Effective density of inhaled environmental and engineered nanoparticles and its impact on the lung deposition and dosimetry.

BACKGROUND: Airborne environmental and engineered nanoparticles (NPs) are inhaled and deposited in the respiratory system. The inhaled dose of such NPs and their deposition location in the lung determines their impact on health. When calculating NP deposition using particle inhalation models, a common approach is to use the bulk material density, ρb, rather than the effective density, ρeff. This neglects though the porous agglomerate structure of NPs and may result in a significant error of their lung-deposited dose and location. RESULTS: Here, the deposition of various environmental NPs (aircraft and diesel black carbon, wood smoke) and engineered NPs (silica, zirconia) in the respiratory system of humans and mice is calculated using the Multiple-Path Particle Dosimetry model accounting for their realistic structure and effective density. This is done by measuring the NP ρeff which was found to be up to one order of magnitude smaller than ρb. Accounting for the realistic ρeff of NPs reduces their deposited mass in the pulmonary region of the respiratory system up to a factor of two in both human and mouse models. Neglecting the ρeff of NPs does not alter significantly the distribution of the deposited mass fractions in the human or mouse respiratory tract that are obtained by normalizing the mass deposited at the head, tracheobronchial and pulmonary regions by the total deposited mass. Finally, the total deposited mass fraction derived this way is in excellent agreement with those measured in human studies for diesel black carbon. CONCLUSIONS: The doses of inhaled NPs are overestimated by inhalation particle deposition models when the ρb is used instead of the real-world effective density which can vary significantly due to the porous agglomerate structure of NPs. So the use of realistic ρeff, which can be measured as described here, is essential to determine the lung deposition and dosimetry of inhaled NPs and their impact on public health.

E-cigarette vaping associated acute lung injury (EVALI): state of science and future research needs.

"E-Cigarette (e-cig) Vaping-Associated Acute Lung Injury" (EVALI) has been linked to vitamin-E-acetate (VEA) and Δ-9-tetrahydrocannabinol (THC), due to their presence in patients' e-cigs and biological samples. Lacking standardized methodologies for patients' data collection and comprehensive physicochemical/toxicological studies using real-world-vapor exposures, very little data are available, thus the underlying pathophysiological mechanism of EVALI is still unknown. This review aims to provide a comprehensive and critical appraisal of existing literature on clinical/epidemiological features and physicochemical-toxicological characterization of vaping emissions associated with EVALI. The literature review of 161 medical case reports revealed that the predominant demographic pattern was healthy white male, adolescent, or young adult, vaping illicit/informal THC-containing e-cigs. The main histopathologic pattern consisted of diffuse alveolar damage with bilateral ground-glass-opacities at chest radiograph/CT, and increased number of macrophages or neutrophils and foamy-macrophages in the bronchoalveolar lavage. The chemical analysis of THC/VEA e-cig vapors showed a chemical difference between THC/VEA and the single THC or VEA. The chemical characterization of vapors from counterfeit THC-based e-cigs or in-house-prepared e-liquids using either cannabidiol (CBD), VEA, or medium-chain triglycerides (MCT), identified many toxicants, such as carbonyls, volatile organic compounds, terpenes, silicon compounds, hydrocarbons, heavy metals, pesticides and various industrial/manufacturing/automotive-related chemicals. There is very scarce published toxicological data on emissions from THC/VEA e-liquids. However, CBD, MCT, and VEA emissions exert varying degrees of cytotoxicity, inflammation, and lung damage, depending on puffing topography and cell line. Major knowledge gaps were identified, including the need for more systematic-standardized epidemiological surveys, comprehensive physicochemical characterization of real-world e-cig emissions, and mechanistic studies linking emission properties to specific toxicological outcomes.

Printer center nanoparticles alter the DNA repair capacity of human bronchial airway epithelial cells.

