Use of a common European approach for nanomaterials' testing to support regulation: a case study on titanium and silicon dioxide representative nanomaterials.

The European Union (EU) continuously takes ensuring the safe use of manufactured nanomaterials (MNMs) in consumer products into consideration. The application of a common approach for testing MNMs, including the use of optimized protocols and methods' selection, becomes increasingly important to obtain reliable and comparable results supporting the regulatory framework. In the present study, we tested four representative MNMs, two titanium dioxides (NM100 and NM101) and two silicon dioxides (NM200 and NM203), using the EU FP7-NANoREG approach, starting from suspension and dispersion preparations, through to their characterization and final evaluation of biological effects. MNM dispersions were prepared following a refined NANOGENOTOX protocol and characterized by dynamic light scattering (DLS) in water/bovine serum albumin and in media used for in vitro testing. Potential genotoxic effects were evaluated on human bronchial BEAS-2B cells using micronucleus and Comet assays, and pro-inflammatory effects by cytokines release. Murine macrophages RAW 264.7 were used to detect potential innate immune responses using two functional endpoints (pro-inflammatory cytokines and nitric oxide [NO] production). The interaction of MNMs with RAW 264.7 cells was studied by electron microscopy. No chromosomal damage and slight DNA damage and an oxidative effect, depending on MNMs, were observed in bronchial cells. In murine macrophages, the four MNMs directly induced tumor necrosis factor α or interleukin 6 secretion, although at very low levels; lipopolysaccharide-induced NO production was significantly decreased by the titania and one silica MNM. The application of this approach for the evaluation of MNM biological effects could be useful for both regulators and industries.

Alkaline earth silicate (AES) wools: Evaluation of potential cyto-genotoxic and inflammatory effects on human respiratory cells.

Biosoluble AES wools are increasingly used since considered not hazardous, however, few toxicity studies are available. We evaluated cytotoxic, genotoxic-oxidative and inflammatory effects of two differently soluble AES wools, AES1 (high MgO percentage) and AES2 (high CaO percentage), on alveolar (A549) and bronchial (BEAS-2B) cells. Fiber dimensions and dissolution in cell media were evaluated by SEM analysis. Cell viability, LDH release, direct/oxidative DNA damage (fpg-comet assay) and IL-6, IL-8 and TNF-α release (ELISA), were analysed after 24 h exposure to 2-200 µg/ml. On A549 cells AES1 induced LDH release, slight direct DNA damage and oxidative DNA damage with very high IL-6 release at 100 µg/ml; AES2 induced higher DNA damage than AES1 and slight oxidative DNA damage. On BEAS-2B cells we found direct DNA damage (higher for AES1) and slight oxidative DNA damage (associated to slight increased IL-6 and IL-8 release for AES1). The higher genotoxicity of more soluble AES2 on A549 cells could be explained by higher respirable fibers % and fiber number/µg found after 24 h in RPMI-medium at 100 µg/ml. The higher membrane damage, oxidative DNA damage and inflammation induced by AES1 in A549 cells could be due to the higher DLG and silica percentage. These findings suggest further investigations on AES toxicity.

Cyto-genotoxic effects of smoke from commercial filter and non-filter cigarettes on human bronchial and pulmonary cells.

Cigarette smoke is a complex mixture of chemicals, some of which are known as carcinogens. The cyto-genotoxic effects of cigarette-smoke extract (CSE) from commercial cigarettes without (A and B) and with filter (C and D) were evaluated at different CSE concentrations on A549 and BEAS-2B cells. The particle content of the cigarette smoke and the metal composition of the CSE were also analyzed. The cells were exposed to 1-10% of the CSE from one cigarette per experiment. Cytotoxicity was evaluated by use of the MTT assay after 24h, and the lactate dehydrogenase (LDH) assay after 30min and 24h. The Fpg-modified comet assay was used to evaluate direct-oxidative DNA damage on cells exposed for 30min. As expected, unfiltered cigarette smoke (particularly from the B cigarette) contained a higher number of particles than filtered smoke. With smoke extract from the B cigarette we found a decrease in cell viability only in BEAS-2B cells. The results of the LDH test showed membrane damage for B-cigarette smoke extract, particularly in BEAS-2B cells. Extracts from unfiltered cigarette smoke induced significant direct DNA damage, to a larger extent in A549 cells. Filtered cigarette-smoke extract induced a significant direct DNA damage at 5-10%. A significant induction of oxidative DNA damage was found at the highest CSE concentration in both cell types (by smoke extracts from B and C cigarettes in A549 cells, and from A and D cigarettes in BEAS-2B cells). Smoke extracts from filter cigarettes induced less direct DNA damage than those from unfiltered cigarettes in A549 cells, probably due to a protective effect of filter. In BEAS-2B cells the smoke extract from the B-cigarette showed the highest genotoxic effect, with a concentration-dependent trend. These findings show a higher cyto-genotoxicity for smoke extracts from the B-cigarette and oxidative effects for those from the A and D cigarettes, particularly in BEAS-2B cells. Moreover, there was a higher responsiveness of A549 cells to genotoxic insult of CSE, and a cigarette-dependent genotoxicity in BEAS-2B cells. Our experimental model demonstrated to be suitable to sensitively detect early genotoxic response of different lung-cell types to non-cytotoxic concentrations of complex inhalable mixtures.

