In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches.

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

Variation in pentose phosphate pathway-associated metabolism dictates cytotoxicity outcomes determined by tetrazolium reduction assays.

Tetrazolium reduction and resazurin assays are the mainstay of routine in vitro toxicity batteries. However, potentially erroneous characterization of cytotoxicity and cell proliferation can arise if verification of baseline interaction of test article with method employed is neglected. The current investigation aimed to demonstrate how interpretation of results from several standard cytotoxicity and proliferation assays vary in dependence on contributions from the pentose phosphate pathway (PPP). Non-tumorigenic Beas-2B cells were treated with graded concentrations of benzo[a]pyrene (B[a]P) for 24 and 48 h prior to cytotoxicity and proliferation assessment with commonly used MTT, MTS, WST1, and Alamar Blue assays. B[a]P caused enhanced metabolism of each dye assessed despite reductions in mitochondrial membrane potential and was reversed by 6-aminonicotinamide (6AN)-a glucose-6-phosphate dehydrogenase inhibitor. These results demonstrate differential sensitivity of standard cytotoxicity assessments on the PPP, thus (1) decoupling "mitochondrial activity" as an interpretation of cellular formazan and Alamar Blue metabolism, and (2) demonstrating the implicit requirement for investigators to sufficiently verify interaction of these methods in routine cytotoxicity and proliferation characterization. The nuances of method-specific extramitochondrial metabolism must be scrutinized to properly qualify specific endpoints employed, particularly under the circumstances of metabolic reprogramming.

Developing a Solution for Nasal and Olfactory Transport of Nanomaterials.

With advances in nanotechnology, engineered nanomaterial applications are a rapidly growing sector of the economy. Some nanomaterials can reach the brain through nose-to-brain transport. This transport creates concern for potential neurotoxicity of insoluble nanomaterials and a need for toxicity screening tests that detect nose-to-brain transport. Such tests can involve intranasal instillation of aqueous suspensions of nanomaterials in dispersion media that limit particle agglomeration. Unfortunately, protein and some elements in existing dispersion media are suboptimal for potential nose-to-brain transport of nanomaterials because olfactory transport has size- and ion-composition requirements. Therefore, we designed a protein-free dispersion media containing phospholipids and amino acids in an isotonic balanced electrolyte solution, a solution for nasal and olfactory transport (SNOT). SNOT disperses hexagonal boron nitride nanomaterials with a peak particle diameter below 100 nm. In addition, multiwalled carbon nanotubes (MWCNTs) in an established dispersion medium, when diluted with SNOT, maintain dispersion with reduced albumin concentration. Using stereomicroscopy and microscopic examination of plastic sections, dextran dyes dispersed in SNOT are demonstrated in the neuroepithelium of the nose and olfactory bulb of B6;129P2-Omptm3Mom/MomJ mice after intranasal instillation in SNOT. These findings support the potential for SNOT to disperse nanomaterials in a manner permitting nose-to-brain transport for neurotoxicity studies.

Histopathology of the broad class of carbon nanotubes and nanofibers used or produced in U.S. facilities in a murine model.