Nano-enabled, toner-based printing equipment emit nanoparticles during operation. The bioactivity of these nanoparticles as documented in a plethora of published toxicological studies raises concerns about their potential health effects. These include pro-inflammatory effects that can lead to adverse epigenetic alterations and cardiovascular disorders in rats. At the same time, their potential to alter DNA repair pathways at realistic doses remains unclear. In this study, size-fractionated, airborne particles from a printer center in Singapore were sampled and characterized. The PM0.1 size fraction (particles with an aerodynamic diameter less than 100 nm) of printer center particles (PCP) were then administered to human lung adenocarcinoma (Calu-3) or lymphoblastoid (TK6) cells. We evaluated plasma membrane integrity, mitochondrial activity, and intracellular reactive oxygen species (ROS) generation. Moreover, we quantified DNA damage and alterations in the cells' capacity to repair 6 distinct types of DNA lesions. Results show that PCP altered the ability of Calu-3 cells to repair 8oxoG:C lesions and perform nucleotide excision repair, in the absence of acute cytotoxicity or DNA damage. Alterations in DNA repair capacity have been correlated with the risk of various diseases, including cancer, therefore further genotoxicity studies are needed to assess the potential risks of PCP exposure, at both occupational settings and at the end-consumer level.

E-Cigarette (E-Cig) Liquid Composition and Operational Voltage Define the In Vitro Toxicity of Δ8Tetrahydrocannabinol/Vitamin E Acetate (Δ8THC/VEA) E-Cig Aerosols.

The 2019 United States outbreak of E-cigarette (e-cig), or Vaping, Associated Acute Lung Injury (EVALI) has been linked to presence of vitamin E acetate (VEA) in Δ8tetrahydrocannabinol (Δ8THC)-containing e-liquids, as supported by VEA detection in patient biological samples. However, the pathogenesis of EVALI and the complex physicochemical properties of e-cig emissions remain unclear, raising concerns on health risks of vaping. This study investigates the effect of Δ8THC/VEA e-liquids and e-cig operational voltage on in vitro toxicity of e-cig aerosols. A novel E-cigExposure Generation System platform was used to generate and characterize e-cig aerosols from a panel of Δ8THC/VEA or nicotine-based e-liquids at 3.7 or 5 V. Human lung Calu-3 cells and THP-1 monocytes were exposed to cell culture media conditioned with collected e-cig aerosol condensate at doses of 85 and 257 puffs/m2 lung surface for 24 h, whereafter specific toxicological endpoints were assessed (including cytotoxicity, metabolic activity, reactive oxygen species generation, apoptosis, and inflammatory cytokines). Higher concentrations of gaseous volatile organic compounds were emitted from Δ8THC/VEA compared with nicotine-based e-liquids, especially at 5 V. Emitted PM2.5 concentrations in aerosol were higher for Δ8THC/VEA at 5 V and averagely for nicotine-based e-liquids at 3.7 V. Overall, aerosols from nicotine-based e-liquids showed higher bioactivity than Δ8THC/VEA aerosols in THP-1 cells, with no apparent differences in Calu-3 cells. Importantly, presence of VEA in Δ8THC and menthol flavoring in nicotine-based e-liquids increased cytotoxicity of aerosols across both cell lines, especially at 5 V. This study systematically investigates the physicochemical and toxicological properties of a model of Δ8THC/VEA and nicotine e-cigarette condensate exposure demonstrating that pyrolysis of these mixtures can generate hazardous toxicants whose synergistic actions potentially drive acute lung injury upon inhalation.

Toxicity, uptake, and nuclear translocation of ingested micro-nanoplastics in an in vitro model of the small intestinal epithelium.

