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.

Constant vs. cyclic flow when testing face masks and respirators as source control devices for simulated respiratory aerosols.

SARS-CoV-2 spreads by infectious aerosols and droplets from the respiratory tract. Masks and respirators can reduce the transmission of infectious respiratory diseases by collecting these aerosols at the source. The ability of source control devices to block aerosols can be tested by expelling an aerosol through a headform using constant airflows, which are simpler, or cyclic airflows, which are more realistic but require more complex methods. Experiments with respirators found that using cyclic vs. constant flows affected the amount of aerosol inhaled, but similar comparisons have not been made for source control devices with exhaled aerosols. We measured the collection efficiencies for exhaled aerosols for two cloth masks, two medical masks with and without an elastic mask brace, a neck gaiter, and an N95 filtering facepiece respirator using 15 L/min and 85 L/min constant and cyclic flows and a headform with pliable skin. The collection efficiencies for the 15 L/min cyclic flow, 15 L/min constant flow, and 85 L/min constant flow were not significantly different in most cases. The apparent collection efficiencies for the 85 L/min cyclic flow were artificially increased by rebreathing and refiltration of the aerosol from the collection chamber. The collection efficiencies correlated well with the fit factors (ρ > 0.95) but not the filtration efficiencies (ρ < 0.54). Our results suggest that the aerosol collection efficiency measurements of source control devices are comparable when testing the devices using either constant or cyclic airflows and that the potential for aerosol rebreathing must be considered when conducting experiments.

Efficacy of Do-It-Yourself air filtration units in reducing exposure to simulated respiratory aerosols.

Many respiratory diseases, including COVID-19, can be spread by aerosols expelled by infected people when they cough, talk, sing, or exhale. Exposure to these aerosols indoors can be reduced by portable air filtration units (air cleaners). Homemade or Do-It-Yourself (DIY) air filtration units are a popular alternative to commercially produced devices, but performance data is limited. Our study used a speaker-audience model to examine the efficacy of two popular types of DIY air filtration units, the Corsi-Rosenthal cube and a modified Ford air filtration unit, in reducing exposure to simulated respiratory aerosols within a mock classroom. Experiments were conducted using four breathing simulators at different locations in the room, one acting as the respiratory aerosol source and three as recipients. Optical particle spectrometers monitored simulated respiratory aerosol particles (0.3-3 µm) as they dispersed throughout the room. Using two DIY cubes (in the front and back of the room) increased the air change rate as much as 12.4 over room ventilation, depending on filter thickness and fan airflow. Using multiple linear regression, each unit increase of air change reduced exposure by 10%. Increasing the number of filters, filter thickness, and fan airflow significantly enhanced the air change rate, which resulted in exposure reductions of up to 73%. Our results show DIY air filtration units can be an effective means of reducing aerosol exposure. However, they also show performance of DIY units can vary considerably depending upon their design, construction, and positioning, and users should be mindful of these limitations.

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.

Reduction of exposure to simulated respiratory aerosols using ventilation, physical distancing, and universal masking.

To limit community spread of SARS-CoV-2, CDC recommends universal masking indoors, maintaining 1.8 m of physical distancing, adequate ventilation, and avoiding crowded indoor spaces. Several studies have examined the independent influence of each control strategy in mitigating transmission in isolation, yet controls are often implemented concomitantly within an indoor environment. To address the influence of physical distancing, universal masking, and ventilation on very fine respiratory droplets and aerosol particle exposure, a simulator that coughed and exhaled aerosols (the source) and a second breathing simulator (the recipient) were placed in an exposure chamber. When controlling for the other two mitigation strategies, universal masking with 3-ply cotton masks reduced exposure to 0.3-3 µm coughed and exhaled aerosol particles by >77% compared to unmasked tests, whereas physical distancing (0.9 or 1.8 m) significantly changed exposure to cough but not exhaled aerosols. The effectiveness of ventilation depended upon the respiratory activity, that is, coughing or breathing, as well as the duration of exposure time. Our results demonstrate that a layered mitigation strategy approach of administrative and engineering controls can reduce personal inhalation exposure to potentially infectious very fine respiratory droplets and aerosol particles within an indoor environment.

U.S. Federal Agency interests and key considerations for new approach methodologies for nanomaterials.

