Anna Maria Fresegna

Exposure to Radiofrequency Radiation (900 MHz, GSM signal) does not Affect Micronucleus Frequency and Cell Proliferation in Human Peripheral Blood Lymphocytes: An Interlaboratory Study

Abstract Scarfì, M. R., Fresegna, A. M., Villani, P., Pinto, R., Marino, C., Sarti, M., Altavista, P., Sannino, A. and Lovisolo, G. A. Exposure to Radiofrequency Radiation (900 MHz, GSM signal) does not Affect Micronucleus Frequency and Cell Proliferation in Human Peripheral Blood Lymphocytes: An Interlaboratory Study. Radiat. Res. 165, 655–663 (2006). 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.

Evaluation of DNA damage in different stages of mouse spermatogenesis after testicular X irradiation

Abstract Cordelli, E., Fresegna, A. M., Leter, G., Eleuteri, P., Spanò, M. and Villani, P. Evaluation of DNA Damage in Different Stages of Mouse Spermatogenesis after Testicular X Irradiation. Radiat. Res. 160, 443–451 (2003). To evaluate whether DNA alterations in mature spermatozoa could stem from DNA damage induced in immature germ cells, testis cells and spermatozoa were analyzed by the comet assay and by the sperm chromatin structure assay 14, 45 and 100 days after in vivo X irradiation of the testes. These times were selected, according to the mouse seminiferous epithelium cycle, to follow the DNA damage induced in different germ cell compartments. The cytotoxic action was assessed by DNA flow cytometric analysis of testicular cells. A dose-dependent increase of DNA damage in testis cells was observed 14 days after irradiation, whereas mature sperm cells were not affected. On the other hand, an increase in DNA strand breaks was seen in spermatozoa 45 days after treatment. DNA damage returned to the control levels 100 days after irradiation. The methods used to evaluate DNA damage gave comparable results, emphasizing the correlation between DNA fragmentation and susceptibility of sperm chromatin to denaturation. Both techniques showed the high radiosensitivity of differentiating spermatogonia. The overall results showed that DNA damage induced in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa.

935 MHz cellular phone radiation. An in vitro study of genotoxicity in human lymphocytes.

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.

Evaluation of cytotoxic, genotoxic and inflammatory response in human alveolar and bronchial epithelial cells exposed to titanium dioxide nanoparticles

The toxicity of titanium dioxide nanoparticles (TiO2‐NPs), used in several applications, seems to be influenced by their specific physicochemical characteristics. Cyto‐genotoxic and inflammatory effects induced by a mixture of 79% anatase/21% rutile TiO2‐NPs were investigated in human alveolar (A549) and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml–1 30 min, 2 and 24 h to assess potential pulmonary toxicity. The specific physicochemical properties such as crystallinity, NP size and shape, agglomerate size, surface charge and specific surface area (SSA) were analysed. Cytotoxic effects were studied by evaluating cell viability using the WST1 assay and membrane damage using LDH analysis. Direct/oxidative DNA damage was assessed by the Fpg‐comet assay and the inflammatory potential was evaluated as interleukin (IL)‐6, IL‐8 and tumour necrosis factor (TNF)‐α release by enzyme‐linked immunosorbant assay (ELISA). In A549 cells no significant viability reduction and moderate membrane damage, only at the highest concentration, were detected, whereas BEAS‐2B cells showed a significant viability reduction and early membrane damage starting from 10 µg ml–1. Direct/oxidative DNA damage at 40 µg ml–1 and increased IL‐6 release at 5 µg ml–1 were found only in A549 cells after 2 h. The secretion of pro‐inflammatory cytokine IL‐6, involved in the early acute inflammatory response, and oxidative DNA damage indicate the promotion of early and transient oxidative‐inflammatory effects of tested TiO2‐NPs on human alveolar cells. The findings show a higher susceptibility of normal bronchial cells to cytotoxic effects and higher responsiveness of transformed alveolar cells to genotoxic, oxidative and early inflammatory effects induced by tested TiO2‐NPs. This different cell behaviour after TiO2‐NPs exposure suggests the use of both cell lines and multiple end‐points to elucidate NP toxicity on the respiratory system. Copyright

Evaluation of uptake, cytotoxicity and inflammatory effects in respiratory cells exposed to pristine and -OH and -COOH functionalized multi-wall carbon nanotubes.

Toxic effects were reported for pristine‐multi‐wall carbon nanotubes (p‐MWCNTs) while the role of the functionalization on MWCNT‐induced toxicity is not yet well defined. We evaluated on human alveolar (A549) epithelial cells and normal bronchial (BEAS‐2B) cells exposed to p‐MWCNTs, MWCNTs‐OH and MWCNTs‐COOH: uptake by TEM, cell viability by different assays, membrane damage by the LDH assay and cytokine release by ELISA. The aims of the present study were to: (i) confirm MWCNT cytotoxicity mechanisms hypothesized in our previous studies; (ii) identify the most reliable viability assay to screen MWCNT toxicity; and (iii) to test our model to clarify the role of functionalization on MWCNT‐induced toxicity. In A549 cells, p‐MWCNTs and MWCNTs‐OH were localized free in the cytoplasm and inside vacuoles whereas MWCNTs‐COOH were confined inside filled cytoplasmic vesicles. WST‐1 and Trypan blue assays showed in A549 cells a similar slight viability reduction for all MWCNTs whereas in BEAS‐2B cells WST1 showed a high viability reduction at the highest concentrations, particularly for MWCNTs‐COOH. The MTT assay showed a false cytotoxicity as a result of MWCNTs‐interference. Pristine and MWCNTs‐COOH induced membrane damage, particularly in BEAS‐2B cells. MWCNTs‐COOH induced interleukin‐6 (IL‐6) and IL‐8 release in A549 cells whereas p‐MWCNTs induced IL‐8 release in BEAS‐2B cells. MWCNTs intracellular localization in A549 cells confirms the toxicity mechanisms previously hypothesized, with p‐MWCNTs disrupting the membrane and vesicle‐confined MWCNTs‐COOH inducing inflammation. WST‐1 was more reliable than MTT to test MWCNT‐toxicity. BEAS‐2B cells were more susceptible then A549 cells, particularly to MWCNT‐COOH cytotoxicity. Our results confirm the toxicity of p‐MWCNTs and demonstrate, also for the two kinds of tested functionalized MWCNTs toxic effects with a different mechanism of action. Copyright

Study of cytotoxic and genotoxic effects of hydroxyl-functionalized multiwalled carbon nanotubes on human pulmonary cells

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.

Investigation on cobalt-oxide nanoparticles cyto-genotoxicity and inflammatory response in two types of respiratory cells.

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. Copyright

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.

X-Ray Induced DNA Damage and Repair in Germ Cells of PARP1 −/− Male Mice

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.

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