[Show abstract][Hide abstract] ABSTRACT: The increasing use of cobalt oxide (Co3O4) nanoparticles (NPs) in several applications and the suggested genotoxic potential of Co-oxide highlight the importance of evaluating Co3O4 NPs toxicity. Cyto-genotoxic and inflammatory effects induced by Co3O4 NPs were investigated in human alveolar (A549), and bronchial (BEAS-2B) cells exposed to 1-40 µg ml-1. The physicochemical properties of tested NPs were analysed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cytotoxicity was studied to analyze cell viability (WST1 test) and membrane damage (LDH assay), direct/oxidative DNA damage was assessed by the Formamido-pyrimidine glycosylase (Fpg)-modified comet assay and inflammation by interleukin (IL)-6, IL-8 and tumor necrosis factor-alpha (TNF-α) release (ELISA). In A549 cells, no cytotoxicity was found, whereas BEAS-2B cells showed a viability reduction at 40 µg ml-1 and early membrane damage at 1, 5 and 40 µg ml-1. In A549 cells, direct and oxidative DNA damage at 20 and 40 µg ml-1 were detected without any effects on cytokine release. In BEAS-2B cells, significant direct DNA damage at 40 µg ml-1 and significant oxidative DNA damage with a peak at 5 µg ml-1, that was associated with increased TNF-α release at 1 µg ml-1 after 2 h and increased IL-8 release at 20 µg ml-1 after 24 h, were detected. The findings show in the transformed alveolar cells no cytotoxicity and genotoxic/oxidative effects at 20 and 40 µg ml-1. In normal bronchial cells, moderate cytotoxicity, direct DNA damage only at the highest concentration and significant oxidative-inflammatory effects at lower concentrations were detected. The findings confirm the genotoxic-oxidative potential of Co3O4 NPs and show greater sensitivity of BEAS-2B cells to cytotoxic and oxidative-inflammatory effects suggesting the use of different cell lines and multiple end-points to elucidate Co3O4 NPs toxicity.
No preview · Article · Mar 2015 · Journal of Applied Toxicology
[Show abstract][Hide abstract] ABSTRACT: Functionalized MWCNTs are used in many commercial and biomedical applications, but their potential health effects are not well defined. We investigated and compared cytotoxic, genotoxic/oxidative, and inflammatory effects of pristine and carboxyl MWCNTs exposing human respiratory (A549 and BEAS-2B) cells to 1-40 μg/mL of CNTs for 24 h. Both MWCNTs induced low viability reduction (by WST1 assay) in A549 cells and only MWCNTs-COOH caused high viability reduction in BEAS-2B cells reaching 28.5% viability at 40 μg/mL. Both CNTs induced membrane damage (by LDH assay) with higher effects in BEAS-2B cells at the highest concentrations reaching 20% cytotoxicity at 40 μg/mL. DNA damage (by Fpg-comet assay) was induced by pristine MWCNTs in A549 cells and by both MWCNTs in BEAS-2B cells reaching for MWCNTs-COOH a tail moment of 22.2 at 40 μg/mL versus 10.2 of unexposed cells. Increases of IL-6 and IL-8 release (by ELISA) were detected in A549 cells exposed to MWCNTs-COOH from 10 μg/mL while IL-8 increased in BEAS-2B cells exposed to pristine MWCNTs at 20 and 40 μg/mL. The results show higher cytogenotoxicity of MWCNTs-COOH in bronchial and of pristine MWCNTs in alveolar cells. Different inflammatory response was also found. The findings suggest the use of in vitro models with different end points and cells to study CNT toxicity.