Engineered nanomaterials (ENMs) come in a wide array of shapes, sizes, surface coatings, and compositions, and often possess novel or enhanced properties compared to larger sized particles of the same elemental composition. To ensure the safe commercialization of products containing ENMs, it is important to thoroughly understand their potential risks. Given that ENMs can be created in an almost infinite number of variations, it is not feasible to conduct in vivo testing on each type of ENM. Instead, new approach methodologies (NAMs) such as in vitro or in chemico test methods may be needed, given their capacity for higher throughput testing, lower cost, and ability to provide information on toxicological mechanisms. However, the different behaviors of ENMs compared to dissolved chemicals may challenge safety testing of ENMs using NAMs. In this study, member agencies within the Interagency Coordinating Committee on the Validation of Alternative Methods were queried about what types of ENMs are of agency interest and whether there is agency-specific guidance for ENM toxicity testing. To support the ability of NAMs to provide robust results in ENM testing, two key issues in the usage of NAMs, namely dosimetry and interference/bias controls, are thoroughly discussed.

Face mask fit modifications that improve source control performance.

BACKGROUND: During the COVID-19 pandemic, face masks are used as source control devices to reduce the expulsion of respiratory aerosols from infected people. Modifications such as mask braces, earloop straps, knotting and tucking, and double masking have been proposed to improve mask fit however the data on source control are limited. METHODS: The effectiveness of mask fit modifications was determined by conducting fit tests on human subjects and simulator manikins and by performing simulated coughs and exhalations using a source control measurement system. RESULTS: Medical masks without modification blocked ≥56% of cough aerosols and ≥42% of exhaled aerosols. Modifying fit by crossing the earloops or placing a bracket under the mask did not increase performance, while using earloop toggles, an earloop strap, and knotting and tucking the mask increased performance. The most effective modifications for improving source control performance were double masking and using a mask brace. Placing a cloth mask over a medical mask blocked ≥85% of cough aerosols and ≥91% of exhaled aerosols. Placing a brace over a medical mask blocked ≥95% of cough aerosols and ≥99% of exhaled aerosols. CONCLUSIONS: Fit modifications can greatly improve the performance of face masks as source control devices for respiratory aerosols.

Efficacy of Ventilation, HEPA Air Cleaners, Universal Masking, and Physical Distancing for Reducing Exposure to Simulated Exhaled Aerosols in a Meeting Room.

There is strong evidence associating the indoor environment with transmission of SARS-CoV-2, the virus that causes COVID-19. SARS-CoV-2 can spread by exposure to droplets and very fine aerosol particles from respiratory fluids that are released by infected persons. Layered mitigation strategies, including but not limited to maintaining physical distancing, adequate ventilation, universal masking, avoiding overcrowding, and vaccination, have shown to be effective in reducing the spread of SARS-CoV-2 within the indoor environment. Here, we examine the effect of mitigation strategies on reducing the risk of exposure to simulated respiratory aerosol particles within a classroom-style meeting room. To quantify exposure of uninfected individuals (Recipients), surrogate respiratory aerosol particles were generated by a breathing simulator with a headform (Source) that mimicked breath exhalations. Recipients, represented by three breathing simulators with manikin headforms, were placed in a meeting room and affixed with optical particle counters to measure 0.3-3 µm aerosol particles. Universal masking of all breathing simulators with a 3-ply cotton mask reduced aerosol exposure by 50% or more compared to scenarios with simulators unmasked. While evaluating the effect of Source placement, Recipients had the highest exposure at 0.9 m in a face-to-face orientation. Ventilation reduced exposure by approximately 5% per unit increase in air change per hour (ACH), irrespective of whether increases in ACH were by the HVAC system or portable HEPA air cleaners. The results demonstrate that mitigation strategies, such as universal masking and increasing ventilation, reduce personal exposure to respiratory aerosols within a meeting room. While universal masking remains a key component of a layered mitigation strategy of exposure reduction, increasing ventilation via system HVAC or portable HEPA air cleaners further reduces exposure.

Efficacy of Portable Air Cleaners and Masking for Reducing Indoor Exposure to Simulated Exhaled SARS-CoV-2 Aerosols - United States, 2021.

