[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.
International Journal of Molecular Sciences 01/2013; 14(9):18078-92. · 2.46 Impact Factor
[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 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 09/2012; · 3.90 Impact Factor
[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.
Toxicology in Vitro 05/2012; 26(6):831-40. · 2.65 Impact Factor
[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.
Journal of Applied Toxicology 01/2012; 32(6):454-64. · 2.60 Impact Factor
[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.
Journal of Nanomaterials 01/2012; 2012. · 1.55 Impact Factor
[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.
Journal of Applied Toxicology 10/2009; 30(3):218-25. · 2.60 Impact Factor
[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.
Journal of Applied Toxicology 06/2009; 29(6):537-44. · 2.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.
[show abstract][hide abstract] ABSTRACT: The trace element vanadium interacts with living cells, in which it exerts a variety of biological effects depending on its chemical form and oxidation state. Tetravalent vanadium was shown to affect several genotoxicity end-points in vitro, but its genotoxic potential in vivo is not elucidated. In this study, the genotoxic effects induced in vivo by subacute oral exposure to vanadyl sulphate (VOSO4), a tetravalent vanadium salt, were investigated. To this aim male CD1 mice were administered with VOSO4 in drinking water over the dose range 2-1000 mg/l for 5 weeks. The incidence of micronucleated blood reticulocytes was measured along treatment period. At the end of treatment, micronuclei in both blood reticulocytes and bone marrow polychromatic erythrocytes were determined; in addition, DNA lesions detectable by comet assay were assessed in marrow and testicular cells. Tissue distribution of vanadium at sacrifice was determined by atomic absorption spectrometry. Comet assays and the analysis of micronuclei in polychromatic erythrocytes did not reveal treatment related effects. A slight increase in micronucleated reticulocytes, with no relationship with the administered dose, was observed in some treated groups. The determination of vanadium content in kidney, liver, spleen, bone, stomach, small intestine and testis highlighted low internal exposure, especially in soft tissues. Overall, data indicate scarce bioavailability for orally administered tetravalent vanadium, and lack of significant genotoxic potential in vivo.
[show abstract][hide abstract] ABSTRACT: The objective of this study was to investigate whether 24 h exposure to radiofrequency electromagnetic fields similar to those emitted by mobile phones induces genotoxic effects and/or effects on cell cycle kinetics in cultured human peripheral blood lymphocytes. The effect of 900 MHz exposure (GSM signal) was evaluated at four specific absorption rates (SARs, 0, 1, 5 and 10 W/kg peak values). The exposures were carried out in wire patch cells under strictly controlled conditions of both temperature and dosimetry, and the induction of genotoxic effects was evaluated in lymphocyte cultures from 10 healthy donors by applying the cytokinesis-block micronucleus assay. Positive controls were provided by using mitomycin C. Two research groups were involved in the study, one at ENEA, Rome, and the other at CNR-IREA, Naples. Each laboratory tested five donors, and the resulting slides were scored by both laboratories. Following this experimental scheme, it was also possible to compare the results obtained by cross-scoring of slides. The results obtained provided no evidence for the existence of genotoxic or cytotoxic effects in the range of SARs investigated. These findings were confirmed in the two groups of five donors examined in the two laboratories and when the same slides were scored by two operators.
Radiation Research 07/2006; 165(6):655-63. · 2.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: The possibility of genotoxicity of radiofrequency radiation (RFR) applied alone or in combination with x-rays was investigated in vitro using several assays on human lymphocytes. The chosen specific absorption rate (SAR) values are near the upper limit of actual energy absorption in localized tissue when persons use some cellular telephones. The purpose of the combined exposures was to examine whether RFR might act epigenetically by reducing the fidelity of repair of DNA damage caused by a well-characterized and established mutagen.
Blood specimens from 14 donors were exposed continuously for 24 h to a Global System for Mobile Communications (GSM) basic 935 MHz signal. The signal was applied at two SAR; 1 and 2 W/Kg, alone or combined with a 1-min exposure to 1.0 Gy of 250 kVp x-rays given immediately before or after the RFR. The assays employed were the alkaline comet technique to detect DNA strand breakage, metaphase analyses to detect unstable chromosomal aberrations and sister chromatid exchanges, micronuclei in cytokinesis-blocked binucleate lymphocytes and the nuclear division index to detect alterations in the speed of in vitro cell cycling.
By comparison with appropriate sham-exposed and control samples, no effect of RFR alone could be found for any of the assay endpoints. In addition RFR did not modify any measured effects of the x-radiation.
This study has used several standard in vitro tests for chromosomal and DNA damage in Go human lymphocytes exposed in vitro to a combination of x-rays and RFR. It has comprehensively examined whether a 24-h continuous exposure to a 935 MHz GSM basic signal delivering SAR of 1 or 2 W/Kg is genotoxic per se or whether, it can influence the genotoxicity of the well-established clastogenic agent; x-radiation. Within the experimental parameters of the study in all instances no effect from the RFR signal was observed.
International Journal of Radiation Biology 06/2006; 82(5):339-46. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: The question whether extremely low frequency magnetic fields (ELFMFs) may contribute to mutagenesis or carcinogenesis is of current interest. In order to evaluate the possible genotoxic effects of ELFMFs, human blood cells from four donors were exposed in vitro for 48 h to 50 Hz, 1 mT uniform magnetic field generated by a Helmholtz coil system. Comet assay (SCGE), sister chromatid exchanges (SCE), chromosome aberrations (CAs), and micronucleus (MN) test were used to assess the DNA damage. ELF pretreated cells were also irradiated with 1 Gy of X-ray to investigate the possible combined effect of ELFMFs and ionizing radiation. Furthermore, nuclear division index (NDI) and proliferation index (PRI) were evaluated. Results do not evidence any DNA damage induced by ELFMF exposure or any effect on cell proliferation. Data obtained from the combined exposure to ELFMFs and ionizing radiation do not suggest any synergistic or antagonistic effect.
[show abstract][hide abstract] ABSTRACT: In the past, epidemiological studies indicated a possible correlation between the exposure to ELF fields and cancer. Public concern over possible hazards associated with exposure to extremely low frequency magnetic fields (ELFMFs) stimulated an increased scientific research effort. More recent research and laboratory studies, however, have not been able to definitively confirm the correlation suggested by epidemiological studies. The aim of this study was to evaluate the effects of 50 Hz magnetic fields in human blood cells exposed in vitro, using several methodological approaches for the detection of genotoxicity. Whole blood samples obtained from five donors were exposed for 2 h to 50 Hz, 1 mT uniform magnetic field generated by a Helmholtz coil system. Comet assay, sister chromatid exchanges (SCE), chromosome aberrations (CA), and micronucleus (MN) tests were used to assess DNA damage, one hallmark of malignant cell transformation. The effects of a combined exposure with X-rays were also evaluated. Results obtained do not show any significant difference between ELFMFs exposed and unexposed samples. Moreover, no synergistic effect with ionizing radiation has been observed. A slight but significant decrease of cell proliferation was evident in ELFMFs treated samples and samples subjected to the combined exposure.