Characterisation of a smoke/ aerosol exposure in vitro system (SAEIVS) for delivery of complex mixtures directly to cells at the air-liquid interface.

In vitro testing is important to characterise biological effects of consumer products, including nicotine delivery products such as cigarettes, e-cigarettes and heated tobacco products. Users' cells are exposed to these products' aerosols, of variant chemical compositions, as they move along the respiratory tract. In vitro exposure systems are available to model such exposures, including delivery of whole aerosols to cells, and at the air-liquid interface. Whilst there are clear advantages of such systems, factors including time to aerosol delivery, aerosol losses and number of cell cultures that can be exposed at one time could be improved. This study aimed to characterise a custom-built smoke/ aerosol exposure in vitro system (SAEIVS) using 1R6F reference cigarette smoke. This system contains five parallel smoking chambers and delivers different dilutions of smoke/ aerosol to two separate cell culture exposure chambers in <10 s. Using two dosimetry measures (optical density 400 nm [OD400 ]; mass spectrometric nicotine quantification), the SAEIVS demonstrated excellent linearity of smoke dilution prior to exposure (R2  = 0.9951 for mass spectrometric quantification; R2  = 0.9965 for OD400 ) and consistent puff-wise exposures across 24 and 96 well plates in cell culture relevant formats (e.g., within inserts). Smoke loss was lower than previously reported for other systems (OD400 : 16%; nicotine measurement: 20%). There was good correlation of OD400 and nicotine measurements, indicating that OD was a useful surrogate for exposure dosimetry for the product tested. The findings demonstrated that the SAEIVS is a fit-for-purpose exposure system for the reproducible dose-wise exposure assessment of nicotine delivery product aerosols.

Inhibition of Autophagy Negates Radiofrequency-Induced Adaptive Response in SH-SY5Y Neuroblastoma Cells.

In the last years, radiofrequency (RF) has demonstrated that it can reduce DNA damage induced by a subsequent treatment with chemical or physical agents in different cell types, resembling the adaptive response, a phenomenon well documented in radiobiology. Such an effect has also been reported by other authors both in vitro and in vivo, and plausible hypotheses have been formulated, spanning from the perturbation of the cell redox status, to DNA repair mechanisms, and stress response machinery, as possible cellular mechanisms activated by RF pre-exposure. These mechanisms may underpin the observed phenomenon, and require deeper investigations. The present study aimed to determine whether autophagy contributes to RF-induced adaptive response. To this purpose, SH-SY5Y human neuroblastoma cells were exposed for 20 h to 1950 MHz, UMTS signal, and then treated with menadione. The results obtained indicated a reduction in menadione-induced DNA damage, assessed by applying the comet assay. Such a reduction was negated when autophagy was inhibited by bafilomycin A1 and E64d. Moreover, CRISPR SH-SY5Y cell lines defective for ATG7 or ATG5 genes did not show an adaptive response. These findings suggest the involvement of autophagy in the RF-induced adaptive response in human neuroblastoma cells; although, further investigation is required to extend such observation at the molecular level.

ELF Magnetic Field Exposure System for In Vitro Studies Based on Lee-Whiting Coils.

In order to run a series of in vitro studies on the effect of extremely low-frequency magnetic fields on cell cultures, developing and characterizing an appropriate exposure system is required. The present design is based on a two-shielded Lee-Whiting coils system. The circular design was chosen because its axial symmetry allowed for both reducing simulation unknowns and measurement points during the characterization, and additionally made the machining of the parts easier. The system can generate magnetic flux densities (B fields) up to 1 mT root-mean-square amplitude (rms) with no active cooling system in the incubator, and up to 3 mTrms with it. The double-wrapped windings with twisted pairs allow for the use of each set of coils either as exposure or control with no detectable parasitic B field in the control. The artifacts have also been analyzed; the B field in the center of the sham control chamber is about 1 µTrms for a maximum of 3 mTrms in the exposure chamber, the parasitic incident electric fields are less than 1 V/m, the temperature difference between sham and exposure chamber is less than or equal to 0.2 °C, and the typical vibration difference between sham and exposure is less than 0.1 m/s2 . © 2020 Bioelectromagnetics Society.

