Fig. 3. Calibration curve of Cr(VI) in the range 0–5 gl −1 after extraction with TBAB and complexation with DPC in water, and in a pool of EBC; the slopes of the curves and Pearson’s r-value are also shown. The intercept represents the value of the blank in the absence of standard Cr(VI). Cr(VI) concentration (5 gl −1 ) was unaffected by the matrix (data not shown), and the simultaneous presence in solution of 5 gl −1 Cr(VI) and 500 gl −1 Fe 2+ (which tends to reduce Cr(VI) at acidic pH) reduced the ETAAS Cr(VI) absorption peak by only about 10%. • The calibration curve of Cr(VI) in a pool of different EBCs (total Cr = 0.1 gl −1 ) showed an approximately 10% decrease in the Cr signal within the range 0–5 gl −1 (Fig. 3). Moreover, as in the case of water, the presence of up to 50 gl −1 of Cr(III) in EBC did not interfere with the Cr(VI) signal. • Considering 3 S.D. of the blank, the LOD of EBC Cr(VI) was about 0.2 gl −1 . • The coefficient of variation (CV) of the measurements of standard samples (0.8, 2 and 5 gl −1 ) in water and a pool of EBCs was
234 M. Goldoni et al. / Analytica Chimica Acta 562 (2006) 229–235 be concentrated in the organic phase simply by changing the volume of the DPC/butanol solution: using a 3:1 ratio of sample:DPC/butanol solution (9:3 ml), tripled the increase in signal intensity and reduced the LOD by a factor of 3 (Fig. 1). This means that about 75% of Cr(VI) complexed with DPC was concentrated in the organic phase. Cr(VI) extraction of some pieces of membrane with the buffer suggested by Hazelwood et al. [29] in order to monitor the soluble and insoluble Cr(VI) compounds did not significantly change the results, the concentration of insoluble Cr(VI) in this study being considered negligible. Fig. 2A and B also shows negligible interferences from other cationic transition elements in environmental samples. The results with and without the SPE step recommended in the literature to clean the samples from other cationic transition elements or impurities were perfectly consistent in the investigated range of concentrations, and this was confirmed by in vitro experiments in which 500 gl −1 of Cr(VI) were incubated with equal absolute quantities of cationic Fe, Pb, Ni, Cd, Cu and Co (data not shown). Another advantage of our method is that the SPE cartridges could be saturated by high concentrations of Cr(VI), whereas a direct colorimetric measure is linear in the range of the linearity of the spectrophotometer (Abs about 1.5). Further work is needed to compare our method with the ISO 16740 speciation method recently published [30]. In determining EBC Cr(VI), we adopted a different and more sensitive method that is useful for small concentrations and small volume samples (the LOD passes from 0.5 to 0.2 gl −1 ). Tetraalkylammonium (tetrapropyl or tetrabutylammonium) bromide tends to form ion pairs with metal cyanides, alkyl sulfonate, sulfur oxide, inorganic anions and oxyanions [31–34] and, in particular, tetrabutylammonium ions form stable ion pairs with chromate at acidic pH in aqueous solutions [17,27]. This ion pair can be selectively extracted in organic solvents as 4-methyl- 2-pentanone or chloroform, or in aqueous two-phase systems [17,27]. We adapted the method of Noroozifar and Khorasani- Motlagh [27] to the small volumes of collected EBC (never more than 2 ml over 15 min), and the use of ETAAS also allowed good sensitivity in the generally diluted EBC samples. The percent recovery of Cr(VI) as an ion pair in 4-methyl-2-pentanone was in line with that observed in previous studies [17,27] despite the small volumes, and did not depend on the Cr(VI) concentration in the considered range (Fig. 3). Moreover, the experiments using drinking water and Fe 2+ , together with the results of interference studies published in the literature [27], make the developed method ideal for diluted samples such as EBC, although the slight decrease in the extraction efficiency of Cr(VI) as an ion pair and then as a complex with DPC (about 10% overall) suggested adding the standard directly to the pools of EBCs and treating them as samples. This can be explained as a probable interference of traces of EBC anions (salts and proteins) present upon the extraction of Cr(VI). The slight effect of the different components on the signal of the blank suggested using highly pure reagents to minimise these effects, but the blank absorbance was in any case acceptable: about 14 mAbs against the 6 mAbs of pure water. The optimisation of the methods of measuring soluble Cr(VI) in environmental samples and EBC allowed us to study the kinet- ics of Cr(VI) in the airways. First of all, total Cr in EBC decreased 15 h after the last exposure, a finding that is in line with previous results observed before