Abstract In order to extend the range of applications and uses of carbon nanotubes (CNT), many have been functionalised. However, due to the diversity of experimental models used in studies, it remains difficult to conclude whether functionalised CNTs are more, less or equally toxic than their native counterparts. In this study, we selected thin multi-walled CNTs (MWCNTs) with hydroxyl or carboxyl functional groups. We studied the toxicity of these MWCNTs on three lung cell lines since the main route of occupational exposure is inhalation. First, bronchial epithelial BEAS-2B cells were used to study the epithelial-mesenchymal transition (EMT). After 6 wk of culture, during which the cells were treated with MWCNTs twice a week, induction of EMT was more pronounced with pristine MWCNTs than with functionalised ones. Secondly, the expression of the cytokine IL-8 in A549 cells was assessed after 24 h of treatment. This cytokine expression was similar or lower after treatment with functionalised MWCNTs than with pristine particles. Finally, analysis of alveolar macrophages NR8383 treated for 24 h showed an increase in the percentage of binucleated cells, an effect that was more marked with pristine MWCNTs than with functionalised CNTs. This study showed that thin MWCNTs have an impact in lung cells: induction of EMT involved in lung diseases, increased expression of IL-8, which can predict lung inflammation, and impaired integrity of macrophages, which constitute the lung’s first line of defence. Although the effects are less pronounced with functionalised MWCNTs than with their pristine counterparts, they still induce adverse effects on lung cells and must be handled with caution.

Functionalisation of carbon nanotubes (CNTs), to make them more hydrophilic, is an essential step in their preparation for use in a wide range of industrial processes. The toxicological impact of these modifications has been investigated. However, due to the diversity of experimental models used in studies, no consensus has yet been reached. Here, we studied the toxicity of multi-walled CNT (MWCNT) with or without hydroxyl or carboxyl functional groups in lung cell models—chosen due to the risk of inhalation exposure for employees during handling of nanomaterials. In this study, we investigated the induction of the epithelial-mesenchymal transition (EMT) of human bronchial epithelial cells BEAS-2B following 6 weeks of treatment with MWCNTs ranging from 0.125 to 1 µg/cm2. Epithelial and mesenchymal markers were modified following MWCNT treatment, confirming the induction of the EMT. The effect was more pronounced with non-functionalised MWCNTs than with functionalised ones. In the lung carcinoma cell line A549, IL-8 expression was measured following treatment for 24 h to 0.5–10 µg/cm2 MWCNTs. An equal to or lower level was observed after treatment with functionalised MWCNTs than non-functionalised MWCNTs. Finally, the rat alveolar macrophage cell line NR8383 were exposed to 0.125–10 µg/cm2 to MWCNTs for 24 h. Non-functionalised MWCNTs led to a higher proportion of binucleation than exposure to their functionalised counterparts. Functionalised MWCNTs induce toxicity in lung cell lines to a lower extent than non-functionalised MWCNTs. Because of this toxicity, all MWCNTs should be handled with care regardless of their functionalisation.

Abstract Interpreting in vitro toxicology data with nanomaterials requires accurate determination of the dose effectively delivered to cells. For carbon nanotubes (CNTs), this remains particularly challenging due to the difficulty of quantifying inorganic carbon within complex biological matrices. Moreover, existing deposition models typically assume spherical particles, which may not adequately reflect the behaviour of fibrous CNTs. We developed an analytical method to quantify CNTs remaining in suspension, loosely associated with cells, or strongly bound/internalized. The approach combines UV-Vis-NIR spectrophotometry with chemical digestion or lyophilization, depending on the fraction analysed. The method was validated for selectivity, linearity, limits of detection and quantification, lyophilization yield, bias, and precision (within- and between-run). It was applied to ten multi-walled CNTs of various morphologies (long or short, thin or thick) and surface chemistries (hydroxyl- or carboxyl-functionalized), dispersed at four to six exposure concentrations in cell culture medium. Human bronchial epithelial cells (BEAS-2B) were exposed for 96 h prior to quantification. The method successfully estimated both delivered and cellular CNT doses, which did not always match nominal concentrations. CNT distribution among fractions varied with morphology: for short CNTs, the delivered dose closely matched the applied dose, whereas for long CNTs (functionalized or not), up to 20% remained suspended in the medium at the highest concentrations, likely due to a “pool noodle” effect that limits sedimentation. The approach also revealed that cell death contributed to the release of nanotube-containing cells into the culture medium.