Proposal of 28 GHz In Vitro Exposure System Based on Field Uniformity for Three-Dimensional Cell Culture Experiments.

This paper proposes a novel in vitro exposure system operating at millimeter-wave (mmWave) 28 GHz, one of the frequency bands under consideration for fifth generation (5G) communication. We employed the field uniformity concept along cross-sectional observation planes at shorter distances from the radiation antenna for better efficiency and a small-size system. A choke-ring antenna was designed for this purpose in consideration of a wider beamwidth (BW) and a symmetric far-field pattern across three principal planes. The permittivity of Dulbecco's modified Eagle's medium solution was measured to examine the specific absorption rate (SAR) of the skin cell layer inside a Petri dish model for a three-dimensional (3D) cell culture in vitro experiment. The best deployment of Petri dishes, taking into account a geometrical field symmetry, was proposed. Local SAR values within the cell layer among the Petri dishes were determined with different polarization angles. It was determined that this polarization effect should be considered when the actual exposure and deployment were conducted. We finally proposed an in vitro exposure system based on the field uniformity including downward exposure from an antenna for 3D cell culture experiments. A small-size chamber system was obtained, and the size was estimated using the planar near-field chamber design rule. Bioelectromagnetics. 2019;40:445-457. © 2019 Bioelectromagnetics Society.

A new cell culture exposure system for studying the toxicity of volatile chemicals at the air-liquid interface.

A cell culture exposure system (CCES) was developed to expose cells established at an air-liquid interface (ALI) to volatile chemicals. We characterized the CCES by exposing indigo dye-impregnated filter inserts inside culture wells to 125 ppb ozone (O3) for 1 h at flow rates of 5 and 25 mL/min/well; the reaction of O3 with an indigo dye produces a fluorescent product. A 5-fold increase in fluorescence at 25 mL/min/well versus 5 mL/min/well was observed, suggesting higher flows were more effective. We then exposed primary human bronchial epithelial cells (HBECs) to 0.3 ppm acrolein for 2 h at 3, 5, and 25 mL/min/well and compared our results against well-established in vitro exposure chambers at the U.S. EPA's Human Studies Facility (HSF Chambers). We measured transcript changes of heme oxygenase-1 (HMOX1) and interleukin-8 (IL-8), as well as lactate dehydrogenase (LDH) release, at 0, 1, and 24 h post-exposure. Comparing responses from HSF Chambers to the CCES, differences were only observed at 1 h post-exposure for HMOX1. Here, the HSF Chamber produced a ∼6-fold increase while the CCES at 3 and 5 mL/min/well produced a ∼1.7-fold increase. Operating the CCES at 25 mL/min/well produced a ∼4.5-fold increase; slightly lower than the HSF Chamber. Our biological results, supported by our comparison against the HSF Chambers, agree with our fluorescence results, suggesting that higher flows through the CCES are more effective at delivering volatile chemicals to cells. This new CCES will be deployed to screen the toxicity of volatile chemicals in EPA's chemical inventories.

A matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies.

Knowledge of biological reactivity and underlying toxicity mechanisms of airborne particulate matter (PM) is central to the characterization of the risk associated with these pollutants. An integrated screening platform consisting of protein profiling of cellular responses and cytotoxic analysis was developed in this study for the estimation of PM potencies. Mouse macrophage (J774A.1) and human lung epithelial cells (A549) were exposed in vitro to Ottawa urban particles (EHC6802) and two reference mineral particles (TiO2 and SiO2 ). Samples from the in vitro exposure experiment were tested following an integrated classical cytotoxicity/toxicoproteomic assessment approach for cellular viability (CellTiter Blue®, lactate dehydrogenase) and proteomic analyses. Cellular proteins were pre-fractionated by molecular weight cut-off filtration, digested enzymatically and were analyzed by matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry for protein profiling and identification. Optimization of detergent removal, pre-fractionation strategies and enzymatic digestion procedures led to increased tryptic peptide (m/z) signals with reduced sample processing times, for small total protein contents. Proteomic analyses using this optimized procedure identified statistically significant (P < 0.05) PM dose-dependent changes at the molecular level. Ranking of PM potencies based on toxicoproteomic analysis were in line with classical cytotoxicity potency-based ranking. The high content toxicoproteomic approach exhibited the potential to add value to risk characterization of environmental PM exposures by complementing and validating existing cytotoxicity testing strategies.