Genotoxic and oxidative effects induced on A549 cells by extract of PM10 collected in an electric steel plant.

The present study was aimed at assessing the carcinogenic risk of occupational exposure to PM10 in electric steel plants. PM10 was collected on cellulose filter respectively outside (site 1) and inside (site 2) the furnace area, was measured, extracted and its metal content was analysed by ICP-MS. Cells were exposed for 30 min, 2 and 4 hours to extract of filter from each site diluted at 0.004, 0.008 and 0.02%. The direct/oxidative DNA damage caused by PM10 was evaluated on A549 cells by Fpg-modified comet assay, analysing Tail moment (TM) and comet percentage. Air samples contained 1.08 mg/m3 of PM10 in site 1 and 5.54 mg/m3in site 2 and different amounts of metals with higher levels of Zn, Al, Ni, Pb, Cd, Cr, Ba in site 2 and of Fe, Mn, Sb in site 1. In cells exposed for 2h to PM10 from both sites, an oxidative DNA damage was found concentrations of 0.008% and 0.02%. For site 2, a direct DNA damage at 0.02% was also found. After 4h a direct/oxidative DNA damage was detected at 0.02% for site 2 and an oxidative DNA damage for site 1. The results indicate a moderate DNA damage induction by used diluitions of PM10 extracts with higher extent for more polluted site 2. These findings show the suitability of this experimental model to evaluate early DNA damage induced by complex mixtures containing metals on target organ, suggesting its use to study biological effects of occupational exposure to such substances.

Evaluation of DNA damage induction on human pulmonary cells exposed to PAHs from organic extract of PM10 collected in a coke-oven plant.

Occupational exposure of coke oven workers, classified by IARC as human carcinogen, is characterized by the presence of PAHs emitted during pyrolysis of coal. We aimed to clarify the mechanism of action of complex mixtures of PAHs and to identify biomarkers of early biological effect, evaluating on lung epithelial cells (A549) genotoxic and oxidative damage of airborne particulate matter collected in a coke plant. Particulate matter was collected in the oven area on glass filter, extract and analysed by GC/MS. Direct/oxidative DNA damage induced by exposure to extract were evaluated by Fpg comet assay. The cells were exposed for 30 min, 2h and 4h to extract of half filter diluted at 0.004%, 0.008% and 0.02%. We evaluated comet percentage and analysed tail moment values of cells treated with Fpg enzyme (TMenz) and untreated (TM) that indicate respectively oxidative and direct DNA damage. Air sample contained 0.328 microg/m3 of pyrene, 0.33 microg/m3 of benzo(a)anthracene, 1.073 microg/m3 of benzo(b)fluoranthene, 0.22 microg/m3 of benzo(k)fluoranthene, 0.35 microg/m3 of benzo(a)pyrene, 0.079 microg/m3 of dibenzo(a,h)anthracene and 0.40 microg/m3 of benzo(g,h,i)perylene. The dose-dependent increase of TM and TMenz in exposed cells was not significant, indicating only a slight direct and oxidative DNA damage in exposed cells. A small dose-time dependent increase of comet percentage was found. The study shows the high sensitivity of comet assay to measure early DNA damage also at low doses suggesting its use on lung epithelial cells to evaluate the effects of complex mixtures of genotoxic substances on target organ.

Cytotoxic and oxidative effects induced by man-made vitreous fibers (MMVFs) in a human mesothelial cell line.

The introduction of man-made vitreous fibers (MMVFs) as a substitute for asbestos in industrial and residential applications raises concerns about their potential health hazards. The aim of our study was to assess cytotoxic and oxidative effects induced on a human mesothelial cell line (MeT-5A) by exposure to glass wool (GW), rock wool (RW) and refractory ceramic fibers (RCF) in comparison with crocidolite asbestos (CR). MeT-5A cells were exposed for 24 h to 2, 5 and 10 microg/cm2 of MMVF and crocidolite fibers and analysed by scanning electron microscope (SEM) for cell surface alterations. Cells were exposed for 2 h to 1, 2, 5 and 10 microg/cm2 of the same fibers and analysed by enzyme Fpg-modified comet test for direct and oxidative DNA damage. SEM revealed loss of microvilli in cells exposed to RCF and numerous blebs in cells exposed to higher doses of RW. Comet test showed significant direct DNA damage in cells exposed to RCF even at the lowest dose. Comet test with Fpg, that permits the detection of oxided DNA bases, showed significant oxidative DNA damage in cells exposed to higher doses of RW. The presence of DNA damage and alterations of cell surface induced by low doses of RCF and the presence of oxidative DNA damage and blebs on cell surface in cells exposed to higher dose of RW suggest possible cytotoxic, oxidative and genotoxic effects for these MMVFs.