Julia Catalán

Genotoxicity of nanomaterials: DNA damage and micronuclei induced by carbon nanotubes and graphite nanofibres in human bronchial epithelial cells in vitro

Despite the increasing industrial use of different nanomaterials, data on their genotoxicity are scant. In the present study, we examined the potential genotoxic effects of carbon nanotubes (CNTs; >50% single-walled, approximately 40% other CNTs; 1.1 nm x 0.5-100 microm; Sigma-Aldrich) and graphite nanofibres (GNFs; 95%; outer diameter 80-200 nm, inner diameter 30-50 nm, length 5-20 microm; Sigma-Aldrich) in vitro. Genotoxicity was assessed by the single cell gel electrophoresis (comet) assay and the micronucleus assay (cytokinesis-block method) in human bronchial epithelial BEAS 2B cells cultured for 24h, 48h, or 72h with various doses (1-100 microg/cm(2), corresponding to 3.8-380 microg/ml) of the carbon nanomaterials. In the comet assay, CNTs induced a dose-dependent increase in DNA damage at all treatment times, with a statistically significant effect starting at the lowest dose tested. GNFs increased DNA damage at all doses in the 24-h treatment, at two doses (40 and 100 microg/cm(2)) in the 48-h treatment (dose-dependent effect) and at four doses (lowest 10 microg/cm(2)) in the 72-h treatment. In the micronucleus assay, no increase in micronucleated cells was observed with either of the nanomaterials after the 24-h treatment or with CNTs after the 72-h treatment. The 48-h treatment caused a significant increase in micronucleated cells at three doses (lowest 10 microg/cm(2)) of CNTs and at two doses (5 and 10 microg/cm(2)) of GNFs. The 72-h treatment with GNFs increased micronucleated cells at four doses (lowest 10 microg/cm(2)). No dose-dependent effects were seen in the micronucleus assay. The presence of carbon nanomaterial on the microscopic slides disturbed the micronucleus analysis and made it impossible at levels higher than 20 microg/cm(2) of GNFs in the 24-h and 48-h treatments. In conclusion, our results suggest that both CNTs and GNFs are genotoxic in human bronchial epithelial BEAS 2B cells in vitro. This activity may be due to the fibrous nature of these carbon nanomaterials with a possible contribution by catalyst metals present in the materials-Co and Mo in CNTs (<5wt.%) and Fe (<3wt.%) in GNFs.

Genotoxic effects of nanosized and fine TiO2.

The in-vitro genotoxicity of nanosized TiO2 rutile and anatase was assessed in comparison with fine TiO2 rutile in human bronchial epithelial BEAS 2B cells using the single-cell gel electrophoresis (comet) assay and the cytokinesis-block micronucleus test. BEAS 2B cells were exposed to eight doses (1—100 μg/cm2) of titanium(IV) oxide nanosized rutile (>95%, <5% amorphous SiO2 coating; 10 × 40 nm), nanosized anatase (99.7%; <25 nm), or fine rutile (99.9%; <5 μm) for 24, 48, and 72 h. Fine rutile reduced cell viability at lower doses than nanosized anatase, which was more cytotoxic than nanosized rutile. In the comet assay, nanosized anatase and fine rutile induced DNA damage at several doses with all treatment times. Dose-dependent effects were seen after the 48- and 72-h treatments with nanosized anatase and after the 24-, 48- (in one out of two experiments), and 72-h treatments (one experiment) with fine rutile. The lowest doses inducing DNA damage were 1 μg/cm2 for fine rutile and 10 μg/cm 2 for nanosized anatase. Nanosized rutile showed a significant induction in DNA damage only at 80 μg/cm2 in the 24-h treatment and at 80 and 100 μg/ cm2 in the 72-h treatment (with a dose-dependent effect). Only nanosized anatase could elevate the frequency of micronucleated BEAS 2B cells, producing a significant increase at 10 and 60 μg/cm 2 after the 72-h treatment (no dose-dependency). At increasing doses of all the particles, MN analysis became difficult due to the presence of TiO2 on the microscopic slides. In conclusion, our studies in human bronchial epithelial BEAS 2B cells showed that uncoated nanosized anatase TiO2 and fine rutile TiO2 are more efficient than SiO 2-coated nanosized rutile TiO2 in inducing DNA damage, whereas only nanosized anatase is able to slightly induce micronuclei.

Age-associated micronuclei containing centromeres and the X chromosome in lymphocytes of women.