SARS-CoV-2, the virus that causes COVID-19, can be spread by exposure to droplets and aerosols of respiratory fluids that are released by infected persons when they cough, sing, talk, or exhale. To reduce indoor transmission of SARS-CoV-2 between persons, CDC recommends measures including physical distancing, universal masking (the use of face masks in public places by everyone who is not fully vaccinated), and increased room ventilation (1). Ventilation systems can be supplemented with portable high efficiency particulate air (HEPA) cleaners* to reduce the number of infectious particles in the air and provide enhanced protection from transmission between persons (2); two recent reports found that HEPA air cleaners in classrooms could reduce overall aerosol particle concentrations by ≥80% within 30 minutes (3,4). To investigate the effectiveness of portable HEPA air cleaners and universal masking at reducing exposure to exhaled aerosol particles, the investigation team used respiratory simulators to mimic a person with COVID-19 and other, uninfected persons in a conference room. The addition of two HEPA air cleaners that met the Environmental Protection Agency (EPA)-recommended clean air delivery rate (CADR) (5) reduced overall exposure to simulated exhaled aerosol particles by up to 65% without universal masking. Without the HEPA air cleaners, universal masking reduced the combined mean aerosol concentration by 72%. The combination of the two HEPA air cleaners and universal masking reduced overall exposure by up to 90%. The HEPA air cleaners were most effective when they were close to the aerosol source. These findings suggest that portable HEPA air cleaners can reduce exposure to SARS-CoV-2 aerosols in indoor environments, with greater reductions in exposure occurring when used in combination with universal masking.

Efficacy of universal masking for source control and personal protection from simulated cough and exhaled aerosols in a room.

Face masks reduce the expulsion of respiratory aerosols produced during coughs and exhalations ("source control"). Factors such as the directions in which people are facing (orientation) and separation distance also affect aerosol dispersion. However, it is not clear how the combined effects of masking, orientation, and distance affect the exposure of individuals to respiratory aerosols in indoor spaces. We placed a respiratory aerosol simulator ("source") and a breathing simulator ("recipient") in a 3 m × 3 m chamber and measured aerosol concentrations for different combinations of masking, orientation, and separation distance. When the simulators were front-to-front during coughing, masks reduced the 15-min mean aerosol concentration at the recipient by 92% at 0.9 and 1.8 m separation. When the simulators were side-by-side, masks reduced the concentration by 81% at 0.9 m and 78% at 1.8 m. During breathing, masks reduced the aerosol concentration by 66% when front-to-front and 76% when side-by-side at 0.9 m. Similar results were seen at 1.8 m. When the simulators were unmasked, changing the orientations from front-to-front to side-by-side reduced the cough aerosol concentration by 59% at 0.9 m and 60% at 1.8 m. When both simulators were masked, changing the orientations did not significantly change the concentration at either distance during coughing or breathing. Increasing the distance between the simulators from 0.9 m to 1.8 m during coughing reduced the aerosol concentration by 25% when no masks were worn but had little effect when both simulators were masked. During breathing, when neither simulator was masked, increasing the separation reduced the concentration by 13%, which approached significance, while the change was not significant when both source and recipient were masked. Our results show that universal masking reduces exposure to respiratory aerosol particles regardless of the orientation and separation distance between the source and recipient.

Maximizing Fit for Cloth and Medical Procedure Masks to Improve Performance and Reduce SARS-CoV-2 Transmission and Exposure, 2021.

Universal masking is one of the prevention strategies recommended by CDC to slow the spread of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19) (1). As of February 1, 2021, 38 states and the District of Columbia had universal masking mandates. Mask wearing has also been mandated by executive order for federal property* as well as on domestic and international transportation conveyances.† Masks substantially reduce exhaled respiratory droplets and aerosols from infected wearers and reduce exposure of uninfected wearers to these particles. Cloth masks§ and medical procedure masks¶ fit more loosely than do respirators (e.g., N95 facepieces). The effectiveness of cloth and medical procedure masks can be improved by ensuring that they are well fitted to the contours of the face to prevent leakage of air around the masks' edges. During January 2021, CDC conducted experimental simulations using pliable elastomeric source and receiver headforms to assess the extent to which two modifications to medical procedure masks, 1) wearing a cloth mask over a medical procedure mask (double masking) and 2) knotting the ear loops of a medical procedure mask where they attach to the mask's edges and then tucking in and flattening the extra material close to the face (knotted and tucked masks), could improve the fit of these masks and reduce the receiver's exposure to an aerosol of simulated respiratory droplet particles of the size considered most important for transmitting SARS-CoV-2. The receiver's exposure was maximally reduced (>95%) when the source and receiver were fitted with modified medical procedure masks. These laboratory-based experiments highlight the importance of good fit to optimize mask performance. Until vaccine-induced population immunity is achieved, universal masking is a highly effective means to slow the spread of SARS-CoV-2** when combined with other protective measures, such as physical distancing, avoiding crowds and poorly ventilated indoor spaces, and good hand hygiene. Innovative efforts to improve the fit of cloth and medical procedure masks to enhance their performance merit attention.