Analysis of nitromethane from samples exposed in vitro to chloropicrin by stable isotope dilution headspace gas chromatography with mass spectrometry.

Chloropicrin (trichloronitromethane) is a widely used soil fumigant and an old chemical warfare agent. The metabolism of chloropicrin is not well known in mammals but nitromethane has been shown to be one of its main metabolites. Here, a fast and simple headspace gas chromatography with mass spectrometry method was applied for the measurement of nitromethane from aqueous samples. The analytical method was validated using stable isotope labeled internal standard and a small sample volume of 260 µL. No conventional sample preparation steps were needed. The method was accurate (relative standard deviations ≤1.5%) and linear (R(2) = 0.9996) within the concentration range of 0.1-6.0 µg/mL. This method was used to measure nitromethane in in vitro incubations with human and pig liver cell fractions containing enzymes for xenobiotic metabolism, exposed to chloropicrin. The results indicate that the presence of glutathione is necessary for the formation of nitromethane from chloropicrin. Also, nitromethane was formed mostly in liver cytosol fractions, but not in microsomal fractions after the incubation with chloropicrin. Our results suggest that although nitromethane is not the unequivocal biomarker of chloropicrin exposure, this method could be applied for screening the elevated levels in humans after chloropicrin exposure.

In vitro and in vivo toxicity of urban and rural particulate matter from California.

Particulate matter (PM) varies in chemical composition and mass concentration based on location, source, and particle size. This study sought to evaluate the in vitro and in vivo toxicity of coarse (PM10-2.5) and fine (PM25) PM samples collected at 5 diverse sites within California. Coarse and fine PM samples were collected simultaneously at 2 rural and 3 urban sites within California during the summer. A human pulmonary microvascular endothelial cell line (HPMEC-ST1.6R) was exposed to PM suspensions (50 µg/mL) and analyzed for reactive oxygen species (ROS) after 5 hours of treatment. In addition, FVB/N mice were exposed by oropharyngeal aspiration to 50 µg PM, and lavage fluid was collected 24 hrs post-exposure and analyzed for total protein and %PMNs. Correlations between trace metal concentrations, endotoxin, and biological endpoints were calculated, and the effect of particle size range, locale (urban vs. rural), and location was determined. Absolute principal factor analysis was used to identify pollution sources of PM from elemental tracers of those sources. Ambient PM elicited an ROS and pro-inflammatory-related response in the cell and mouse models, respectively. These responses were dependent on particle size, locale, and location. Trace elements associated with soil and traffic markers were most strongly linked to the adverse effects in vitro and in vivo. Particle size, location, source, and composition of PM collected at 5 locations in California affected the ROS response in human pulmonary endothelial cells and the inflammatory response in mice.

Global gene expression analysis reveals novel transcription factors associated with long-term low-level exposure of EA.hy926 human endothelial cells to bisphenol A.