Despite mounting evidence of increasing micro- and nanoplastics (MNPs) in natural environments, food, and drinking water, little is known of the potential health hazards of MNPs ingestion. We assessed toxicity and uptake of environmentally relevant MNPs in an in vitro small intestinal epithelium (SIE). Test MNPs included 25 and 1000 nm polystyrene (PS) microspheres (PS25 and PS1K); 25, 100, and 1000 nm carboxyl modified PS spheres (PS25C, PS100C, and PS1KC), and secondary MNPs from incinerated polyethylene (PEI). MNPs were subjected to 3-phase digestion to mimic transformations in the gastrointestinal tract (GIT) and digestas applied to the SIE. Carboxylated MNPs significantly reduced viability and increased permeability to 3 kD dextran. Uptake of carboxyl PS materials was size dependent, with significantly greater uptake of PS25C. Fluorescence confocal imaging showed some PS25C agglomerates entering cells independent of endosomes (suggesting diffusion), others within actin shells (suggesting phagocytosis), and many free within the epithelial cells, including agglomerates within nuclei. Pre-treatment with the dynamin inhibitor Dyngo partially reduced PS25 translocation, suggesting a potential role for endocytosis. These findings suggest that ingestion exposures to MNPs could have serious health consequences and underscore the urgent need for additional detailed studies of the potential hazards of ingested MNPs.

Oxidized carbon black nanoparticles induce endothelial damage through C-X-C chemokine receptor 3-mediated pathway.

Oxidation of engineered nanomaterials during application in various industrial sectors can alter their toxicity. Oxidized nanomaterials also have widespread industrial and biomedical applications. In this study, we evaluated the cardiopulmonary hazard posed by these nanomaterials using oxidized carbon black (CB) nanoparticles (CBox) as a model particle. Particle surface chemistry was characterized by X-ray photo electron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). Colloidal characterization and in vitro dosimetry modeling (particle kinetics, fate and transport modeling) were performed. Lung inflammation was assessed following oropharyngeal aspiration of CB or oxidized CBox particles (20 µg per mouse) in C57BL/6J mice. Toxicity and functional assays were also performed on murine macrophage (RAW 264.7) and endothelial cell lines (C166) with and without pharmacological inhibitors. Oxidant generation was assessed by electron paramagnetic resonance spectroscopy (EPR) and via flow cytometry. Endothelial toxicity was evaluated by quantifying pro-inflammatory mRNA expression, monolayer permeability, and wound closure. XPS and FTIR spectra indicated surface modifications, the appearance of new functionalities, and greater oxidative potential (both acellular and in vitro) of CBox particles. Treatment with CBox demonstrated greater in vivo inflammatory potentials (lavage neutrophil counts, secreted cytokine, and lung tissue mRNA expression) and air-blood barrier disruption (lavage proteins). Oxidant-dependent pro-inflammatory signaling in macrophages led to the production of CXCR3 ligands (CXCL9,10,11). Conditioned medium from CBox-treated macrophages induced significant elevation in endothelial cell pro-inflammatory mRNA expression, enhanced monolayer permeability and impairment of scratch healing in CXCR3 dependent manner. In summary, this study mechanistically demonstrated an increased biological potency of CBox particles and established the role of macrophage-released chemical mediators in endothelial damage.

Fate, cytotoxicity and cellular metabolomic impact of ingested nanoscale carbon dots using simulated digestion and a triculture small intestinal epithelial model.

Carbon dots (CDs) are a promising material currently being explored in many industrial applications in the biomedical and agri-food areas; however, studies supporting the environmental health risk assessment of CDs are needed. This study focuses on various CD forms including iron (FeCD) and copper (CuCD) doped CDs synthesized using hydrothermal method, their fate in gastrointestinal tract, and their cytotoxicity and potential changes to cellular metabolome in a triculture small intestinal epithelial model. Physicochemical characterization revealed that 75% of Fe in FeCD and 95% of Cu in CuCD were dissolved during digestion. No significant toxic effects were observed for pristine CDs and FeCDs. However, CuCD induced significant dose-dependent toxic effects including decreases in TEER and cell viability, increases in cytotoxicity and ROS production, and alterations in important metabolites, including D-glucose, L-cysteine, uridine, citric acid and multiple fatty acids. These results support the current understanding that pristine CDs are relatively non-toxic and the cytotoxicity is dependent on the doping molecules.