Chronological aging of women is clearly associated with an increase in both X-chromosome loss and micronuclei formation in peripheral lymphocytes. It has been suggested that micronucleus formation is an important mechanism of chromosome loss. In the present study, fluorescence in situ hybridization was used to study micronuclei content in two age groups (women below 30 and above 50 years old). A probe for centromeric alphoid consensus sequences (SO-αAllCen) and a cloned X-specific centromeric probe (pXBR) were separately used to detect the presence of any chromosomes and the X chromosome, respectively. The presence of centromere-positive micronuclei was significantly higher among the older donors (51.5%) than among the younger donors (34.3%). The X chromosome was highly overrepresented in the micronuclei, the older women showing a higher proportion of X-positive micronuclei (24.0%) than the younger women (14.0%). Assuming that the rest of the centromere-positive micronuclei contained autosomes, a significant age-dependent difference was also noted for micronuclei harboring autosomes (27.5 % among the older women and 20.3 % among the younger women). These findings suggest that both the X chromosome and autosomes are responsible for the age-dependent increase of micronuclei in women’s peripheral lymphocytes.

Genotoxicity of inhaled nanosized TiO2 in mice

In vitro studies have suggested that nanosized titanium dioxide (TiO(2)) is genotoxic. The significance of these findings with respect to in vivo effects is unclear, as few in vivo studies on TiO(2) genotoxicity exist. Recently, nanosized TiO(2) administered in drinking water was reported to increase, e.g., micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs) and DNA damage in leukocytes. Induction of micronuclei in mouse PCEs was earlier also described for pigment-grade TiO(2) administered intraperitoneally. The apparent systemic genotoxic effects have been suggested to reflect secondary genotoxicity of TiO(2) due to inflammation. However, a recent study suggested that induction of DNA damage in mouse bronchoalveolar lavage (BAL) cells after intratracheal instillation of nanosized or fine TiO(2) is independent of inflammation. We examined here, if inhalation of freshly generated nanosized TiO(2) (74% anatase, 26% brookite; 5 days, 4 h/day) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5 mg/m(3) could induce genotoxic effects in C57BL/6J mice locally in the lungs or systematically in peripheral PCEs. DNA damage was assessed by the comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. MN were analyzed by acridine orange staining in blood PCEs collected 48 h after the last exposure. A dose-dependent deposition of Ti in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to nanosized TiO(2) anatase. Our inhalation exposure resulted in much lower systemic TiO(2) doses than the previous oral and intraperitoneal treatments, and lung epithelial cells probably received considerably less TiO(2) than BAL cells in the earlier intratracheal study.

Induction of micronuclei in peripheral blood and bone marrow erythrocytes of rats and mice exposed to 1,3-butadiene by inhalation

Female CB6F1 mice and male Wistar rats were exposed to different concentrations of 1,3-butadiene (1,3-BD) by inhalation. Micronucleus tests using both peripheral blood erythrocytes and femoral marrow cells of these animals were performed. Cells were stained either using conventional acridine orange (AO) staining or supravitally using AO-coated slides. Dose-dependent increases in the frequency of micronuclei (MN) were observed both in blood and in bone marrow cells in mice. 1,3-BD did not, however, increase the frequency of MN in either blood or bone marrow cells of rats at any of the tested concentrations.

Induction of chromosomal aberrations by carbon nanotubes and titanium dioxide nanoparticles in human lymphocytes in vitro

Abstract We examined if three commercially available nanomaterials – short singlewall carbon nanotubes (SWCNTs), short multiwall carbon nanotubes (MWCNTs) and nanosized titanium dioxide anatase (TiO2; primary particle size <25 nm) – can induce structural chromosomal aberrations (CAs) in cultures of isolated human lymphocytes. To find a suitable sampling time, the cells were treated with 6.25–300 μg/ml of the nanomaterials for 24, 48 and 72 h. The 48-h treatment was the most effective, inducing a dose-dependent increase in chromosome-type CAs (all materials) and chromatid-type CAs (SWCNTs and TiO2 anatase). The 72-h treatment yielded a positive result with SWCNTs. None of the treatments significantly affected cell count or the mitotic index. Our results suggest that with nanomaterials a continuous treatment for about two cell cycles is needed for CA induction, possibly reflecting access of nanomaterials to the nucleus during the first mitosis or delayed secondary genotoxic effect associated with the inflammatory process.

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Abstract Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24u2009h after a single pharyngeal aspiration of MWCNT-S (1–200u2009μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8u2009mg/m3) for 4 d, 4u2009h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200u2009μg/mouse) and in MNPCEs after inhalation exposure (17.5u2009mg/m3). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10u2009μg/cm2), while MWCNT-T increased strand breakage only at 200u2009μg/cm2. Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.

Influence of culture time on the frequency and contents of human lymphocyte micronuclei with and without cytochalasin B.