Bisphenol A (BPA) is an endocrine disruptor that binds to estrogen receptors (ER); however, studies have shown that the ER pathway was not always the primary molecular mechanism of BPA's action in cells and that gene transcription could be altered by different exposure times and doses. Here, we sought to understand the correlation between the BPA-responsive genes that have associated biological functions and the transcription factors (TFs) involved in their regulation by repeatedly exposing human endothelial cells EA.hy926 to three nanomolar concentrations of BPA (10-9 M, 10-8 M, and 10-7 M) for 14 weeks, after which changes in global gene expression were determined by RNA sequencing. Cytoscape plug-in iRegulon was used to infer TFs involved in the control of BPA-deregulated genes. The results show a minimal overlap in deregulated genes between three concentrations of BPA, with 10-9 M BPA having the highest number of deregulated genes. TF analysis suggests that all three concentrations of BPA were active in the absence of an ER-mediated pathway. A unique set of TFs (NES≥4) has been identified for each BPA concentration, including the NFκB family and CEBPB for 10-9 M BPA, MEF family, AHR/ARNT, and ZBTB33 for 10-8 M BPA, and IRF1-7 and OVOL1/OVOL2 for 10-7 M BPA, whereas STAT1/STAT2 were common TFs for 10-9 M and 10-7 M BPA. Overall, our data suggest that long-term low-level exposure of EA.hy926 cells to BPA leads to concentration-specific changes in gene expression that are not controlled by the ER-mediated signaling but rather by other mechanisms.

Biodiesel feedstock determines exhaust toxicity in 20% biodiesel: 80% mineral diesel blends.

To address climate change concerns, and reduce the carbon footprint caused by fossil fuel use, it is likely that blend ratios of renewable biodiesel with commercial mineral diesel fuel will steadily increase, resulting in biodiesel use becoming more widespread. Exhaust toxicity of unblended biodiesels changes depending on feedstock type, however the effect of feedstock on blended fuels is less well known. The aim of this study was to assess the impact of biodiesel feedstock on exhaust toxicity of 20% blended biodiesel fuels (B20). Primary human airway epithelial cells were exposed to exhaust diluted 1/15 with air from an engine running on conventional ultra-low sulfur diesel (ULSD) or 20% blends of soy, canola, waste cooking oil (WCO), tallow, palm or cottonseed biodiesel in diesel. Physico-chemical exhaust properties were compared between fuels and the post-exposure effect of exhaust on cellular viability and media release was assessed 24 h later. Exhaust properties changed significantly between all fuels with cottonseed B20 being the most different to both ULSD and its respective unblended biodiesel. Exposure to palm B20 resulted in significantly decreased cellular viability (96.3 ± 1.7%; p < 0.01) whereas exposure to soy B20 generated the greatest number of changes in mediator release (including IL-6, IL-8 and TNF-α, p < 0.05) when compared to air exposed controls, with palm B20 and tallow B20 closely following. In contrast, canola B20 and WCO B20 were the least toxic with only mediators G-CSF and TNF-α being significantly increased. Therefore, exposure to palm B20, soy B20 and tallow B20 were found to be the most toxic and exposure to canola B20 and WCO B20 the least. The top three most toxic and the bottom three least toxic B20 fuels are consistent with their unblended counterparts, suggesting that feedstock type greatly impacts exhaust toxicity, even when biodiesel only comprises 20% of the fuel.

The effect of airborne Palladium nanoparticles on human lung cells, endothelium and blood - A combinatory approach using three in vitro models.

A better understanding of the mechanisms behind adverse health effects caused by airborne fine particles and nanoparticles (NP) is essential to improve risk assessment and identification the most critical particle exposures. While the use of automobile catalytic converters is decreasing the exhausts of harmful gases, concentrations of fine airborne particles and nanoparticles (NPs) from catalytic metals such as Palladium (Pd) are reaching their upper safe level. Here we used a combinatory approach with three in vitro model systems to study the toxicity of Pd particles, to infer their potential effects on human health upon inhalation. The three model systems are 1) a lung system with human lung cells (ALI), 2) an endothelial cell system and 3) a human whole blood loop system. All three model systems were exposed to the exact same type of Pd NPs. The ALI lung cell exposure system showed a clear reduction in cell growth from 24 h onwards and the effect persisted over a longer period of time. In the endothelial cell model, Pd NPs induced apoptosis, but not to the same extent as the most aggressive types of NPs such as TiO2. Similarly, Pd triggered clear coagulation and contact system activation but not as forcefully as the highly thrombogenic TiO2 NPs. In summary, we show that our 3-step in vitro model of the human lung and surrounding vessels can be a useful tool for studying pathological events triggered by airborne fine particles and NPs.