BACKGROUND: Multi-walled carbon nanotubes and nanofibers (CNT/F) have been previously investigated for their potential toxicities; however, comparative studies of the broad material class are lacking, especially those with a larger diameter. Additionally, computational modeling correlating physicochemical characteristics and toxicity outcomes have been infrequently employed, and it is unclear if all CNT/F confer similar toxicity, including histopathology changes such as pulmonary fibrosis. Male C57BL/6 mice were exposed to 40 µg of one of nine CNT/F (MW #1-7 and CNF #1-2) commonly found in exposure assessment studies of U.S. facilities with diameters ranging from 6 to 150 nm. Human fibroblasts (0-20 µg/ml) were used to assess the predictive value of in vitro to in vivo modeling systems. RESULTS: All materials induced histopathology changes, although the types and magnitude of the changes varied. In general, the larger diameter MWs (MW #5-7, including Mitsui-7) and CNF #1 induced greater histopathology changes compared to MW #1 and #3 while MW #4 and CNF #2 were intermediate in effect. Differences in individual alveolar or bronchiolar outcomes and severity correlated with physical dimensions and how the materials agglomerated. Human fibroblast monocultures were found to be insufficient to fully replicate in vivo fibrosis outcomes suggesting in vitro predictive potential depends upon more advanced cell culture in vitro models. Pleural penetrations were observed more consistently in CNT/F with larger lengths and diameters. CONCLUSION: Physicochemical characteristics, notably nominal CNT/F dimension and agglomerate size, predicted histopathologic changes and enabled grouping of materials by their toxicity profiles. Particles of greater nominal tube length were generally associated with increased severity of histopathology outcomes. Larger particle lengths and agglomerates were associated with more severe bronchi/bronchiolar outcomes. Spherical agglomerated particles of smaller nominal tube dimension were linked to granulomatous inflammation while a mixture of smaller and larger dimensional CNT/F resulted in more severe alveolar injury.

Carbon nanotube filler enhances incinerated thermoplastics-induced cytotoxicity and metabolic disruption in vitro.

BACKGROUND: Engineered nanomaterials are increasingly being incorporated into synthetic materials as fillers and additives. The potential pathological effects of end-of-lifecycle recycling and disposal of virgin and nano-enabled composites have not been adequately addressed, particularly following incineration. The current investigation aims to characterize the cytotoxicity of incinerated virgin thermoplastics vs. incinerated nano-enabled thermoplastic composites on two in vitro pulmonary models. Ultrafine particles released from thermally decomposed virgin polycarbonate or polyurethane, and their carbon nanotube (CNT)-enabled composites were collected and used for acute in vitro exposure to primary human small airway epithelial cell (pSAEC) and human bronchial epithelial cell (Beas-2B) models. Post-exposure, both cell lines were assessed for cytotoxicity, proliferative capacity, intracellular ROS generation, genotoxicity, and mitochondrial membrane potential. RESULTS: The treated Beas-2B cells demonstrated significant dose-dependent cellular responses, as well as parent matrix-dependent and CNT-dependent sensitivity. Cytotoxicity, enhancement in reactive oxygen species, and dissipation of ΔΨm caused by incinerated polycarbonate were significantly more potent than polyurethane analogues, and CNT filler enhanced the cellular responses compared to the incinerated parent particles. Such effects observed in Beas-2B were generally higher in magnitude compared to pSAEC at treatments examined, which was likely attributable to differences in respective lung cell types. CONCLUSIONS: Whilst the effect of the treatments on the distal respiratory airway epithelia remains limited in interpretation, the current in vitro respiratory bronchial epithelia model demonstrated profound sensitivity to the test particles at depositional doses relevant for occupational cohorts.

Potential antitumor activity of digitoxin and user-designed analog administered to human lung cancer cells.

BACKGROUND: Cardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes. METHODS: We treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration. RESULTS: Digitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling. CONCLUSIONS: Our study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity. SIGNIFICANCE: Discovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.

Characterization of aerosolized particles from nanoclay-enabled composites during manipulation processes.

Manufacturing, processing, use, and disposal of nanoclay-enabled composites potentially lead to the release of nanoclay particles from the polymer matrix in which they are embedded; however, exposures to airborne particles are poorly understood. The present study was conducted to characterize airborne particles released during sanding of nanoclay-enabled thermoplastic composites. Two types of nanoclay, Cloisite® 25A and Cloisite® 93A, were dispersed in polypropylene at 0%, 1%, and 4% loading by weight. Zirconium aluminum oxide (P100/P180 grits) and silicon carbide (P120/P320 grits) sandpapers were used to abrade composites in controlled experiments followed by real-time and offline particle analyses. Overall, sanding the virgin polypropylene with zirconium aluminum oxide sandpaper released more particles compared to silicon carbide sandpaper, with the later exhibiting similar or lower concentrations than that of polypropylene. Thus, a further investigation was performed for the samples collected using the zirconium aluminum oxide sandpaper. The 1% 25A, 1% 93A, and 4% 93A composites generated substantially higher particle number concentrations (1.3-2.6 times) and respirable mass concentrations (1.2-2.3 times) relative to the virgin polypropylene, while the 4% 25A composite produced comparable results, regardless of sandpaper type. It was observed that the majority of the inhalable particles were originated from composite materials with a significant number of protrusions of nanoclay (18-59%). These findings indicate that the percent loading and dispersion of nanoclay in the polypropylene modified the mechanical properties and thus, along with sandpaper type, affected the number of particles released during sanding, implicating the cause of potential adverse health effects.