High-Throughput Screening Platform for Nanoparticle-Mediated Alterations of DNA Repair Capacity.

The potential genotoxic effects of engineered nanomaterials (ENMs) may occur through the induction of DNA damage or the disruption of DNA repair processes. Inefficient DNA repair may lead to the accumulation of DNA lesions and has been linked to various diseases, including cancer. Most studies so far have focused on understanding the nanogenotoxicity of ENM-induced damages to DNA, whereas the effects on DNA repair have been widely overlooked. The recently developed fluorescence multiplex-host-cell reactivation (FM-HCR) assay allows for the direct quantification of multiple DNA repair pathways in living cells and offers a great opportunity to address this methodological gap. Herein an FM-HCR-based method is developed to screen the impact of ENMs on six major DNA repair pathways using suspended or adherent cells. The sensitivity and efficiency of this DNA repair screening method were demonstrated in case studies using primary human small airway epithelial cells and TK6 cells exposed to various model ENMs (CuO, ZnO, and Ga2O3) at subcytotoxic doses. It was shown that ENMs may inhibit nucleotide-excision repair, base-excision repair, and the repair of oxidative damage by DNA glycosylases in TK6 cells, even in the absence of significant genomic DNA damage. It is of note that the DNA repair capacity was increased by some ENMs, whereas it was suppressed by others. Overall, this method can be part of a multitier, in vitro hazard assessment of ENMs as a functional, high-throughput platform that provides insights into the interplay of the properties of ENMs, the DNA repair efficiency, and the genomic stability.

Effects of Ingested Nanomaterials on Tissue Distribution of Co-ingested Zinc and Iron in Normal and Zinc-Deficient Mice.

Cellulose nanofibers (CNF) reduced serum triglyceride levels in rats when co-administered with heavy cream by gavage. Do CNF and other nanomaterials (NMs) alter the tissue distribution and retention of co-administered metal ions? We evaluated whether 5 different NMs affected tissue distribution of co-ingested 65Zn++ and 59Fe+++ in zinc-replete versus zinc-deficient mice. Male C57BL/6J mice were fed either zinc-replete or zinc-deficient diets for 3 weeks, followed by gavage with NM suspensions in water containing both 65ZnCl2 and 59FeCl3. Urine and feces were measured for 48 h post-gavage. Mice were euthanized and samples of 22 tissues were collected and analyzed for 65Zn and 59Fe in a gamma counter. Our data show that zinc deficiency alters the tissue distribution of 65Zn but not of 59Fe, indicating that zinc and iron homeostasis are regulated by distinct mechanisms. Among the tested NMs, soluble starch-coated chitosan nanoparticles, cellulose nanocrystals, and TiO2 reduced Zn and Fe tissue retention in zinc-deficient but not in zinc-replete animals.

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.

Inflammation Increases Susceptibility of Human Small Airway Epithelial Cells to Pneumonic Nanotoxicity.

Exposure to inhaled anthropogenic nanomaterials (NM) with dimension <100 nm has been implicated in numerous adverse respiratory outcomes. Although studies have identified key NM physiochemical determinants of pneumonic nanotoxicity, the complex interactive and cumulative effects of NM exposure, especially in individuals with preexisting inflammatory respiratory diseases, remain unclear. Herein, the susceptibility of primary human small airway epithelial cells (SAEC) exposed to a panel of reference NM, namely, CuO, ZnO, mild steel welding fume (MSWF), and nanofractions of copier center particles (Nano-CCP), is examined in normal and tumor necrosis factor alpha (TNF-α)-induced inflamed SAEC. Compared to normal SAEC, inflamed cells display an increased susceptibility to NM-induced cytotoxicity by 15-70% due to a higher basal level of intracellular reactive oxygen species (ROS). Among the NM screened, ZnO, CuO, and Nano-CCP are observed to trigger an overcompensatory response in normal SAEC, resulting in an increased tolerance against subsequent oxidative insults. However, the inflamed SAEC fails to adapt to the NM exposure due to an impaired nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated cytoprotective response. The findings reveal that susceptibility to pulmonary nanotoxicity is highly dependent on the interplay between NM properties and inflammation of the alveolar milieu.