Toxicological characterization of produced water from the Permian Basin.

Produced water (PW) is a hypersaline waste stream generated from the shale oil and gas industry, consisting of numerous anthropogenic and geogenic compounds. Despite prior geochemical characterization, the comprehensive toxicity assessment is lacking for evaluating treatment technologies and the beneficial use of PW. In this study, a suite of in vitro toxicity assays using various aquatic organisms (luminescent bacterium Vibrio fischeri, fish gill cell line RTgill-W1, and microalgae Scenedesmus obliquus) were developed to investigate the toxicological characterizations of PW from the Permian Basin. The exposure to PW, PW inorganic fraction (PW-IF), and PW salt control (PW-SC) at 30-50% dilutions caused significant toxicological effects in all model species, revealing the high salinity was the foremost toxicological driver in PW. In addition, the toxicity level of PW was usually higher than that of PW-IF, suggesting that organic contaminants might also play a critical role in PW toxicity. When comparing the observed toxicity with associated chemical characterizations in different PW samples, strong correlations were found between them since higher concentrations of contaminants could generally result in higher toxicity towards exposed organisms. Furthermore, the toxicity results from the pretreated PW indicated that those in vitro toxicity assays had different sensitives to the chemical components present in PW. As expected, the combination of multiple pretreatments could lead to a more significant decrease in toxicity compared to the single pretreatment since the mixture of contaminants in PW might exhibit synergistic toxicity. Overall, the current work is expected to enhance our understanding of the potential toxicological impacts of PW to aquatic ecosystems and the relationships between the chemical profiles and observed toxicity in PW, which might be conducive to the establishment of monitoring, remediation, treatment, and reuse protocols for PW.

In Vitro Systems for Toxicity Evaluation of Microbial Volatile Organic Compounds on Humans: Current Status and Trends.

Microbial volatile organic compounds (mVOC) are metabolic products and by-products of bacteria and fungi. They play an important role in the biosphere: They are responsible for inter- and intra-species communication and can positively or negatively affect growth in plants. But they can also cause discomfort and disease symptoms in humans. Although a link between mVOCs and respiratory health symptoms in humans has been demonstrated by numerous studies, standardized test systems for evaluating the toxicity of mVOCs are currently not available. Also, mVOCs are not considered systematically at regulatory level. We therefore performed a literature survey of existing in vitro exposure systems and lung models in order to summarize the state-of-the-art and discuss their suitability for understanding the potential toxic effects of mVOCs on human health. We present a review of submerged cultivation, air-liquid-interface (ALI), spheroids and organoids as well as multi-organ approaches and compare their advantages and disadvantages. Furthermore, we discuss the limitations of mVOC fingerprinting. However, given the most recent developments in the field, we expect that there will soon be adequate models of the human respiratory tract and its response to mVOCs.

Emission Factors of CO2 and Airborne Pollutants and Toxicological Potency of Biofuels for Airplane Transport: A Preliminary Assessment.

Aviation is one of the sectors affecting climate change, and concerns have been raised over the increase in the number of flights all over the world. To reduce the climate impact, efforts have been dedicated to introducing biofuel blends as alternatives to fossil fuels. Here, we report environmentally relevant data on the emission factors of biofuel/fossil fuel blends (from 13 to 17% v/v). Moreover, in vitro direct exposure of human bronchial epithelial cells to the emissions was studied to determine their potential intrinsic hazard and to outline relevant lung doses. The results show that the tested biofuel blends do not reduce the emissions of particles and other chemical species compared to the fossil fuel. The blends do reduce the elemental carbon (less than 40%) and total volatile organic compounds (less than 30%) compared to fossil fuel emissions. The toxicological outcomes show an increase in oxidative cellular response after only 40 min of exposure, with biofuels causing a lower response compared to fossil fuels, and lung-deposited doses show differences among the fuels tested. The data reported provide evidence of the possibility to reduce the climate impact of the aviation sector and contribute to the risk assessment of biofuels for aviation.