Impacts of Organomodified Nanoclays and Their Incinerated Byproducts on Bronchial Cell Monolayer Integrity.

Incorporation of engineered nanomaterials (ENMs) into nanocomposites using advanced manufacturing strategies is set to revolutionize diverse technologies. Of these, organomodified nanoclays (ONCs; i.e., smectite clays with different organic coatings) act as nanofillers in applications ranging from automotive to aerospace and biomedical systems. Recent toxicological evaluations increased awareness that exposure to ONC can occur along their entire life cycle, namely, during synthesis, handling, use, manipulation, and disposal. Compared to other ENMs, however, little information exists describing which physicochemical properties contribute to induced health risk. This study conducted high content screening on bronchial epithelial cell monolayers for coupled high-throughput in vitro assessment strategies aimed to evaluate acute toxicity of a library of ONCs (all of prevalent use) prior to and after simulated disposal by incineration. Coating-, incineration status-, and time-dependent effects were considered to determine changes in the pulmonary monolayer integrity, cell transepithelial resistance, apoptosis, and cell metabolism. Results showed that after exposure to each ONC at its half-maximal inhibitory concentration (IC50) there is a material-induced toxicity effect with pristine nanoclay, for instance, displaying acute loss of monolayer coverage, resistance, and metabolism, coupled with increased number of apoptotic cells. Conversely, the other three ONCs tested displayed little loss of monolayer integrity; however, they exhibited differential coating-dependent increased apoptosis and up to 40-45% initial reduction in cell metabolism. Moreover, incinerated byproducts of ONCs exhibited significant loss of monolayer coverage and integrity, increased necrosis, with little evidence of monolayer re-establishment. These findings indicate that characteristics of organic coating type largely determine the mechanism of cytotoxicity and the ability of the monolayer to recover. Use of high content screening coupled with traditional in vitro assays proves to serve as a rapid pulmonary toxicity assessment tool to help define prevention by targeted physicochemical material properties design strategies.

Iron Oxide Nanoparticle-Induced Neoplastic-Like Cell Transformation in Vitro Is Reduced with a Protective Amorphous Silica Coating.

Iron oxide nanoparticles (IONP) have recently surged in production and use in a wide variety of biomedical and environmental applications. However, their potential long-term health effects, including carcinogenesis, are unknown. Limited research suggests IONP can induce genotoxicity and neoplastic transformation associated with particle dissolution and release of free iron ions. "Safe by design" strategies involve the modification of particle physicochemical properties to affect subsequent adverse outcomes, such as an amorphous silica coating to reduce IONP dissolution and direct interaction with cells. We hypothesized that long-term exposure to a specific IONP (nFe2O3) would induce neoplastic-like cell transformation, which could be prevented with an amorphous silica coating (SiO2-nFe2O3). To test this hypothesis, human bronchial epithelial cells (Beas-2B) were continuously exposed to a 0.6 µg/cm2 administered a dose of nFe2O3 (∼0.58 µg/cm2 delivered dose), SiO2-nFe2O3 (∼0.55 µg/cm2 delivered dose), or gas metal arc mild steel welding fumes (GMA-MS, ∼0.58 µg/cm2 delivered dose) for 6.5 months. GMA-MS are composed of roughly 80% iron/iron oxide and were recently classified as a total human carcinogen. Our results showed that low-dose/long-term in vitro exposure to nFe2O3 induced a time-dependent neoplastic-like cell transformation, as indicated by increased cell proliferation and attachment-independent colony formation, which closely matched that induced by GMA-MS. This transformation was associated with decreases in intracellular iron, minimal changes in reactive oxygen species (ROS) production, and the induction of double-stranded DNA damage. An amorphous silica-coated but otherwise identical particle (SiO2-nFe2O3) did not induce this neoplastic-like phenotype or changes in the parameters mentioned above. Overall, the presented data suggest the carcinogenic potential of long-term nFe2O3 exposure and the utility of an amorphous silica coating in a "safe by design" hazard reduction strategy, within the context of a physiologically relevant exposure scenario (low-dose/long-term), with model validation using GMA-MS.