and after a weekend, and confirms that Cr can pass through airway lining fluid [11]: the kinetics of pulmonary lung desorption is relatively slow [35], and Cr accumulation has recently been observed in the bronchi and lungs of chromate workers [36]. Most importantly, the EBC Cr(VI) data confirmed that Cr(VI) was reduced to Cr(III) by airway lining fluid, but not completely so (Fig. 4A and B), even in workers exposed to smaller environmental Cr(VI) concentrations than the limits suggested by ACGIH [37], and those exposed to lower Cr concentrations than those reported in our previous study [11]. About 33.5% of Cr was still in the hexavalent form immediately after chromate exposure (about 67% of the environmental samples were hexavalent): 9/10 workers had clearly detectable EBC Cr(VI) levels, and 4/10 had detectable levels about 15 h after the last exposure. The persistence of Cr(VI) in the airways can justify the previously reported increase in biomarkers of inflammation in EBC [11], because unreduced Cr(VI) can be absorbed at pulmonary level and reveal its toxicity in pulmonary cells. We are currently attempting to relate the reduction kinetics of EBC Cr(VI) to individual susceptibility and EBC biomarkers of inflammation in a larger number of subjects. 5. Conclusions In conclusion, we have developed a method for measuring EBC Cr(VI) based on ion pair extraction followed by ETAAS determination. The results of this study highlight the potential of EBC as a medium for assessing lung dose and effects after exposure to inhaled pneumotoxic substances (particularly transition elements) with different oxidation states. The integrated use of EBC and classic biological matrices such as urine and blood, which reflect systemic exposure, may therefore allow the fundamental completion of the biological monitoring of pneumotoxic compounds. Acknowledgements This study was supported by the National Heart, Blood and Lung Institute (NHLBI), Bethesda, MD, USA (grant R01 HL72323), and the Italian Ministry of Education, University and Research (PRIN 200306145). References [1] ATSDR, Toxicological profile for Chromium (final report), NTIS Accession No. PB2000-108022. Agency for Toxic Substances and Disease Registry, Atlanta, GA, 2000, 461pp. [2] International Agency for Research on Cancer, Chromium, nickel and welding, IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, vol. 49, IARC Scientific Pubblications, IARC, Lyon, 1999. [3] S. De Flora, M. Bagnasco, D. Serra, P. Zanacchi, Mutat. Res. 238 (1990) 99–172. [4] S. De Flora, K.E. Wetterhahn, Life Chem. Rep. 7 (1989) 169–244. [5] D. Bagchi, S.J. Stohs, B.W. Downs, M. Bagchi, H.G. Preuss, Toxicology 180 (2002) 5–22.
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UNIVERSITÀ DEGLI STUDI DI PARMA DO
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Riassunto La raccolta e l’analisi
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Sommario CAPITOLO 1: Introduzione .
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5.3.1 Scopi dello studio ..........
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Congressi Nazionali ed Internaziona
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ischio consiste nel caratterizzare
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1.1 I biomarcatori Il termine “bi
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Forschungsgemainschaft (DFG, 2004).
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elettrotermico (ETAAS) fino ad arri
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circostanze, sarebbe più appropria
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3.3.2 Miscele di inquinanti comples
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Molti studi in vivo e in vitro hann
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sigaretta (Rustemeier et al., 2002;
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Amorim, L.C., de Cardeal, L.Z., 200
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Bernard, A., 2004. Renal dysfunctio
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Coogan, T.P., Squibb, K.S., Motz, J
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Effros, R.M., Hoagland, K.W., Bosbo
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Microscopic analysis of chromium ac
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McGarvey, L.P., Dunbar, K., Martin,
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NIOSH, 2003. NIOSH Method 7703: Hex
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Rahman, I., Kelly, F., 2003. Biomar
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Controlli Lavoratori N° soggetti 2
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N° soggetti 10 Sesso (M/F) 10/0 La
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