Continuous Long-Term Exposure to Low Concentrations of MWCNTs Induces an Epithelial-Mesenchymal Transition in BEAS-2B Cells.

In the field of nanotechnology, the use of multi-walled carbon nanotubes (MWCNTs) is growing. Pulmonary exposure during their production, use, and handling is raising concerns about their potential adverse health effects. The purpose of this study is to assess how the physical characteristics of MWCNTs, such as diameter and/or length, can play a role in cellular toxicity. Our experimental design is based on the treatment of human bronchial epithelial cells (BEAS-2B) for six weeks with low concentrations (0.125-1 µg/cm2) of MWCNTs having opposite characteristics: NM-403 and Mitsui-7. Following treatment with both MWCNTs, we observed an increase in mitotic abnormalities and micronucleus-positive cells. The cytotoxic effect was delayed in cells treated with NM-403 compared to Mitsui-7. After 4-6 weeks of treatment, a clear cellular morphological change from epithelial to fibroblast-like phenotype was noted, together with a change in the cell population composition. BEAS-2B cells underwent a conversion from the epithelial to mesenchymal state as we observed a decrease in the epithelial marker E-cadherin and an increased expression of mesenchymal markers N-cadherin, Vimentin, and Fibronectin. After four weeks of recovery, we showed that the induced epithelial-mesenchymal transition is reversible, and that the degree of reversibility depends on the MWCNT.

Pulmonary toxicity of two different multi-walled carbon nanotubes in rat: Comparison between intratracheal instillation and inhalation exposure.

Multi-walled carbon nanotubes (MWCNTs), which vary in length, diameter, functionalization and specific surface area, are used in diverse industrial processes. Since these nanomaterials have a high aspect ratio and are biopersistant in the lung, there is a need for a rapid identification of their potential health hazard. We assessed in Sprague-Dawley rats the pulmonary toxicity of two pristine MWCNTs (the "long and thick" NM-401 and the "short and thin" NM-403) following either intratracheal instillation or 4-week inhalation in order to gain insights into the predictability and intercomparability of the two methods. The deposited doses following inhalation were lower than the instilled doses. Both types of carbon nanotube induced pulmonary neutrophil influx using both exposure methods. This influx correlated with deposited surface area across MWCNT types and means of exposure at two different time points, 1-3 days and 28-30 days post-exposure. Increased levels of DNA damage were observed across doses and time points for both exposure methods, but no dose-response relationship was observed. Intratracheal instillation of NM-401 induced fibrosis at the highest dose while lower lung deposited doses obtained by inhalation did not induce such lung pathology. No fibrosis was observed following NM-403 exposure. When the deposited dose was taken into account, sub-acute inhalation and a single instillation of NM-401 and NM-403 produced very similar inflammation and DNA damage responses. Our data suggest that the dose-dependent inflammatory responses observed after intratracheal instillation and inhalation of MWCNTs are similar and were predicted by the deposited surface area.

Is ventilatory therapy combined with exercise training effective in patients with heart failure and sleep-disordered breathing? Results of a randomized trial during a cardiac rehabilitation programme (SATELIT-HF).

