Assessing the relevance of in vitro studies in nanotoxicology by examining correlations between in vitro and in vivo data (original) (raw)
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Toxicity of TiO2 Nanoparticles: Validation of Alternative Models
International Journal of Molecular Sciences
There are many studies concerning titanium dioxide (TiO2) nanoparticles (NP) toxicity. Nevertheless, there are few publications comparing in vitro and in vivo exposure, and even less comparing air–liquid interface exposure (ALI) with other in vitro and in vivo exposures. The identification and validation of common markers under different exposure conditions are relevant for the development of smart and quick nanotoxicity tests. In this work, cell viability was assessed in vitro by WST-1 and LDH assays after the exposure of NR8383 cells to TiO2 NP sample. To evaluate in vitro gene expression profile, NR8383 cells were exposed to TiO2 NP during 4 h at 3 cm2 of TiO2 NP/cm2 of cells or 19 μg/mL, in two settings—submerged cultures and ALI. For the in vivo study, Fischer 344 rats were exposed by inhalation to a nanostructured aerosol at a concentration of 10 mg/m3, 6 h/day, 5 days/week for 4 weeks. This was followed immediately by gene expression analysis. The results showed a low cytotox...
Cellular and Molecular Biology, 2020
nvestigations on adverse biological effects of nanoparticles (NP) are performed usually either in vivo on rodents or in vitro under submerged conditions where NP are in suspension into cell culture media. However, sedimentation of NP in vitro is a continuous process and to assess the exact deposited cellular dose remains difficult, as the cellular internal dose is a function of time. Moreover, the cellular responses to NP under submerged culture conditions or by exposing rodents by nose-only to NP aerosols might differ from those observed at physiological settings at the air-liquid interface (ALI). Rat alveolar NR8383 macrophages were exposed to aerosols at the air-liquid interface. We studied TiO2 NM105, a mixture of anatase and rutile. NR8383 cells were exposed to a single dose of 3.0 cm2/cm2 of TiO2 aerosol. Following RNA extraction, transcriptome allowing full coverage of the rat genome was performed, and differentially expressed genes were retrieved. Their products were analyze...
Evaluating the Use of TiO2 Nanoparticles for Toxicity Testing in Pulmonary A549 Cells
International Journal of Nanomedicine
Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO 2 P25), are among the most produced nanomaterials worldwide. The broad use of TiO 2 P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO 2 P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO 2 P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO 2 P25 for biological testing in A549 cells and to evaluate their biological effects. Methods: We determined the size and crystallinity of TiO 2 P25. We used four techniques for TiO 2 P25 dispersion. We estimated the colloid stability of TiO 2 P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO 2 P25 (0-100 µg.mL −1) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. Results: We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO 2 P25. Under these conditions, TiO 2 P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO 2 P25 in A549 cells were met, similar results on the biological effects of TiO 2 P25 were obtained in two independent cell laboratories. Conclusion: We optimized the experimental conditions of TiO 2 P25 preparation for toxicity testing in A549 cells. The results presented here on TiO 2 P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO 2 P25 as a reference material.