In many industrial activities, workers may be exposed by inhalation to particles that are aerosolized, To predict the human health hazard of these materials, we propose to develop a co-culture model (macrophages, granulocytes, and alveolar epithelial cells) designed to be more representative of the inflammatory pulmonary response occurring in vivo. Phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells were used as macrophages, All-trans retinoic acid (ATRA)-differentiated HL60 were used as granulocytes and A549 were used as epithelial alveolar type II cells. A crystalline silica sample DQ12 was used as a prototypical particle for its capabilities to induce DNA damage, inflammatory response, and oxidative stress in epithelial cells; its polyvinylpyridine-N-oxide (PVNO)-surface modified counterpart was also used as a negative particulate control. Cells in mono-, bi- or tri-culture were exposed to DQ12 or DQ12-PVNO for 24 h. DQ12 but not DQ12-PVNO induced a significant increase in DNA damage in A549 cells. The presence of differentiated THP-1 reduced the genotoxic effects of this crystalline silica sample. The exposure of A549 to DQ12 but not DQ12-PVNO induced a significant change in interleukin-8 (IL-8) protein levels which was exacerbated when differentiated THP-1, and HL-60, were added. In addition, while no production of TNFα was detected in the A549 monoculture, elevated levels of this cytokine were observed in the co-culture systems. This work shows that a cell culture model that takes into consideration the complexity of the pulmonary inflammatory response might be more dependable to study the toxicological properties of particles than "simple" monoculture models.

Interpreting in vitro toxicology results with nanomaterials requires precise knowledge of the amount of materials actually delivered to cells. This dose is particularly challenging to accurately measure or estimate for carbon nanotubes (CNTs) due to difficulties quantifying inorganic carbon within an organic matrix. In addition, existing deposition models often assume spherical particles, and may thus not adequately reflect the behaviour of fibrous CNTs. We present a method to quantify the amounts of CNTs remaining in suspension, loosely bound to cells, or strongly bound/internalized. The approach uses UV-Vis-NIR spectroscopy, preceded by chemical digestion or lyophilization as necessary depending on the fractions investigated. The method was implemented and validated with three CNT types—MWNT-7 (long and thick), NM-403 (short and thin), and A2-6-COOH (short, thin, and functionalized with carboxyl groups)—dispersed at six exposure concentrations in cell culture medium. Human bronchial epithelial cells (BEAS-2B) were exposed to CNTs for 96 h. The analytical method was validated for selectivity, linearity, detection and quantification limits, lyophilization yield, bias, and precision (within- and between-run). This method successfully estimated delivered and cellular amounts, which did not always match the nominal exposure concentration. CNT distribution across fractions varies depending on the type of CNT. For functionalized nanotubes, the amount delivered equals the amount applied, whereas for non-functionalized nanotubes, a significant proportion (up to 20–30