BACKGROUND: Sleep-related disordered breathing is common in patients with chronic heart failure. AIM: To assess the efficacy of short-term nocturnal ventilatory therapy combined with exercise training (V+ET) compared with exercise training alone (ET) in patients with chronic heart failure with sleep-disordered breathing. METHODS: Patients in New York Heart Association functional class II-IIIb, with an apnoea-hypopnoea index (AHI)>15/h, and enrolled in a cardiac rehabilitation programme, were centrally randomized to V+ET or ET. Subjects were classified as having obstructive sleep apnoea (OSA) (n=49) or central sleep apnoea (CSA)/mixed (n=69). The primary outcome was the change in the 10-second average oxygen consumption at maximum exercise (VO2peak) at the end of the cardiac rehabilitation programme. RESULTS: Fifty-eight patients were randomized to V+ET and 60 patients to ET. The median increase in VO2peak was 15% [interquartile range 6-36%] in the V+ET group and 16% [0-31%] in the ET group (P=0.34). AHI decreased in both groups, but significantly more in the V+ET group (P=0.006). The decrease in the ventilatory efficiency (VE/VCO2) slope was not statistically different between the two-randomization groups (P=0.10). In subjects with CSA, the VE/VCO2 slope decreased significantly more in the V+ET group (P=0.03), while there was no difference between the two-randomization groups in subjects with OSA (P=0.75). Six cardiovascular events occurred in patients with OSA (all randomized to the ET group); in subjects with CSA, two events occurred in the V+ET group and three in the ET group. CONCLUSIONS: Short-term nocturnal ventilation combined with exercise training does not increase the exercise capacity of patients with chronic heart failure.

In vitro cell transformation induced by synthetic amorphous silica nanoparticles.

Synthetic amorphous silica nanoparticles (SAS) are among the most widely produced and used nanomaterials, but little is known about their carcinogenic potential. This study aims to evaluate the ability of four different SAS, two precipitated, NM-200 and NM-201, and two pyrogenic, NM-202 and NM-203, to induce the transformation process. For this, we used the recently developed in vitro Bhas 42 cell transformation assay (CTA). The genome of the transgenic Bhas 42 cells contains several copies of the v-Ha-ras gene, making them particularly sensitive to tumor-promoter agents. The Bhas 42 CTA, which includes an initiation assay and a promotion assay, was validated in our laboratory using known soluble carcinogenic substances. Its suitability for particle-type substances was verified by using quartz Min-U-Sil 5 (Min-U-Sil) and diatomaceous earth (DE) microparticles. As expected given their known transforming properties, Min-U-Sil responded positively in the Bhas 42 CTA and DE responded negatively. Transformation assays were performed with SAS at concentrations ranging from 2µg/cm2 to 80µg/cm2. Results showed that all SAS have the capacity to induce transformed foci, interestingly only in the promotion assay, suggesting a mode of action similar to tumor-promoter substances. NM-203 exhibited transforming activity at a lower concentration than the other SAS. In conclusion, this study showed for the first time the transforming potential of different SAS, which act as tumor-promoter substances in the Bhas 42 model of cell transformation.

Epigenetic changes in the early stage of silica-induced cell transformation.

The increasing use of nanomaterials in numerous domains has led to growing concern about their potential toxicological properties, and the potential risk to human health posed by silica nanoparticles remains under debate. Recent studies proposed that these particles could alter gene expression through the modulation of epigenetic marks, and the possible relationship between particle exposure and these mechanisms could represent a critical factor in carcinogenicity. In this study, using the Bhas 42 cell model, we compare the effects of exposure to two transforming particles, a pyrogenic amorphous silica nanoparticle NM-203 to those of the crystalline silica particle Min-U-Sil® 5. Short-term treatment by Min-U-Sil® 5 decreased global DNA methylation and increased the expression of the two de novo DNMTs, DNMT3a and DNMT3b. NM-203 treatment affected neither the expression of these enzymes nor DNA methylation. Moreover, modified global histone H4 acetylation status and HDAC protein levels were observed only in the Min-U-Sil® 5-treated cells. Finally, both types of particle treatment induced strong c-Myc expression in the early stage of cell transformation and this correlated with enrichment in RNA polymerase II as well as histone active marks on its promoter. Lastly, almost all parameters that were modulated in the early stage were restored in transformed cells suggesting their involvement mainly in the first steps of cell transformation.

In vitro comet and micronucleus assays do not predict morphological transforming effects of silica particles in Syrian Hamster Embryo cells.