Asian Pacific Journal of Cancer Prevention, 2014
Two types of nanosized titanium dioxide, anatase (anTiO 2) and rutile (rnTiO 2), are widely used in industry, commercial products and biosystems. TiO 2 has been evaluated as a Group 2B carcinogen. Previous reports indicated that anTiO 2 is less toxic than rnTiO 2 , however, under ultraviolet irradiation anTiO 2 is more toxic than rnTiO 2 in vitro because of differences in their crystal structures. In the present study, we compared the in vivo and in vitro toxic effects induced by anTiO 2 and rnTiO 2. Female SD rats were treated with 500 mg/ml of anTiO 2 or rnTiO 2 suspensions by intra-pulmonary spraying 8 times over a two week period. In the lung, treatment with anTiO 2 or rnTiO 2 increased alveolar macrophage numbers and levels of 8-hydroxydeoxyguanosine (8-OHdG); these increases tended to be lower in the anTiO 2 treated group compared to the rnTiO 2 treated group. Expression of MIP1a mRNA and protein in lung tissues treated with anTiO 2 and rnTiO 2 was also significantly up-regulated, with MIP1a mRNA and protein expression significantly lower in the anTiO 2 group than in the rnTiO 2 group. In cell culture of primary alveolar macrophages (PAM) treated with anTiO 2 and rnTiO 2 , expression of MIP1a mRNA in the PAM and protein in the culture media was significantly higher than in control cultures. Similarly to the in vivo results, MIP1a mRNA and protein expression was significantly lower in the anTiO 2 treated cultures compared to the rnTiO 2 treated cultures. Furthermore, conditioned cell culture media from PAM cultures treated with anTiO 2 had less effect on A549 cell proliferation compared to conditioned media from cultures treated with rnTiO 2. However, no significant difference was found in the toxicological effects on cell viability of ultra violet irradiated anTiO 2 and rnTiO 2. In conclusion, our results indicate that anTiO 2 is less potent in induction of alveolar macrophage infiltration, 8-OHdG and MIP1a expression in the lung, and growth stimulation of A549 cells in vitro than rnTiO 2 .
Nanomaterials
Titanium dioxide nanoparticles (TiO2NPs) are increasingly used in consumer products, industrial and medical applications, raising concerns on their potential toxicity. The available in vitro and in vivo studies on these NPs show controversial results. Crystalline structure is the physicochemical characteristic that seems to influence mainly TiO2NPs toxicity, so its effect needs to be further studied. We aimed to study whether and how crystalline form influences potential cyto-genotoxic and inflammatory effects induced by two commercial TiO2NPs (TiO2-A, mainly anatase; TiO2-B, mainly rutile) in human alveolar A549 and bronchial BEAS-2B cells exposed to 1–40 µg/mL. Cell viability (WST-1), membrane damage (LDH release), IL-6, IL-8 and TNF-α release (ELISA) and direct/oxidative DNA damage (fpg-comet assay) were evaluated. Physicochemical characterization included analysis of crystalline form (TEM and XRD), specific surface area (BET), agglomeration (DLS) and Z-potential (ELS). Our resul...
Int J Mol Sci, 2014
The alveolar epithelium of the lung is by far the most permeable epithelial barrier of the human body. The risk for adverse effects by inhaled nanoparticles (NPs) depends on their hazard (negative action on cells and organism) and on exposure (concentration in the inhaled air and pattern of deposition in the lung). With the development of advanced in vitro models, not only in vivo, but also cellular studies can be used for toxicological testing. Advanced in vitro studies use combinations of cells cultured in the air-liquid interface. These cultures are useful for particle uptake and mechanistic studies. Whole-body, nose-only, and lung-only exposures of animals could help to determine retention of NPs in the body. Both approaches also have their limitations; cellular studies cannot mimic the entire organism and data obtained by inhalation exposure of rodents have limitations due to differences in the respiratory system from that of humans. Simulation programs for lung deposition in humans could help to determine the relevance of the biological findings. Combination of biological data generated in different biological models and in silico modeling appears suitable for a realistic estimation of potential risks by inhalation exposure to NPs.
2014
Abstract: The alveolar epithelium of the lung is by far the most permeable epithelial barrier of the human body. The risk for adverse effects by inhaled nanoparticles (NPs) depends on their hazard (negative action on cells and organism) and on exposure (concentration in the inhaled air and pattern of deposition in the lung). With the development of advanced in vitro models, not only in vivo, but also cellular studies can be used for toxicological testing. Advanced in vitro studies use combinations of cells cultured in the air-liquid interface. These cultures are useful for particle uptake and mechanistic studies. Whole-body, nose-only, and lung-only exposures of animals could help to determine retention of NPs in the body. Both approaches also have their limitations; cellular studies cannot mimic the entire organism and data obtained by inhalation exposure of rodents have limitations due to differences in the respiratory system from that of humans. Simulation programs for lung depos...