Mitsui-7, heat-treated, and nitrogen-doped multi-walled carbon nanotubes elicit genotoxicity in human lung epithelial cells.

BACKGROUND: The unique physicochemical properties of multi-walled carbon nanotubes (MWCNT) have led to many industrial applications. Due to their low density and small size, MWCNT are easily aerosolized in the workplace making respiratory exposures likely in workers. The International Agency for Research on Cancer designated the pristine Mitsui-7 MWCNT (MWCNT-7) as a Group 2B carcinogen, but there was insufficient data to classify all other MWCNT. Previously, MWCNT exposed to high temperature (MWCNT-HT) or synthesized with nitrogen (MWCNT-ND) have been found to elicit attenuated toxicity; however, their genotoxic and carcinogenic potential are not known. Our aim was to measure the genotoxicity of MWCNT-7 compared to these two physicochemically-altered MWCNTs in human lung epithelial cells (BEAS-2B & SAEC). RESULTS: Dose-dependent partitioning of individual nanotubes in the cell nuclei was observed for each MWCNT material and was greatest for MWCNT-7. Exposure to each MWCNT led to significantly increased mitotic aberrations with multi- and monopolar spindle morphologies and fragmented centrosomes. Quantitative analysis of the spindle pole demonstrated significantly increased centrosome fragmentation from 0.024-2.4 µg/mL of each MWCNT. Significant aneuploidy was measured in a dose-response from each MWCNT-7, HT, and ND; the highest dose of 24 µg/mL produced 67, 61, and 55%, respectively. Chromosome analysis demonstrated significantly increased centromere fragmentation and translocations from each MWCNT at each dose. Following 24 h of exposure to MWCNT-7, ND and/or HT in BEAS-2B a significant arrest in the G1/S phase in the cell cycle occurred, whereas the MWCNT-ND also induced a G2 arrest. Primary SAEC exposed for 24 h to each MWCNT elicited a significantly greater arrest in the G1 and G2 phases. However, SAEC arrested in the G1/S phase after 72 h of exposure. Lastly, a significant increase in clonal growth was observed one month after exposure to 0.024 µg/mL MWCNT-HT & ND. CONCLUSIONS: Although MWCNT-HT & ND cause a lower incidence of genotoxicity, all three MWCNTs cause the same type of mitotic and chromosomal disruptions. Chromosomal fragmentation and translocations have not been observed with other nanomaterials. Because in vitro genotoxicity is correlated with in vivo genotoxic response, these studies in primary human lung cells may predict the genotoxic potency in exposed human populations.

Acrylonitrile butadiene styrene (ABS) and polycarbonate (PC) filaments three-dimensional (3-D) printer emissions-induced cell toxicity.

During extrusion of some polymers, fused filament fabrication (FFF) 3-D printers emit billions of particles per minute and numerous organic compounds. The scope of this study was to evaluate FFF 3-D printer emission-induced toxicity in human small airway epithelial cells (SAEC). Emissions were generated from a commercially available 3-D printer inside a chamber, while operating for 1.5 h with acrylonitrile butadiene styrene (ABS) or polycarbonate (PC) filaments, and collected in cell culture medium. Characterization of the culture medium revealed that repeat print runs with an identical filament yield various amounts of particles and organic compounds. Mean particle sizes in cell culture medium were 201 ±â€¯18 nm and 202 ±â€¯8 nm for PC and ABS, respectively. At 24 h post-exposure, both PC and ABS emissions induced a dose dependent significant cytotoxicity, oxidative stress, apoptosis, necrosis, and production of pro-inflammatory cytokines and chemokines in SAEC. Though the emissions may not completely represent all possible exposure scenarios, this study indicate that the FFF could induce toxicological effects. Further studies are needed to quantify the detected chemicals in the emissions and their corresponding toxicological effects.

