.

Aldehydes in exhaled breath condensate of patients with chronic obstructive pulmonary disease.

The aims of the present study were (1) to evaluate whether individual aldehydes resulting from lipid peroxidation can be measured in exhaled breath condensate, (2) to assess the influence of sampling procedures on aldehyde concentrations, and (3) to compare aldehyde levels of patients with stable, moderate to severe, chronic obstructive pulmonary disease with those of smoking and nonsmoking control subjects. Aldehydes (malondialdehyde, hexanal, heptanal, and nonanal) were measured by liquid chromatography-tandem mass spectrometry in all samples and overlapping results were obtained by different sampling procedures. Malondialdehyde (57.2 +/- 2.4 nmol/L), hexanal (63.5 +/- 4.4 nmol/L), and heptanal (26.6 +/- 3.9 nmol/L) were increased in patients as compared with nonsmoking control subjects (17.7 +/- 5.5 nmol/L, p < 0.0001; 14.2 +/- 3.5 nmol/L, p = 0.004; and 18.7 +/- 0.9 nmol/L, p = 0.002, respectively). Only malondialdehyde was increased in patients compared with smoking control subjects (35.6 +/- 4.0 nmol/L, p = 0.0007). In conclusion, different classes of aldehydes were identified in exhaled breath condensate of humans. Whereas all aldehydes but nonanal were lower in control subjects as compared with other groups, only malondialdehyde distinguished smoking control subjects from patients with chronic obstructive pulmonary disease and could be envisaged as a biomarker potentially useful to monitor the disease and its response to therapy.