Emilia Paba

Multi‐walled carbon nanotubes induce cytotoxicity and genotoxicity in human lung epithelial cells

The increasing use of nanomaterials in consumer products highlights the importance of understanding their potential toxic effects. We evaluated cytotoxic and genotoxic/oxidative effects induced by commercial multi‐walled carbon nanotubes (MWCNTs) on human lung epithelial (A549) cells treated with 5, 10, 40 and 100 µg ml−1 for different exposure times. Scanning electron microscopy (SEM) analysis, MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays were performed to evaluate cytotoxicity. Fpg‐modified comet assay was used to evaluate direct‐oxidative DNA damage. LDH leakage was detected after 2, 4 and 24 h of exposure and viability reduction was revealed after 24 h. SEM analysis, performed after 4 and 24 h exposure, showed cell surface changes such as lower microvilli density, microvilli structure modifications and the presence of holes in plasma membrane. We found an induction of direct DNA damage after each exposure time and at all concentrations, statistically significant at 10 and 40 µg ml−1 after 2 h, at 5, 10, 100 µg ml−1 after 4 h and at 10 µg ml−1 after 24 h exposure. However, oxidative DNA damage was not found. The results showed an induction of early cytotoxic effects such as loss of membrane integrity, surface morphological changes and MWCNT agglomerate entrance at all concentrations. We also demonstrated the ability of MWCNTs to induce early genotoxicity. This study emphasizes the suitability of our approach to evaluating simultaneously the early response of the cell membrane and DNA to different MWCNT concentrations and exposure times in cells of target organ. The findings contribute to elucidation of the mechanism by which MWCNTs cause toxic effects in an in vitro experimental model. Copyright

Evaluation of cytotoxic concentration–time response in A549 cells exposed to respirable α‐quartz

A causal pathway between quartz, silicosis and lung cancer has been postulated. The aim of our study was to assess cytotoxic effects induced in a human lung epithelial cell line (A549) by exposure to α‐quartz. Cells were exposed to respirable α‐quartz (SRM1878a, NIST) at 25, 50 or 100 µg ml−1 for 24 h and at 50 or 100 µg ml−1 for 48 h. Cytotoxic effects were analyzed by scanning electron microscopy (SEM), apoptotic morphology analysis with Hoechst staining and lactate dehydrogenase (LDH) release assay. In cells exposed to α‐quartz for 24 h, a concentration‐dependent bleb development and in particular the localization of blebs at the cell edge at higher concentrations were observed. The blebbing phenomenon was more evident after 48 h of exposure to 50 or to 100 µg ml−1 of α‐quartz and large blebs were localized at the cell edge. At the same concentrations surface smoothing was also observed. Moreover the presence of holes and tears was detected at the highest concentration both at 24 and 48 h. Results of morphological analysis with Hoechst stain evidenced an increase concentration–time dependent of apoptotic cell percentage that was more marked after 48 h exposure to 100 µg ml−1 and a prevalence of late apoptosis stage with the increase of exposure time and concentration. Cells exposed to 50 or 100 µg ml−1 of α‐quartz for 24 and 48 h produced a significant increase in LDH release. The concentration–time‐dependent bleb induction evidenced by SEM correlates with the increase of apoptotic cells and LDH activity release, demonstrating the onset of cytotoxic effects in human lung cells exposed to α‐quartz. Copyright

Exposure to Airborne Culturable Microorganisms and Endotoxin in Two Italian Poultry Slaughterhouses

Even if slaughterhouses’ workers handle large amounts of organic material and are potentially exposed to a wide range of biological agents, relatively little and not recent data are available. The main objective of this study was to characterize indoor concentrations of airborne bacteria, fungi, and endotoxin mod = Im (endotoxin∼Gram-negative*plant*filter) in two Italian poultry slaughterhouses. Air samples near air handling units inlets were also collected. Since there are not standardized protocols for endotoxin sampling and extraction procedures, an additional aim of the study was to compare the extraction efficiency of three different filter.. The study was also aimed at determining the correlation between concentrations of Gram-negative bacteria and endotoxin. In Plant A bacterial levels ranged from 17.5 to 2.6×103 CFU/m3. The highest concentrations were observed in evisceration area of chickens, between the automatic detachment of the neck and washing offal, and near birds coupling before hair-chilling. The highest mean value of Gram-negative (266.5 CFU/m3) was found near the washing offal of turkeys. In Plant B bacterial concentration ranged from 35 to 8×103 CFU/m3. The highest concentration. with the highest value of Gram-negative (248 CFU/m3), was found after defeathering. Fungal concentrations were overall lower than those found for bacteria (range: 0–205 CFU/m3 in Plant A and 0–146.2 CFU/m3 in Plant B). The microbial flora was dominated by Gram-negative and coagulase-negative staphylococci for bacteria and by species belonging to Cladosporium, Penicillium and Aspergillus genera for molds. The highest endotoxin concentrations were measured in washing offal for Plant A (range: 122.7–165.9 EU/m3) and after defeathering for Plant B (range: 0.83–38.85 EU/m3). In this study airborne microorganisms concentrations were lower than those found in similar occupational settings and below the occupational limits proposed by some authors. However, these microorganisms may exert adverse effects on exposed workers, in particular for those engaged in the early slaughtering stages, as evidenced by the presence of pathogenic species. The detection of pathogenic bacteria near AHU inlet may constitute a risk to public health and environmental pollution.

Validation of a high performance liquid chromatography-tandem mass spectrometry method for β-hydroxy fatty acids as environmental markers of lipopolysaccharide

The most commonly analytical method used for the assessment of the occupational exposure to airborne endotoxins is the LAL test, but other methods are reported in the literature. The objective of this work was to develop and validate a method using HPLC coupled to triple quadrupole mass spectrometer for the quantitative determination of β-hydroxy-dodecanoic and β-hydroxy-tetradecanoic acids as chemical markers of lipopolysaccharide in Gram-negative strains. Matrix effect was significant but compensated by the use of internal standard; accuracy was always more than 85% and variability always less than 10%. LOD and LLOQ were respectively 2.5 μg/l and 10 μg/l for β-hydroxy-dodecanoic acid, 1 μg/l and 5 μg/l for β-hydroxy-tetradecanoic acid. The analysis of different bacterial strains showed that samples contain β-hydroxy-dodecanoic and β-hydroxy-tetradecanoic acids in variable ratio, and therefore the sum of their concentrations was considered the chemical marker. The method showed a significant correlation of results with those of LAL-test on four bacterial strains.

Exposure to airborne endotoxin in Italian greenhouses: environmental analyses

The peculiar characteristics of the greenhouses as confined spaces, microclimate and poor air exchange with the outside environment, encourage the development of a large number of biological agents. Endotoxin, is probably a major causative agent of occupational health problems. The objective of this study was to measure the concentrations of airborne endotoxin in greenhouses with different cultures. The influence of microclimate was studied in correlation with endotoxin levels and type of cultured vegetables. The data indicate that workers employed greenhouses are exposed to low levels of inhalable endotoxins; endotoxin concentrations do not correlate with the temperature and relative humidity values. A strong correlation between the leaf size and endotoxin concentration was observed. The mean concentration of endotoxins in the air of greenhouses is relatively low, however, there could be peaks of exposure during harvesting and eradication of broadleaf plants.