Full-text · Article · Jul 2014 · BioMed Research International
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to identify sensitive and noninvasive biomarkers of early carcinogenic effect at target organ to use in biomonitoring studies of workers at risk for previous occupational exposure to potential carcinogens. Standard urine cytology (Papanicolaou staining test), comet assay, and quantitative telomerase repeat amplification protocol (TRAP) assay were performed in 159 ex-rubber workers employed in tyres production and 97 unexposed subjects. In TRAP positive cases, a second level analysis using FISH (Urovysion) was done. Cystoscopy results were available for 11 individuals whose 6 FISH/TRAP/comet positive showed in 3 cases a dysplastic condition confirmed by biopsy, 1 comet positive resulted in infiltrating UBC to the biopsy and with hyperplasia and slight dysplasia to the urinary cytology, 1 comet positive resulted in papillary superficial UBC to the biopsy, 1 FISH/TRAP positive showed a normal condition, and 2 TRAP positive showed in one case a phlogosis condition. The results evidenced good concordance of TRAP, comet, and FISH assays as early biomarkers of procarcinogenic effect confirmed by the dysplastic condition and UBC found by cystoscopy-biopsy analysis. The analysis of these markers in urine cells could be potentially more accurate than conventional cytology in monitoring workers exposed to mixture of bladder potential carcinogens.
Full-text · Article · Apr 2014 · BioMed Research International
[Show abstract][Hide abstract] ABSTRACT: Poly(ADP-ribose)polymerase-1 (PARP1) is a nuclear protein implicated in DNA repair, recombination, replication, and chromatin remodeling. The aim of this study was to evaluate possible differences between PARP1-/- and wild-type mice regarding induction and repair of DNA lesions in irradiated male germ cells. Comet assay was applied to detect DNA damage in testicular cells immediately, and two hours after 4 Gy X-ray irradiation. A similar level of spontaneous and radiation-induced DNA damage was observed in PARP1-/- and wild-type mice. Conversely, two hours after irradiation, a significant level of residual damage was observed in PARP1-/- cells only. This finding was particularly evident in round spermatids. To evaluate if PARP1 had also a role in the dynamics of H2AX phosphorylation in round spermatids, in which γ-H2AX foci had been shown to persist after completion of DNA repair, we carried out a parallel analysis of γ-H2AX foci at 0.5, 2, and 48 h after irradiation in wild-type and PARP1-/- mice. No evidence was obtained of an effect of PARP1 depletion on H2AX phosphorylation induction and removal. Our results suggest that, in round spermatids, under the tested experimental conditions, PARP1 has a role in radiation-induced DNA damage repair rather than in long-term chromatin modifications signaled by phosphorylated H2AX.
Full-text · Article · Sep 2013 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: 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 24 h, and the lactate dehydrogenase (LDH) assay after 30 min and 24 h. The Fpg-modified comet assay was used to evaluate direct-oxidative DNA damage on cells exposed for 30 min. 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.
No preview · Article · Sep 2012 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
[Show abstract][Hide abstract] ABSTRACT: Chemical functionalization of multiwalled carbon nanotubes (MWCNTs) increases their solubility, dispersion, and biological applications. Since there are only a few studies on the toxicity of functionalized MWCNTs, we investigated the cytotoxic and genotoxic-oxidative effects of OH-functionalized MWCNTs on human lung epithelial cells (A549) in order to obtain information on their biological effects. We exposed the cells to 10, 20, 40, and 100 μg/mL of commercial MWCNT-OH for 24 h. Cytotoxicity was then evaluated as the reduction in cell viability, membrane damage, and apoptosis, assessed by MTT and LDH assays and fluorescence microscopic analysis, respectively. The Fpg-modified comet assay was used to assess direct/oxidative DNA damage. We found a concentration-dependent reduction in cell viability and an increase of percentage of apoptotic cells, with no significant cellular LDH release. There was also concentration-dependent direct DNA damage but no oxidative DNA damage. These findings demonstrate the cytotoxicity of MWCNT-OH, through reduction of cell viability and induction of apoptosis without cell membrane damage, and the genotoxicity, by direct DNA damage induction, suggesting that the MWCNTs enter the cell without damaging its membrane and directly interact with the nucleus. This preliminary study highlights the need for further research to examine the potential toxicity of functionalized MWCNTs before starting to use them in biological applications.