A comparison of performance metrics for cloth face masks as source control devices for simulated cough and exhalation aerosols.

Universal mask wearing is recommended by the Centers for Disease Control and Prevention to help control the spread of COVID-19. Masks reduce the expulsion of respiratory aerosols (called source control) and offer some protection to the wearer. However, masks vary greatly in their designs and construction materials, and it is not clear which are most effective. Our study tested 15 reusable cloth masks (which included face masks, neck gaiters, and bandanas), two medical masks, and two N95 filtering facepiece respirators as source control devices for aerosols ≤ 7 µm produced during simulated coughing and exhalation. These measurements were compared with the mask filtration efficiencies, airflow resistances, and fit factors. The source control collection efficiencies for the cloth masks ranged from 17% to 71% for coughing and 35% to 66% for exhalation. The filtration efficiencies of the cloth masks ranged from 1.4% to 98%, while the fit factors were 1.3 to 7.4 on an elastomeric manikin headform and 1.0 to 4.0 on human test subjects. The correlation coefficients between the source control efficacies and the other performance metrics ranged from 0.31 to 0.66 and were significant in all but one case. However, none of the alternative metrics were strong predictors of the source control performance of cloth masks. Our results suggest that a better understanding of the relationships between source control performance and metrics like filtration efficiency, airflow resistance, and fit factor are needed to develop simple methods to estimate the effectiveness of masks as source control devices for respiratory aerosols.

A comparison of performance metrics for cloth masks as source control devices for simulated cough and exhalation aerosols.

Universal mask wearing is recommended to help control the spread of COVID-19. Masks reduce the expulsion of aerosols of respiratory fluids into the environment (called source control) and offer some protection to the wearer. Masks are often characterized using filtration efficiency, airflow resistance, and manikin or human fit factors, which are standard metrics used for personal protective devices. However, none of these metrics are direct measurements of how effectively a mask blocks coughed and exhaled aerosols. We studied the source control performance of 15 cloth masks (face masks, neck gaiters, and bandanas), two medical masks, and two N95 filtering facepiece respirators by measuring their ability to block aerosols ≤ 7 µm expelled during simulated coughing and exhalation (called source control collection efficiency). These measurements were compared with filtration efficiencies, airflow resistances, and fit factors measured on manikin headforms and humans. Collection efficiencies for the cloth masks ranged from 17% to 71% for coughing and 35% to 66% for exhalation. Filtration efficiencies for the cloth masks ranged from 1.4% to 98%, while the fit factors were 1.3 to 7.4 on headforms and 1.0 to 4.0 on human subjects. The Spearman's rank correlation coefficients between the source control collection efficiencies and the standard metrics ranged from 0.03 to 0.68 and were significant in all but two cases. However, none of the standard metrics were strongly correlated with source control performance. A better understanding of the relationships between source control collection efficiency, filtration efficiency, airflow resistance, and fit factor is needed.

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.

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.

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.

Direct stimulation of human fibroblasts by nCeO2 in vitro is attenuated with an amorphous silica coating.

BACKGROUND: Nano-scaled cerium oxide (nCeO2) is used in a variety of applications, including use as a fuel additive, catalyst, and polishing agent, yet potential adverse health effects associated with nCeO2 exposure remain incompletely understood. Given the increasing utility and demand for engineered nanomaterials (ENMs) such as nCeO2, "safety-by-design" approaches are currently being sought, meaning that the physicochemical properties (e.g., size and surface chemistry) of the ENMs are altered in an effort to maximize functionality while minimizing potential toxicity. In vivo studies have shown in a rat model that inhaled nCeO2 deposited deep in the lung and induced fibrosis. However, little is known about how the physicochemical properties of nCeO2, or the coating of the particles with a material such as amorphous silica (aSiO2), may affect the bio-activity of these particles. Thus, we hypothesized that the physicochemical properties of nCeO2 may explain its potential to induce fibrogenesis, and that a nano-thin aSiO2 coating on nCeO2 may counteract that effect. RESULTS: Primary normal human lung fibroblasts were treated at occupationally relevant doses with nCeO2 that was either left uncoated or was coated with aSiO2 (amsCeO2). Subsequently, fibroblasts were analyzed for known hallmarks of fibrogenesis, including cell proliferation and collagen production, as well as the formation of fibroblastic nodules. The results of this study are consistent with this hypothesis, as we found that nCeO2 directly induced significant production of collagen I and increased cell proliferation in vitro, while amsCeO2 did not. Furthermore, treatment of fibroblasts with nCeO2, but not amsCeO2, significantly induced the formation of fibroblastic nodules, a clear indicator of fibrogenicity. Such in vitro data is consistent with recent in vivo observations using the same nCeO2 nanoparticles and relevant doses. This effect appeared to be mediated through TGFß signaling since chemical inhibition of the TGFß receptor abolished these responses. CONCLUSIONS: These results indicate that differences in the physicochemical properties of nCeO2 may alter the fibrogenicity of this material, thus highlighting the potential benefits of "safety-by-design" strategies. In addition, this study provides an efficient in vitro method for testing the fibrogenicity of ENMs that strongly correlates with in vivo findings.

