Nicola Sannolo

Effects of low concentrations of benzene on human lung cells in vitro.

Exposure to benzene causes health hazards to humans. The airway epithelium is a physical barrier to inhaled toxicants and particulates. This is an in vitro basic science study to evaluate the effects of benzene on lung cells without the inflammatory responses triggered by inhalation. Dose-response cytotoxicity was assessed using two cell lines: alveolar derived (A549) human epithelial adenocarcinoma and human lung (LL24) fibroblast. A549 cells were more resistant than LL24 fibroblast lung cells to benzene. LL24 cells demonstrated enhanced proliferation with diluted benzene solutions. Moreover, low concentrations of benzene enhanced telomerase activity in LL24 cells while no effects were observed in the adenocarcinoma cells. Proteolysis of lung matrix by matrix metalloproteinases (MMPs) is an early event observed in lung pathologies. Benzene increased MMP-2 and MMP-3 mRNA. Using the ratio (MMP-1+MMP-2+MMP-3)/(TIMP-1+TIMP-2), as an index of prodestructive activity, we observed a dose-dependent increase. The overall higher expression levels of MMPs in benzene treated cells did not appear to be controlled by TIMPs, which are negatively correlated. Comparing different cell lines, we demonstrated how crucial is the targets susceptibility. Our observations may represent early functional alterations that occur in the airways of exposed people.

Proteomic approach for the analysis of acrylamide-hemoglobin adducts. Perspectives for biological monitoring.

The formation of adducts between acrylamide and hemoglobin in vitro was investigated by using mass spectrometric methodologies to identify the amino acid residues sensitive to alkylation. Liquid chromatography-electrospray ionisation mass spectrometry analysis of either intact or trypsin-digested alpha- and beta-globin chains isolated from hemolysate samples incubated in vitro with acrylamide at different molecular ratios allowed us to identify Cys93 of beta-globin as the most reactive site in hemoglobin, according to a Michael-type addition reaction between acrylamide and the sulphydryl group of cysteine. The only other reactive sites were Cys104 of alpha-globin and the N-terminal amino groups of both chains. The method developed, based on electrospray ionisation quadrupole time-of-flight tandem mass spectrometry analysis of intact globin chains was able to specifically detect low levels of adducts. In this way, rapid identification of alkylated portion of Hb was achieved to be potentially used as a biomarker for high-sensitivity biological monitoring.

Mass spectrometric analysis of haemoglobin adducts formed by methyl bromide in vitro.

An analytical procedure is described for the identification of the adducts formed by interaction of methyl bromide and haemoglobin. The reaction products of in vitro incubation of haemoglobin with methyl bromide have been characterised by electrospray mass spectrometry and gas chromatography-mass spectrometry. A prominent reactivity of several potential nucleophilic sites of haemoglobin was observed. Analogous results were recorded on blood samples of workers exposed to methyl bromide. The results obtained represent the basis for the complete structural characterisation of the modified haemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of alkylation degree and the identification of modified amino acids in proteins.

Gas chromatographic quantitation of breath hydrogen and carbon monoxide for clinical investigation in adults and in children

A simple and suitable method of breath sampling and the gas chromatographic determination of H2 and CO in breath of children as well as of adults are described. The analytical system allows the measurement of concentrations of H2 and CO as low as a few ppm. The proposed method of breath collection (tedlar bag with low resistance valve face mask) appears particularly useful for long storage purposes, because it eliminates the need for additional manipulations and the possible associated sample dilution. Normalization of H2 breath amounts to internal standard O2 to obtain reliable and precise measurements is particularly useful in non-collaborating patients whose observed H2 peak values increase by 15% after the correction. The overall procedure is fast, inexpensive and accurately recognizes adult or children lactose malabsorbers as well as subjects exposed even to low levels of CO.