Full-text · Article · Jul 2012 · Journal of Nanomaterials
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jun 2012 · Journal of Applied Toxicology
[Show abstract][Hide abstract] ABSTRACT: Chemical functionalization extends CNT applications conferring them new functions, but could modify their toxicity. We compared cytotoxicity and genotoxic/oxidative effects of -OH functionalized and pristine MWCNTs to evaluated the influence of the functionalization exposing A549 cells to 1-40μg/ml of both MWCNTs for 2, 4 and 24h. Cytotoxicity was evaluated by MTT and LDH tests and apoptosis induction, direct/oxidative DNA damage by Fpg-modified comet assay. After 24h we found viability reduction significant at 20 and 40μg/ml for both the MWCNTs with a detectable viability reduction already at lower concentrations for MWCNTs. A significant LDH release was found only for MWCNTs. Significant apoptosis induction was found from 10μg/ml of MWCNT-OH. A concentration-dependent increase of direct DNA damage, significant at 40μg/ml of MWCNTs and beginning from 5μg/ml of MWCNT-OH was detected at all exposure times. Oxidative DNA damage was not observed for both CNTs. The results indicate a different cytotoxic mechanism, by membrane damage for MWCNTs and apoptosis for MWCNT-OH, that could be explained by a different cellular uptake. Moreover, we found an earlier genotoxic effect for MWCNT-OH. The findings suggest that further studies on functionalized CNTs are necessary before using them in several applications particularly in biomedical field.
No preview · Article · May 2012 · Toxicology in Vitro
[Show abstract][Hide abstract] ABSTRACT: 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 alpha-quartz. Cells were exposed to respirable alpha-quartz (SRM1878a, NIST) at 25, 50 or 100 microg ml(-1 )for 24 h and at 50 or 100 microg 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 alpha-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 microg ml(-1) of alpha-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 microg ml(-1) and a prevalence of late apoptosis stage with the increase of exposure time and concentration. Cells exposed to 50 or 100 microg ml(-1) of alpha-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 alpha-quartz.
No preview · Article · Aug 2009 · Journal of Applied Toxicology
[Show abstract][Hide abstract] ABSTRACT: The mechanism of Cr(VI) genotoxicity has still not been elucidated. We used Fpg-modified comet assay to assess direct-oxidative DNA damage on human lung (A549) and bronchial (BEAS-2B) cells exposed to 0.1, 0.5, 1.0 and 10 microm sodium chromate for 0.5, 1 and 4 h. Moreover we evaluated apoptosis by morphological analysis and caspase-3 activity, also after 24 h. On A549 cells a time-dependent DNA damage, expressed as tail DNA%, beginning from 0.5 microm was found. For oxidative DNA damage an induction after 30 min to 0.5 microm decreasing with time, and a time-dependent increase at 10 microm was found, indicating for low Cr(VI) concentration the oxidative stress as the first event followed by direct DNA damage and for the highest concentration a time-dependent increase in oxidative DNA damage. On BEAS-2B cells DNA damage was induced within 1 h at 0.5-10 microm, without changes with time, showing that BEAS-2B cells are able to resist to Cr(VI) genotoxicity. Early oxidative DNA damage at 0.1 microm decreasing with time was also found. Significant apoptosis was observed by morphological analysis in A549 cells and to a lower extent in BEAS-2B at 10 microm. The exposure to 10 microm induced caspase-3 activity after 4 h in BEAS-2B and after 24 h in A549 cells. The findings show a higher responsiveness of A549 cells to genotoxic effect of Cr(VI) and early transient oxidative DNA damage in BEAS-2B. The results emphasize the suitability of this experimental model to evaluate the early genotoxic response of different cells to non-cytotoxic concentrations of Cr(VI) on target organ.
No preview · Article · Jan 2009 · Journal of Applied Toxicology