Crystalline silica particles and asbestos have both been classified as carcinogenic by the International Agency for Research on Cancer (IARC). However, because of the limited data available, amorphous silica was not classifiable. In vitro, the carcinogenic potential of natural crystalline and amorphous silica particles has been revealed by the Syrian Hamster Embryo (SHE) cell transformation assay. On the other hand, the genotoxic potential of those substances has not been investigated in SHE cells. And yet, genotoxicity assays are commonly used for hazard evaluation and they are often used as in vitro assays of reference to predict a possible carcinogenic potential. The main objective of this study was to compare the genotoxic potential and the carcinogenic potential of different crystalline and amorphous silica particles in SHE cells. Three silica samples of different crystallinity were used: natural amorphous silica, partially crystallized silica and quartz silica particles. Their genotoxicity were tested through the in vitro micronucleus assay and the comet assay in SHE, and their carcinogenic potential through the SHE transformation assay. In addition, silica samples were also tested with the same genotoxicity assays in V79 hamster-lung cells, a common in vitro model for particle exposure. Results obtained in the micronucleus and the comet assays show that none of the silica was capable of inducing genotoxic effects in SHE cells and only the amorphous silica induced genotoxic effects in V79 cells. However in the SHE cell transformation assays, the partially crystallized and quartz silica were able to induce morphological cell transformation. Together, these data suggest that, in vitro, the short-term genotoxic assays alone are not sufficient to predict the hazard and the carcinogenic potential of this type of particles; SHE transformation assay appears a more reliable tool for this purpose and should be included in the "in vitro battery assays" for hazard assessment.

Genotoxicity of synthetic amorphous silica nanoparticles in rats following short-term exposure. Part 2: intratracheal instillation and intravenous injection.

Synthetic amorphous silica nanomaterials (SAS) are extensively used in food and tire industries. In many industrial processes, SAS may become aerosolized and lead to occupational exposure of workers through inhalation in particular. However, little is known about the in vivo genotoxicity of these particulate materials. To gain insight into the toxicological properties of four SAS (NM-200, NM-201, NM-202, and NM-203), rats are treated with three consecutive intratracheal instillations of 3, 6, or 12 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection (cumulative doses of 9, 18, and 36 mg/kg). Deoxyribonucleic acid (DNA) damage was assessed using erythrocyte micronucleus test and the standard and Fpg-modified comet assays on cells from bronchoalveolar lavage fluid (BALF), lung, blood, spleen, liver, bone marrow, and kidney. Although all of the SAS caused increased dose-dependent changes in lung inflammation as demonstrated by BALF neutrophilia, they did not induce any significant DNA damage. As the amount of SAS reaching the blood stream and subsequently the internal organs is probably to be low following intratracheal instillation, an additional experiment was performed with NM-203. Rats received three consecutive intravenous injections of 5, 10, or 20 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection. Despite the hepatotoxicity, thrombocytopenia, and even animal death induced by this nanomaterial, no significant increase in DNA damage or micronucleus frequency was observed in SAS-exposed animals. It was concluded that under experimental conditions, SAS induced obvious toxic effects but did cause any genotoxicity following intratracheal instillation and intravenous injection.

Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: a 4-week inhalation study.

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.

Cytotoxicity and genotoxicity of nanosized and microsized titanium dioxide and iron oxide particles in Syrian hamster embryo cells.