Acquisition of Cancer Stem Cell-like Properties in Human Small Airway Epithelial Cells after a Long-term Exposure to Carbon Nanomaterials.

Cancer stem cells (CSCs) are a key driver of tumor formation and metastasis, but how they are affected by nanomaterials is largely unknown. The present study investigated the effects of different carbon-based nanomaterials (CNMs) on neoplastic and CSC-like transformation of human small airway epithelial cells and determined the underlying mechanisms. Using a physiologically relevant exposure model (long-term/low-dose) with system validation using a human carcinogen, asbestos, we demonstrated that single-walled carbon nanotubes, multi-walled carbon nanotubes, ultrafine carbon black, and crocidolite asbestos induced particle-specific anchorage-independent colony formation, DNA-strand break, and p53 downregulation, indicating genotoxicity and carcinogenic potential of CNMs. The chronic CNM-exposed cells exhibited CSC-like properties as indicated by 3D spheroid formation, anoikis resistance, and CSC markers expression. Mechanistic studies revealed specific self-renewal and epithelial-mesenchymal transition (EMT)-related transcription factors that are involved in the cellular transformation process. Pathway analysis of gene signaling networks supports the role of SOX2 and SNAI1 signaling in CNM-mediated transformation. These findings support the potential carcinogenicity of high aspect ratio CNMs and identified molecular targets and signaling pathways that may contribute to the disease development.

Incineration of Nanoclay Composites Leads to Byproducts with Reduced Cellular Reactivity.

Addition of nanoclays into a polymer matrix leads to nanocomposites with enhanced properties to be used in plastics for food packaging applications. Because of the plastics' high stored energy value, such nanocomposites make good candidates for disposal via municipal solid waste plants. However, upon disposal, increased concerns related to nanocomposites' byproducts potential toxicity arise, especially considering that such byproducts could escape disposal filters to cause inhalation hazards. Herein, we investigated the effects that byproducts of a polymer polylactic acid-based nanocomposite containing a functionalized montmorillonite nanoclay (Cloisite 30B) could pose to human lung epithelial cells, used as a model for inhalation exposure. Analysis showed that the byproducts induced toxic responses, including reductions in cellular viability, changes in cellular morphology, and cytoskeletal alterations, however only at high doses of exposure. The degree of dispersion of nanoclays in the polymer matrix appeared to influence the material characteristics, degradation, and ultimately toxicity. With toxicity of the byproduct occurring at high doses, safety protocols should be considered, along with deleterious effects investigations to thus help aid in safer, yet still effective products and disposal strategies.

Short-Term Pulmonary Toxicity Assessment of Pre- and Post-incinerated Organomodified Nanoclay in Mice.