Identification of TGF-ß receptor-1 as a key regulator of carbon nanotube-induced fibrogenesis.

Carbon nanotubes (CNTs) induce rapid interstitial lung fibrosis, but the underlying mechanisms are unclear. Previous studies indicated that the ability of CNTs to penetrate lung epithelium, enter interstitial tissue, and stimulate fibroblasts to produce collagen matrix is important to lung fibrosis. In this study, we investigated the activation of transforming growth factor-ß receptor-1 [TGF-ß R1; i.e., activin receptor-like kinase 5 (ALK5) receptor] and TGF-ß/Smad signaling pathway in CNT-induced collagen production in human lung fibroblasts. Human lung fibroblasts and epithelial cells were exposed to low, physiologically relevant concentrations (0.02-0.6 µg/cm(2)) of single-walled CNTs (SWCNT) and multiwalled CNTs (MWCNT) in culture and analyzed for collagen, TGF-ß1, TGF-ß R1, and SMAD proteins by Western blotting and immunofluorescence. Chemical inhibition of ALK5 and short-hairpin (sh) RNA targeting of TGF-ß R1 and Smad2 were used to probe the fibrogenic mechanism of CNTs. Both SWCNT and MWCNT induced an overexpression of TGF-ß1, TGF-ß R1 and Smad2/3 proteins in lung fibroblasts compared with vehicle or ultrafine carbon black-exposed controls. SWCNT- and MWCNT-induced collagen production was blocked by ALK5 inhibitor or shRNA knockdown of TGF-ß R1 and Smad2. Our results indicate the critical role of TGF-ß R1/Smad2/3 signaling in CNT-induced fibrogenesis by upregulating collagen production in lung fibroblasts. This novel finding may aid in the design of mechanism-based risk assessment and development of rapid screening tests for nanomaterial fibrogenicity.

Potential in vitro model for testing the effect of exposure to nanoparticles on the lung alveolar epithelial barrier.

Pulmonary barrier function plays a pivotal role in protection from inhaled particles. However, some nano-scaled particles, such as carbon nanotubes (CNT), have demonstrated the ability to penetrate this barrier in animal models, resulting in an unusual, rapid interstitial fibrosis. To delineate the underlying mechanism and specific bio-effect of inhaled nanoparticles in respiratory toxicity, models of lung epithelial barriers are required that allow accurate representation of in vivo systems; however, there is currently a lack of consistent methods to do so. Thus, this work demonstrates a well-characterized in vitro model of pulmonary barrier function using Calu-3 cells, and provides the experimental conditions required for achieving tight junction complexes in cell culture, with trans-epithelial electrical resistance measurement used as a biosensor for proper barrier formation and integrity. The effects of cell number and serum constituents have been examined and we found that changes in each of these parameters can greatly affect barrier formation. Our data demonstrate that use of 5.0 × 104 Calu-3 cells/well in the Transwell cell culture system, with 10% serum concentrations in culture media is optimal for assessing epithelial barrier function. In addition, we have utilized CNT exposure to analyze the dose-, time-, and nanoparticle property-dependent alterations of epithelial barrier permeability as a means to validate this model. Such high throughput in vitro cell models of the epithelium could be used to predict the interaction of other nanoparticles with lung epithelial barriers to mimic respiratory behavior in vivo, thus providing essential tools and bio-sensing techniques that can be uniformly employed.