2013
Background: Hazard identification for risk assessment of nanoparticles (NPs) is mainly composed of in vitro cellbased assays and in vivo animal experimentation. The rapidly increasing number and functionalizations of NPs makes in vivo toxicity tests undesirable on both ethical and financial grounds, creating an urgent need for development of in vitro cell-based assays that accurately predict in vivo toxicity and facilitate safe nanotechnology.
Toxicological Sciences, 2007
Previous studies have reported little correlation between the relative toxicity of particle types when comparing lung toxicity rankings following in vivo instillation versus in vitro cell culture exposures. This study was designed to assess the capacity of in vitro screening studies to predict in vivo pulmonary toxicity of several fine or nanoscale particle types in rats. In the in vivo component of the study, rats were exposed by intratracheal instillation to 1 or 5 mg/kg of the following particle types: (1) carbonyl iron (CI), (2) crystalline silica (CS) (Min-U-Sil 5, a-quartz), (3) precipitated amorphous silica (AS), (4) nano-sized zinc oxide (NZO), or (5) fine-sized zinc oxide (FZO). Depending on particle type and solution state, these particles range in size from 90 to 500 nm in size. Following exposures, the lungs of exposed rats were lavaged and inflammation (neutrophil recruitment) and cytotoxicity end points (bronchoalveolar lavage [BAL] fluid lactate dehydrogenase [LDH] values) were measured at 24 h, 1 week, 1 and 3 months postexposure. For the in vitro component of the study, three different culture conditions were utilized. Cultures of (1) rat L2 lung epithelial cells, (2) primary alveolar macrophages (AMs) (collected via BAL from unexposed rats), as well as (3) AM-L2 lung epithelial cell cocultures were incubated with the particle types listed above, and the culture fluids were evaluated for cytotoxicity end points (LDH, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan [MTT]) as well as inflammatory cytokines (macrophage inflammatory 2 protein [MIP-2], tumor necrosis factor alpha [TNF-a], and interleukin-6 [IL-6]) at one (i.e., cytokines) or several (cytotoxicity) time periods. Results of in vivo pulmonary toxicity studies demonstrated that instilled CI particles produced little toxicity. CS particles produced sustained inflammation and cytotoxicity. AS particles produced reversible and transient inflammatory responses. NZO or FZO particles produced potent but reversible inflammation which was resolved by 1 month postinstillation exposure. Results of in vitro pulmonary cytotoxicity studies demonstrated a variety of responses to the different particle types, primarily at high doses. With respect to the LDH results, L2 cells were the most sensitive and exposures to nano-or fine-sized ZnO for 4 or 24 h were more cytotoxic than exposures to CS or AS particles. Macrophages essentially were resistant and epithelial macrophage cocultures generally reflected the epithelial results at 4 and 24 h incubation, but not at 48 h incubation. MTT results were also interesting but, except for nano-and fine-sized ZnO, did not correlate well with LDH results. Results of in vitro pulmonary inflammation studies demonstrated that L2 cells did not produce MIP-2 cytokines, but CS-or AS-exposed AMs and, to a lesser degree, cocultures secreted these chemotactic factors into the culture media. Measurements of TNF-a in the culture media by particle-exposed cells demonstrated little activity. In addition, IL-6 secretion was measured in CS, AS, and nano-sized ZnO-exposed cocultures. When considering the range of toxicity end points to five different particle types, the comparisons of in vivo and in vitro measurements demonstrated little correlation, particularly when considering many of the variables assessed in this study-such as cell types to be utilized, culture conditions and time course of exposure, as well as measured end points. It seems clear that in vitro cellular systems will need to be further developed, standardized, and validated (relative to in vivo effects) in order to provide useful screening data on the relative toxicity of inhaled particle types.