Organomodified nanoclays (ONCs) are increasingly used as filler materials to improve nanocomposite strength, wettability, flammability, and durability. However, pulmonary risks associated with exposure along their chemical lifecycle are unknown. This study's objective was to compare pre- and post-incinerated forms of uncoated and organomodified nanoclays for potential pulmonary inflammation, toxicity, and systemic blood response. Mice were exposed via aspiration to low (30 µg) and high (300 µg) doses of preincinerated uncoated montmorillonite nanoclay (CloisNa), ONC (Clois30B), their respective incinerated forms (I-CloisNa and I-Clois30B), and crystalline silica (CS). Lung and blood tissues were collected at days 1, 7, and 28 to compare toxicity and inflammation indices. Well-dispersed CloisNa caused a robust inflammatory response characterized by neutrophils, macrophages, and particle-laden granulomas. Alternatively, Clois30B, I-Clois30B, and CS high-dose exposures elicited a low grade, persistent inflammatory response. High-dose Clois30B exposure exhibited moderate increases in lung damage markers and a delayed macrophage recruitment cytokine signature peaking at day 7 followed by a fibrotic tissue signature at day 28, similar to CloisNa. I-CloisNa exhibited acute, transient inflammation with quick recovery. Conversely, high-dose I-Clois30B caused a weak initial inflammatory signal but showed comparable pro-inflammatory signaling to CS at day 28. The data demonstrate that ONC pulmonary toxicity and inflammatory potential relies on coating presence and incineration status in that coated and incinerated nanoclay exhibited less inflammation and granuloma formation than pristine montmorillonite. High doses of both pre- and post-incinerated ONC, with different surface morphologies, may harbor potential pulmonary health hazards over long-term occupational exposures.

Carbon nanotubes physicochemical properties influence the overall cellular behavior and fate.

The unique properties of single walled carbon nanotubes (SWCNTs) make them viable candidates for versatile implementation in the next generation of biomedical devices for targeted delivery of chemotherapeutic agents or cellular-sensing probes. Such implementation requires user-tailored changes in SWCNT's physicochemical characteristics to allow for efficient cellular integration while maintaining nanotubes' functionality. However, isolated reports showed that user-tailoring could induce deleterious effects in exposed cells, from decrease in cellular proliferation, to changes in cellular adhesion, generation of reactive oxygen species or phenotypical variations, just to name a few. Before full implementation of SWCNTs is achieved, their toxicological profiles need to be mechanistically correlated with their physicochemical properties to determine how the induced cellular fate is related to the exposure conditions or samples' characteristics. Our study provides a comprehensive analysis of the synergistic cyto- and genotoxic effects resulted from short-term exposure of human lung epithelial cells to pristine (as manufactured) and user-tailored SWCNTs, as a function of their physicochemical properties. Specifically, through a systematic approach we are correlating the nanotube uptake and nanotube-induced cellular changes to the sample's physicochemical characteristics (e.g., metal impurities, length, agglomerate size, surface area, dispersion, and surface functionalization). By identifying changes in active hallmarks involved in cell-cell connections and maintaining epithelial layer integrity, we also determine the role that short-term exposure to SWCNTs plays in the overall cellular fate and cellular transformation. Lastly, we assess cellular structure-function relationships to identify non-apoptotic pathways induced by SWCNTs exposure that could however lead to changes in cellular behavior and cellular transformation. Our results show that the degree of cell transformation is a function of the physicochemical properties of the SWCNT, with the nanotube with higher length, higher metal content and larger agglomerate size reducing cell viability to a larger extent. Such changes in cell viability are also complemented by changes in cell structure, cycle and cell-cell interactions, all responsible for maintaining cell fate.

Potential Toxicity and Underlying Mechanisms Associated with Pulmonary Exposure to Iron Oxide Nanoparticles: Conflicting Literature and Unclear Risk.

Fine/micron-sized iron oxide particulates are incidentally released from a number of industrial processes, including iron ore mining, steel processing, welding, and pyrite production. Some research suggests that occupational exposure to these particulates is linked to an increased risk of adverse respiratory outcomes, whereas other studies suggest that iron oxide is biologically benign. Iron oxide nanoparticles (IONPs), which are less than 100 nm in diameter, have recently surged in use as components of novel drug delivery systems, unique imaging protocols, as environmental catalysts, and for incorporation into thermoplastics. However, the adverse outcomes associated with occupational exposure to IONPs remain relatively unknown. Relevant in vivo studies suggest that pulmonary exposure to IONPs may induce inflammation, pulmonary fibrosis, genotoxicity, and extra-pulmonary effects. This correlates well with in vitro studies that utilize relevant dose, cell type(s), and meaningful end points. A majority of these adverse outcomes are attributed to increased oxidative stress, most likely caused by particle internalization, dissolution, release of free iron ions, and disruption of iron homeostasis. However, because the overall toxicity profile of IONPs is not well understood, it is difficult to set safe exposure limit recommendations that would be adequate for the protection of at-risk workers. This review article will focus on known risks following IONPs exposure supported by human, animal, and cell culture-based studies, the potential challenges intrinsic to IONPs toxicity assessment, and how these may contribute to the poorly characterized IONPs toxicity profile.