Fuel feedstock determines biodiesel exhaust toxicity in a human airway epithelial cell exposure model.

BACKGROUND: Biodiesel is promoted as a sustainable replacement for commercial diesel. Biodiesel fuel and exhaust properties change depending on the base feedstock oil/fat used during creation. The aims of this study were, for the first time, to compare the exhaust exposure health impacts of a wide range of biodiesels made from different feedstocks and relate these effects with the corresponding exhaust characteristics. METHOD: Primary airway epithelial cells were exposed to diluted exhaust from an engine running on conventional diesel and biodiesel made from Soy, Canola, Waste Cooking Oil, Tallow, Palm and Cottonseed. Exhaust properties and cellular viability and mediator release were analysed post exposure. RESULTS: The exhaust physico-chemistry of Tallow biodiesel was the most different to diesel as well as the most toxic, with exposure resulting in significantly decreased cellular viability (95.8 ± 6.5%) and increased release of several immune mediators including IL-6 (+223.11 ± 368.83 pg/mL) and IL-8 (+1516.17 ± 2908.79 pg/mL) above Air controls. In contrast Canola biodiesel was the least toxic with exposure only increasing TNF-α (4.91 ± 8.61). CONCLUSION: This study, which investigated the toxic effects for the largest range of biodiesels, shows that exposure to different exhausts results in a spectrum of toxic effects in vitro when combusted under identical conditions.

Effect of Different Species of Prorocentrum Genus on the Japanese Oyster Crassostrea gigas Proteomic Profile.

This paper assesses the effects of exposure to toxic concentrations (1200 to 6000 cells/mL) of the dinoflagellates Prorocentrum lima, Prorocentrum minimum, and Prorocentrum rhathymum and several concentrations of aqueous and organic extracts obtained from the same species (0 to 20 parts per thousand) on the Crassostrea gigas (5-7 mm) proteomic profile. Through comparative proteomic map analyses, several protein spots were detected with different expression levels, of which eight were selected to be identified by liquid chromatography-mass spectrometry (LC-MS/MS) analyses. The proteomic response suggests that, after 72 h of exposure to whole cells, the biological functions of C. gigas affected proteins in the immune system, stress response, contractile systems and cytoskeletal activities. The exposure to organic and aqueous extracts mainly showed effects on protein expressions in muscle contraction and cytoskeleton morphology. These results enrich the knowledge on early bivalve developmental stages. Therefore, they may be considered a solid base for new bioassays and/or generation of specific analytical tools that allow for some of the main effects of algal proliferation phenomena on bivalve mollusk development to be monitored, characterized and elucidated.

ECAM: A low-cost vaping device for generating and collecting electronic cigarette condensate for in vitro studies.

The use of electronic cigarettes (ECs) has become widespread despite many unknowns around their long-term health impact. ECs work by vapourising a liquid, known as an e-liquid, typically consisting of propylene glycol, glycerol, flavourings and nicotine. The chemical constituents and resultant impact on cells and tissue are dependent on several factors, including the flavourings used, the vaping topography/use pattern, and the device used. ECAM (Electronic Cigarette Aerosol Machine) is an open source, portable device for creating EC aerosol - for condensate collection and in vitro studies - using a controlled methodology. ECAM was developed as a low cost, automated, and customisable alternative to commercial devices. ECAM consists of a micro diaphragm gas pump to draw air/EC aerosol through the system. The device is automated using an Arduino and solenoid pinch valves are used to alternate between air and EC vapour. Condensate is collected in a vial within a cold-water bath. Each ECAM unit uses a temperature/humidity sensor to measure ambient air conditions and a differential pressure sensor to determine the pressure within the system. ECAM is programmed to adhere to International Standards Organisation 20768:2018. The design files, source code, and build instructions for this device can be found at https://dx.doi.org/10.17605/OSF.IO/3NGU4.

Comparison of passive-dosed and solvent spiked exposures of pro-carcinogen, benzo[a]pyrene, to human lymphoblastoid cell line, MCL-5.