The effects of culture time (52, 64 and 76 h) and cytochalasin B (Cyt-B, 3 micrograms/ml) on the frequency of micronuclei (MN) harbouring whole chromosomes and acentric fragments was investigated in purified lymphocyte cultures of five nonsmoking male donors aged 41-50 years. Centromere-positive (C+) MN were identified by fluorescence in situ hybridization, using an alphoid DNA oligomer probe (SO-alpha AllCen) hybridizing to all human centromeres. For each culture time, 2000 cells and 60 MN were scored per donor, both with and without Cyt-B, making a total of 60,000 cells and 1800 MN. The frequency of MN and the proportion of C+ MN were higher at 64 h and 76 h than at 52 h, irrespective of Cyt-B. The culture time-dependent increase in MN frequency was mainly due to C+ MN which were about 1.5-times more frequent at 64 h and 72 h than at 52 h. The frequencies of C+ MN, expressed per 1000 nuclei, were similar with and without Cyt-B, although the prevalence of C+ MN was consistently about 10 percent units higher in the former type of culture. This effect was due to a decreased frequency of centromere-negative (C-) MN in the binucleate cells, possibly reflecting, e.g. increased inclusion of acentric chromosomal fragments within the main nuclei of such cells, enhanced expulsion of C- MN, or selection against binucleate cells carrying such MN. In conclusion, the present findings indicate that MN harbouring whole chromosomes become more frequent at long culture times with and without Cyt-B and that Cyt-B-induced binucleate cells show a reduced frequency of MN containing acentric fragments. Due to the high background of whole-chromosome-containing MN (mean C+ MN proportions ranged from 42.3% to 62.7%), it may be recommended that centromeric fluorescence in situ hybridization is routinely applied when lymphocyte MN are used as a biomarker of human exposure to clastogens.

Extensive temporal transcriptome and microRNA analyses identify molecular mechanisms underlying mitochondrial dysfunction induced by multi-walled carbon nanotubes in human lung cells

Abstract Understanding toxicity pathways of engineered nanomaterials (ENM) has recently been brought forward as a key step in twenty-first century ENM risk assessment. Molecular mechanisms linked to phenotypic end points is a step towards the development of toxicity tests based on key events, which may allow for grouping of ENM according to their modes of action. This study identified molecular mechanisms underlying mitochondrial dysfunction in human bronchial epithelial BEAS 2B cells following exposure to one of the most studied multi-walled carbon nanotubes (Mitsui MWCNT-7). Asbestos was used as a positive control and a non-carcinogenic glass wool material was included as a negative fibre control. Decreased mitochondrial membrane potential (MMP↓) was observed for MWCNTs at a biologically relevant dose (0.25u2009μg/cm2) and for asbestos at 2u2009μg/cm2, but not for glass wool. Extensive temporal transcriptomic and microRNA expression analyses identified a 330-gene signature (including 26 genes with known mitochondrial function) related to MWCNT- and asbestos-induced MMP↓. Forty-nine of the MMP↓-associated genes showed highly similar expression patterns over time (six time points) and the majority was found to be regulated by two transcription factors strongly involved in mitochondrial homeostasis, APP and NRF1. In addition, four miRNAs were correlated with MMP↓ and one of them, miR-1275, was found to negatively correlate with a large part of the MMP↓-associated genes. Cellular processes such as gluconeogenesis, mitochondrial LC-fatty acid β-oxidation and spindle microtubule function were enriched among the MMP↓-associated genes and miRNAs. These results are expected to be useful in the identification of key events in ENM-related toxicity pathways for the development of molecular screening techniques.

Nanomaterials and Human Health

In assessing health hazards of engineered nanomaterials (ENM), the challenge is which of the various materials’ features need to be considered in adjusting the correct dose for the studies, mass, number concentration or surface area of the materials. The small size of the ENM is especially important for their absorption, distribution, metabolism and excretion because it determines their ability to penetrate biological barriers and reach different organs and cells. Other metrics such as surface area of morphology, for example, aspect ratio in the case of carbon nanotubes (CNT), may be of high importance. The most important target organ of ENM is the lung, where especially CNT may evoke strong pulmonary inflammation and granuloma formation at low doses. Metal oxides such are titanium dioxide can also provoke pulmonary inflammation. Other important targets are the central nervous system, where metal oxide nanoparticles may induce oxidative damage in brain cells at high doses, and the cardiovascular system, where they may disturb the microcirculation. A striking feature of many ENM including CNT and metal oxides is their ability in some cases to induce genotoxic effects and also occasionally increase the likelihood of cancer, especially subsequent to exposure to CNT in sensitive experimental systems. The main challenge of toxicity studies exploring ENM toxicity is so far the small amount of knowledge, and hence more systematic and mechanistic studies of ENM are urgently needed.