Human brain microvascular endothelial cell pairs model tissue-level blood-brain barrier function.

The blood-brain barrier plays a critical role in delivering oxygen and nutrients to the brain while preventing the transport of neurotoxins. Predicting the ability of potential therapeutics and neurotoxicants to modulate brain barrier function remains a challenge due to limited spatial resolution and geometric constraints offered by existing in vitro models. Using soft lithography to control the shape of microvascular tissues, we predicted blood-brain barrier permeability states based on structural changes in human brain endothelial cells. We quantified morphological differences in nuclear, junction, and cytoskeletal proteins that influence, or indicate, barrier permeability. We established a correlation between brain endothelial cell pair structure and permeability by treating cell pairs and tissues with known cytoskeleton-modulating agents, including a Rho activator, a Rho inhibitor, and a cyclic adenosine monophosphate analog. Using this approach, we found that high-permeability cell pairs showed nuclear elongation, loss of junction proteins, and increased actin stress fiber formation, which were indicative of increased contractility. We measured traction forces generated by high- and low-permeability pairs, finding that higher stress at the intercellular junction contributes to barrier leakiness. We further tested the applicability of this platform to predict modulations in brain endothelial permeability by exposing cell pairs to engineered nanomaterials, including gold, silver-silica, and cerium oxide nanoparticles, thereby uncovering new insights into the mechanism of nanoparticle-mediated barrier disruption. Overall, we confirm the utility of this platform to assess the multiscale impact of pharmacological agents or environmental toxicants on blood-brain barrier integrity.

Pilot deep RNA sequencing of worker blood samples from Singapore printing industry for occupational risk assessment.

Several engineered nanomaterials (ENMs) are used in toner-based printing equipment (TPE) including laser printers and photocopiers to improve toner performance. High concentration of airborne nanoparticles due to TPE emissions has been documented in copy centers and chamber studies. Recent animal inhalation studies by our group suggested exposure to laser printer-emitted nanoparticles (PEPs) increased cardiovascular risk by impairing ventricular performance and inducing hypertension and arrhythmia, consistent with global transcriptomic and metabolomic profiling results. There has been no genome-wide transcriptomic analysis of workers exposed to TPE emissions to systematically assess the occupational exposure health risks. In this pilot study, deep RNA sequencing of blood samples of workers in two printing companies in Singapore was performed. The genome-scale analysis of the blood samples from TPE exposed workers revealed perturbed transcriptional activities related to inflammatory and immune responses, metabolism, cardiovascular impairment, neurological diseases, oxidative stress, physical morphogenesis/deformation, and cancer, when compared with the control peers (office workers). Many of these disease risks associated with particle inhalation exposures in such work environments were consistent with the observation from the PEPs rat inhalation studies. In particular, the cell adhesion molecules (CAMs) was a top significantly perturbed pathway in blood samples from exposed workers compared with the office workers in both companies. The protein expression of sICAM was verified in plasma of exposed workers, showing a positive correlation with daily average nanoparticle concentration in indoor air measured in these two companies. Larger scale genomic and molecular epidemiology studies in copier operators are warranted in order to assess potential risks from such particulate matter exposures.

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.

Safer-by-design flame-sprayed silicon dioxide nanoparticles: the role of silanol content on ROS generation, surface activity and cytotoxicity.