Genotoxicity testing methods in vitro provide a means to predict the DNA damaging effects of chemicals on human cells. This is hindered in the case of hydrophobic test compounds, however, which will partition to in vitro components such as plastic-ware and medium proteins, in preference to the aqueous phase of the exposure medium. This affects the freely available test chemical concentration, and as this freely dissolved aqueous concentration is that bioavailable to cells, it is important to define and maintain this exposure. Passive dosing promises to have an advantage over traditional 'solvent spiking' exposure methods and involves the establishment and maintenance of known chemical concentrations in the in vitro medium, and therefore aqueous phase. Passive dosing was applied in a novel format to expose the MCL-5 human lymphoblastoid cell line to the pro-carcinogen, benzo[a]pyrene (B[a]P) and was compared to solvent (dimethyl sulphoxide) spiked B[a]P exposures over 48 h. Passive dosing induced greater changes, at lower concentrations, to micronucleus frequency, p21 mRNA expression, cell cycle abnormalities, and cell and nuclear morphology. This was attributed to a maintained, definable, free chemical concentration using passive dosing and the presence or absence of solvent, and highlights the influence of exposure choice on genotoxic outcomes.

Delivery efficiencies of constituents of combustion-derived aerosols across the air-liquid interface during in vitro exposures.

In vitro aerosol exposure of epithelial cells grown at the air-liquid interface is an experimental methodology widely used in respiratory toxicology. The exposure depends to a large part on the physicochemical properties of individual aerosol constituents, as they determine the transfer kinetics from the aerosol into the cells. We characterized the transfer of 70 cigarette smoke constituents from the smoke into aqueous samples exposed in the Vitrocell® 24/48 aerosol exposure system. The amounts of these compounds in the applied smoke were determined by trapping whole smoke in N,N-dimethylformamide and then compared with their amounts in smoke-exposed, phosphate-buffered saline, yielding compound specific delivery efficiencies. Delivery efficiencies of different smoke constituents differed by up to five orders of magnitude, which indicates that the composition of the applied smoke is not necessarily representative for the delivered smoke. Therefore, dose metrics for in vitro exposure experiments should, if possible, be based on delivered and not applied doses. A comparison to literature on in vivo smoke retention in the respiratory tract indicated that the same applies for smoke retention in the respiratory tract.

Cytotoxicity of carbon nanotube variants: a comparative in vitro exposure study with A549 epithelial and J774 macrophage cells.

While production of engineered carbon nanotubes (CNTs) has escalated in recent years, knowledge of risk associated with exposure to these materials remains unclear. We report on the cytotoxicity of four CNT variants in human lung epithelial cells (A549) and murine macrophages (J774). Morphology, metal content, aggregation/agglomeration state, pore volume, surface area and modifications were determined for the pristine and oxidized single-walled (SW) and multi-walled (MW) CNTs. Cytotoxicity was evaluated by cellular ATP content, BrdU incorporation, lactate dehydrogenase (LDH) release, and CellTiter-Blue (CTB) reduction assays. All CNTs were more cytotoxic than respirable TiO2 and SiO2 reference particles. Oxidation of CNTs removed most metallic impurities but introduced surface polar functionalities. Although slopes of fold changes for cytotoxicity endpoints were steeper with J774 compared to A549 cells, CNT cytotoxicity ranking in both cell types was assay-dependent. Based on CTB reduction and BrdU incorporation, the cytotoxicity of the polar oxidized CNTs was higher compared to the pristine CNTs. In contrast, pristine CNTs were more cytotoxic than oxidized CNTs when assessed for cellular ATP and LDH. Correlation analyses between CNTs' physico-chemical properties and average relative potency revealed the impact of metal content and surface area on the potency values estimated using ATP and LDH assays, while surface polarity affected the potency values estimated from CTB and BrdU assays. We show that in order to reliably estimate the risk posed by these materials, in vitro toxicity assessment of CNTs should be conducted with well characterized materials, in multiple cellular models using several cytotoxicity assays that report on distinct cellular processes.