Early Assessment and Correlations of Nanoclay's Toxicity to Their Physical and Chemical Properties.

Nanoclays' functionalization with organic modifiers increases their individual barrier properties, thermal stability, and mechanical properties and allows for ease of implementation in food packaging materials or medical devices. Previous reports have shown that, while organic modifiers integration between the layered mineral silicates leads to nanoclays with different degrees of hydrophobicity that become easily miscible in polymers, they could also pose possible effects at inhalation or ingestion routes of exposure. Through a systematic analysis of three organically modified and one pristine nanoclay, we aimed to relate for the first time the physical and chemical characteristics, determined via microscopical and spectroscopical techniques, with the potential of these nanoclays to induce deleterious effects in in vitro cellular systems, i.e. in immortalized and primary human lung epithelial cell lines. To derive information on how functionalization could lead to toxicological profiles throughout nanoclays' life cycle, both as-received and thermally degraded nanoclays were evaluated. Our analysis showed that the organic modifiers chemical composition influenced both the physical and chemical characteristics of the nanoclays as well as their toxicity. Overall, when cells were exposed to nanoclays with organic modifiers containing bioreactive groups, they displayed lower cellular numbers as well more elongated cellular morphologies relative to the pristine nanoclay and the nanoclay containing a modifier with long carbon chains. Additionally, thermal degradation caused loss of the organic modifiers as well as changes in size and shape of the nanoclays, which led to changes in toxicity upon exposure to our model cellular systems. Our study provides insight into the synergistic effects of chemical composition, size, and shape of the nanoclays and their toxicological profiles in conditions that mimic exposure in manufacturing and disposal environments, respectively, and can help aid in safe-by-design manufacturing of nanoclays with user-controlled functionalization and lower toxicity levels when food packaging applications are considered.

Effect of surface functionalizations of multi-walled carbon nanotubes on neoplastic transformation potential in primary human lung epithelial cells.

Functionalized multi-walled carbon nanotube (fMWCNT) development has been intensified to improve their surface activity for numerous applications, and potentially reduce toxic effects. Although MWCNT exposures are associated with lung tumorigenesis in vivo, adverse responses associated with exposure to different fMWCNTs in human lung epithelium are presently unknown. This study hypothesized that different plasma-coating functional groups determine MWCNT neoplastic transformation potential. Using our established model, human primary small airway epithelial cells (pSAECs) were continuously exposed for 8 and 12 weeks at 0.06 µg/cm2 to three-month aged as-prepared-(pMWCNT), carboxylated-(MW-COOH), and aminated-MWCNTs (MW-NHx). Ultrafine carbon black (UFCB) and crocidolite asbestos (ASB) served as particle controls. fMWCNTs were characterized during storage, and exposed cells were assessed for several established cancer cell hallmarks. Characterization analyses conducted at 0 and 2 months of aging detected a loss of surface functional groups over time due to atmospheric oxidation, with MW-NHx possessing less oxygen and greater lung surfactant binding affinity. Following 8 weeks of exposure, all fMWCNT-exposed cells exhibited significant increased proliferation compared to controls at 7 d post-treatment, while UFCB- and ASB-exposed cells did not differ significantly from controls. UFCB, pMWCNT, and MW-COOH exposure stimulated significant transient invasion behavior. Conversely, aged MW-NHx-exposed cells displayed moderate increases in soft agar colony formation and morphological transformation potential, while UFCB cells showed a minimal effect compared to all other treatments. In summary, surface properties of aged fMWCNTs can impact cell transformation events in vitro following continuous, occupationally relevant exposures.

Evaluation of tumorigenic potential of CeO2 and Fe2O3 engineered nanoparticles by a human cell in vitro screening model.