Multi-walled carbon nanotubes (MWCNTs) are promising nanomaterials because of their various physicochemical properties. However, their production and handling can lead to their aerosolization. Then, they can be inhaled, reach the deep lung and induce health effects. The respiratory system is composed of different cell types, including epithelial cells and alveolar macrophages, which are on the front line following inhalation of MWCNTs. The aim of this study was to evaluate the influence of their physicochemical properties on their toxic effects. To address this objective, we use two in vitro models, NR8383 rat alveolar macrophages and BEAS-2B human bronchial epithelial cells. BEAS-2B cells are treated with low concentrations of nanomaterials for 6 weeks in order to evaluate a potential epithelial-mesenchymal transition (EMT). NR8383 macrophages are treated for up to 24 hours in order to study their polarization and cell signalling. The first results show that the treatment of BEAS-2B cells induced a modification of their phenotype in a differential time-scale and concentration depending on the MWCNT. This modification is accompanied by a change in the expression of genes specific to EMT. In addition, the treatment of NR8383 induced a modification of cell signalling, which can be observed in particular through the expression of transmembrane receptors and that of pro-inflammatory cytokines. The differential effects observed following treatment of the cells with the different types of MWCNTs can be considered regarding their physical properties, such as their length, diameter and specific surface area, as well as their chemical properties, in other terms their functionalization.

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.

Toxicity induced by multi-walled carbon nanotubes and their metal impurities.

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.

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.

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.

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. Environ. Mol. Mutagen. 56:228–244, 2015. © 2014 Wiley Periodicals, Inc.

The nature of occupational risks and hazards in industries that produce or use synthetic amorphous silica (SAS) nanoparticles is still under discussion. Manufactured SAS occur in amorphous form and can be divided into two main types according to the production process, namely, pyrogenic silica (powder) and precipitated silica (powder, gel or colloid). The physical and chemical properties of SAS may vary in terms of particle size, surface area, agglomeration state or purity, and differences in their toxicity potential might therefore be expected. The aim of this study was to compare the cytotoxicity and genotoxicity of representative manufactured SAS samples in Chinese hamster lung fibroblasts (V79 cells). Five samples from industrial SAS producers were evaluated, that is, two pyrogenic SAS powders (with primary particle sizes of 20 nm and 25/70 nm), one precipitated SAS powder (20 nm) and two precipitated SAS colloids (15 and 40/80 nm). V79 cell cultures were treated with different concentrations of SAS pre-dispersed in bovine serum albumin –water medium. Pyr (pyrogenic) 20, Pre (precipitated) 20 and Col (colloid) 15 significantly decreased the cell viability after 24 h of exposure, whilst Pyr 25/70 and Col 40/80 had negligible effects. The cytotoxicity of Pyr 20, Pre 20 and Col 15 was revealed by the induction of apoptosis, and Pyr 20 and Col 15 also produced DNA damage. However, none of the SAS samples generated intracellular reactive oxidative species, micronuclei or genomic mutations in V79 cells after 24 h of exposure. Overall, the results of this study show that pyrogenic, precipitated and colloidal manufactured SAS of around 20 nm primary particle size can produce significant cytotoxic and genotoxic effects in V79 cells. In contrast, the coarser-grained pyrogenic and colloid SAS (approximately 50 nm) yielded negligible toxicity, despite having been manufactured by same processes as their finer-grained equivalents. To explain these differences, the influence of particle agglomeration and oxidative species formation is discussed.

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.

Carbon nanotubes (CNTs) belong to a specific class of nanomaterials with unique properties. Because of their anticipated use in a wide range of industrial applications, their toxicity is of increasing concern. In order to determine whether specific physicochemical characteristics of CNTs are responsible for their toxicological effects, we investigated the cytotoxic and genotoxic effects of eight CNTs representative of each of the commonly encountered classes: single- SW-, double- DW-, and multiwalled (MW) CNTs, purified and raw. In addition, because most previous studies of CNT toxicity were conducted on immortalized cell lines, we decided to compare results obtained from V79 cells, an established cell line, with results from SHE (Syrian hamster embryo) cells, an easy-to-handle normal cell model. After 24 hours of treatment, MWCNTs were generally found to be more cytotoxic than SW- or DWCNTs. MWCNTs also provoked more genotoxic effects. No correlation could be found between CNT genotoxicity and metal impurities, length, surface area, or induction of cellular oxidative stress, but genotoxicity was seen to increase with CNT width. The toxicity observed for some CNTs leads us to suggest that they might also act by interfering with the cell cycle, but no significant differences were observed between normal and immortalized cells.