Potential differences in the toxicological properties of nanosized and non-nanosized particles have been notably pointed out for titanium dioxide (TiO(2)) particles, which are currently widely produced and used in many industrial areas. Nanoparticles of the iron oxides magnetite (Fe(3)O(4)) and hematite (Fe(2)O(3)) also have many industrial applications but their toxicological properties are less documented than those of TiO(2). In the present study, the in vitro cytotoxicity and genotoxicity of commercially available nanosized and microsized anatase TiO(2), rutile TiO(2), Fe(3)O(4), and Fe(2)O(3) particles were compared in Syrian hamster embryo (SHE) cells. Samples were characterized for chemical composition, primary particle size, crystal phase, shape, and specific surface area. In acellular assays, TiO(2) and iron oxide particles were able to generate reactive oxygen species (ROS). At the same mass dose, all nanoparticles produced higher levels of ROS than their microsized counterparts. Measurement of particle size in the SHE culture medium showed that primary nanoparticles and microparticles are present in the form of micrometric agglomerates of highly poly-dispersed size. Uptake of primary particles and agglomerates by SHE exposed for 24 h was observed for all samples. TiO(2) samples were found to be more cytotoxic than iron oxide samples. Concerning primary size effects, anatase TiO(2), rutile TiO(2), and Fe(2)O(3) nanoparticles induced higher cytotoxicity than their microsized counterparts after 72 h of exposure. Over this treatment time, anatase TiO(2) and Fe(2)O(3) nanoparticles also produced more intracellular ROS compared to the microsized particles. However, similar levels of DNA damage were observed in the comet assay after 24 h of exposure to anatase nanoparticles and microparticles. Rutile microparticles were found to induce more DNA damage than the nanosized particles. However, no significant increase in DNA damage was detected from nanosized and microsized iron oxides. None of the samples tested showed significant induction of micronuclei formation after 24 h of exposure. In agreement with previous size-comparison studies, we suggest that in vitro cytotoxicity and genotoxicity induced by metal oxide nanoparticles are not always higher than those induced by their bulk counterparts.

In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts.

Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres.

Surface reactivity, cytotoxic, and morphological transforming effects of diatomaceous Earth products in Syrian hamster embryo cells.

In order to evaluate the effect of thermal treatments on the surface reactivity and carcinogenic potential of diatomaceous earth (DE) products, the physicochemical features of some specimens--derived by heating the same original material--were compared with their cytotoxic and transforming potency. The samples were an untreated DE (amorphous) progressively heated in the laboratory at 900 degrees C (DE 900) and 1200 degrees C (DE 1200) and a commercial product manufactured from the same DE (Chd) from which the finer fraction (< 10-microm diameter) was separated (Chd-F). Quartz (Min-U-Sil 5) and a vitreous silica (amorphous) smoothed up with hydrofluoric acid and were used as positive and negative controls, respectively. All samples were analyzed for their degree of crystallization, for their ability to release free radicals and reactive oxygen species, and for their cytotoxic and transforming potencies in Syrian hamster embryo (SHE) cells. X-ray diffractometry showed that DE 900, like DE, was still amorphous, whereas DE 1200 as well as the commercial product (Chd) were partially crystallized into cristobalite. The ability of the dust to release hydroxyl (*OH) radicals in the presence of hydrogen peroxide, as revealed by the spin-trapping technique, was as follows: Chd-F, DE 1200 > Chd > DE 900 > DE, suggesting that on heating, the surface acquires a higher potential for free radical release. Most of the silica samples generated COO* radicals from the formate ion, following homolytic rupture of the carbon-hydrogen bond, in the presence of ascorbic acid. A concentration-dependent decrease in cell proliferation and colony-forming efficiency was observed in SHE cultures treated with Chd-F, Chd, and DE. Heating abolished DE cytotoxicity but conferred a transforming ability to thermal treated particles. DE was the only sample that did not induce morphological transformation of cells. According to their transformation capacity, the samples were classified as follows: Chd-F > Chd, DE 1200 > DE 900 >> DE. Taken together, the reported results suggest that (1) the transforming potential of a biogenic amorphous silica is related to the thermal treatment that transforms the original structure in cristobalite and generates surface active sites; (2) the reactivity of samples in releasing *OH radicals correlates to their transforming ability; (3) the finer fraction of the commercial product is significantly more toxic and transforming than the coarse dust; and (4) opposite to silica dusts of mineral origin, which loose both cytotoxicity and transforming ability upon heating, heated diatomite acquires a cell-transforming potency. DE products should be thus considered a set apart of silica-based potentially toxic materials.