With rapid development of novel nanotechnologies that incorporate engineered nanomaterials (ENMs) into manufactured products, long-term, low dose ENM exposures in occupational settings is forecasted to occur with potential adverse outcomes to human health. Few ENM human health risk assessment efforts have evaluated tumorigenic potential of ENMs. Two widely used nano-scaled metal oxides (NMOs), cerium oxide (nCeO2) and ferric oxide (nFe2O3) were screened in the current study using a sub-chronic exposure to human primary small airway epithelial cells (pSAECs). Multi-walled carbon nanotubes (MWCNT), a known ENM tumor promoter, was used as a positive control. Advanced dosimetry modeling was employed to ascertain delivered vs. administered dose in all experimental conditions. Cells were continuously exposed in vitro to deposited doses of 0.18 µg/cm2 or 0.06 µg/cm2 of each NMO or MWCNT, respectively, over 6 and 10 weeks, while saline- and dispersant-only exposed cells served as passage controls. Cells were evaluated for changes in several cancer hallmarks, as evidence for neoplastic transformation. At 10 weeks, nFe2O3- and MWCNT-exposed cells displayed a neoplastic-like transformation phenotype with significant increased proliferation, invasion and soft agar colony formation ability compared to controls. nCeO2-exposed cells showed increased proliferative capacity only. Isolated nFe2O3 and MWCNT clones from soft agar colonies retained their respective neoplastic-like phenotypes. Interestingly, nFe2O3-exposed cells, but not MWCNT cells, exhibited immortalization and retention of the neoplastic phenotype after repeated passaging (12 - 30 passages) and after cryofreeze and thawing. High content screening and protein expression analyses in acute exposure ENM studies vs. immortalized nFe2O3 cells, and isolated ENM clones, suggested that long-term exposure to the tested ENMs resulted in iron homeostasis disruption, an increased labile ferrous iron pool, and subsequent reactive oxygen species generation, a well-established tumorigenesis promotor. In conclusion, sub-chronic exposure to human pSAECs with a cancer hallmark screening battery identified nFe2O3 as possessing neoplastic-like transformation ability, thus suggesting that further tumorigenic assessment is needed.

Toxicity evaluations of nanoclays and thermally degraded byproducts through spectroscopical and microscopical approaches.

BACKGROUND: Montmorillonite is a type of nanoclay that originates from the clay fraction of the soil and is incorporated into polymers to form nanocomposites with enhanced mechanical strength, barrier, and flammability properties used for food packaging, automotive, and medical devices. However, with implementation in such consumer applications, the interaction of montmorillonite-based composites or derived byproducts with biological systems needs to be investigated. METHODS: Herein we examined the potential of Cloisite Na+ (pristine) and Cloisite 30B (organically modified montmorillonite nanoclay) and their thermally degraded byproducts' to induce toxicity in model human lung epithelial cells. The experimental set-up mimicked biological exposure in manufacturing and disposal areas and employed cellular treatments with occupationally relevant doses of nanoclays previously characterized using spectroscopical and microscopical approaches. For nanoclay-cellular interactions and for cellular analyses respectively, biosensorial-based analytical platforms were used, with induced cellular changes being confirmed via live cell counts, viability assays, and cell imaging. RESULTS: Our analysis of byproducts' chemical and physical properties revealed both structural and functional changes. Real-time high throughput analyses of exposed cellular systems confirmed that nanoclay induced significant toxic effects, with Cloisite 30B showing time-dependent decreases in live cell count and cellular viability relative to control and pristine nanoclay, respectively. Byproducts produced less toxic effects; all treatments caused alterations in the cell morphology upon exposure. CONCLUSIONS: Our morphological, behavioral, and viability cellular changes show that nanoclays have the potential to produce toxic effects when used both in manufacturing or disposal environments. GENERAL SIGNIFICANCE: The reported toxicological mechanisms prove the extensibility of a biosensorial-based platform for cellular behavior analysis upon treatment with a variety of nanomaterials.