BACKGROUND: Amorphous silica nanoparticles (SiO2 NPs) have been regarded as relatively benign nanomaterials, however, this widely held opinion has been questioned in recent years by several reports on in vitro and in vivo toxicity. Surface chemistry, more specifically the surface silanol content, has been identified as an important toxicity modulator for SiO2 NPs. Here, quantitative relationships between the silanol content on SiO2 NPs, free radical generation and toxicity have been identified, with the purpose of synthesizing safer-by-design fumed silica nanoparticles. RESULTS: Consistent and statistically significant trends were seen between the total silanol content, cell membrane damage, and cell viability, but not with intracellular reactive oxygen species (ROS), in the macrophages RAW264.7. SiO2 NPs with lower total silanol content exhibited larger adverse cellular effects. The SAEC epithelial cell line did not show any sign of toxicity by any of the nanoparticles. Free radical generation and surface reactivity of these nanoparticles were also influenced by the temperature of combustion and total silanol content. CONCLUSION: Surface silanol content plays an important role in cellular toxicity and surface reactivity, although it might not be the sole factor influencing fumed silica NP toxicity. It was demonstrated that synthesis conditions for SiO2 NPs influence the type and quantity of free radicals, oxidative stress, nanoparticle interaction with the biological milieu they come in contact with, and determine the specific mechanisms of toxicity. We demonstrate here that it is possible to produce much less toxic fumed silicas by modulating the synthesis conditions.

Integrated Transcriptomics, Metabolomics, and Lipidomics Profiling in Rat Lung, Blood, and Serum for Assessment of Laser Printer-Emitted Nanoparticle Inhalation Exposure-Induced Disease Risks.

Laser printer-emitted nanoparticles (PEPs) generated from toners during printing represent one of the most common types of life cycle released particulate matter from nano-enabled products. Toxicological assessment of PEPs is therefore important for occupational and consumer health protection. Our group recently reported exposure to PEPs induces adverse cardiovascular responses including hypertension and arrythmia via monitoring left ventricular pressure and electrocardiogram in rats. This study employed genome-wide mRNA and miRNA profiling in rat lung and blood integrated with metabolomics and lipidomics profiling in rat serum to identify biomarkers for assessing PEPs-induced disease risks. Whole-body inhalation of PEPs perturbed transcriptional activities associated with cardiovascular dysfunction, metabolic syndrome, and neural disorders at every observed time point in both rat lung and blood during the 21 days of exposure. Furthermore, the systematic analysis revealed PEPs-induced transcriptomic changes linking to other disease risks in rats, including diabetes, congenital defects, auto-recessive disorders, physical deformation, and carcinogenesis. The results were also confirmed with global metabolomics profiling in rat serum. Among the validated metabolites and lipids, linoleic acid, arachidonic acid, docosahexanoic acid, and histidine showed significant variation in PEPs-exposed rat serum. Overall, the identified PEPs-induced dysregulated genes, molecular pathways and functions, and miRNA-mediated transcriptional activities provide important insights into the disease mechanisms. The discovered important mRNAs, miRNAs, lipids and metabolites may serve as candidate biomarkers for future occupational and medical surveillance studies. To the best of our knowledge, this is the first study systematically integrating in vivo, transcriptomics, metabolomics, and lipidomics to assess PEPs inhalation exposure-induced disease risks using a rat model.

Nano-TiO2 Drives Epithelial-Mesenchymal Transition in Intestinal Epithelial Cancer Cells.

The majority of cancer mortality is associated with cancer metastasis. Epithelial-to-mesenchymal transition (EMT) is a process by which cells attain migratory and invasive properties, eventually leading to cancer metastasis. Here, it is shown that titanium dioxide nanoparticles (nano-TiO2 ), a common food additive, can induce the EMT process in colorectal cancer cells. Nano-TiO2 exposure is observed to activate transforming growth factor-ß (TGF-ß)/mitogen-activated protein kinase (MAPK) and wingless (Wnt) pathways, and drive the EMT process. Similarly, silica nanoparticles (nano-SiO2 ) and hydroxyapatite nanoparticles (nano-HA), as food-based additives, can be ingested and accumulated in the stomach, and are found to be able to induce the EMT progression. The implication of this work can be profound for colorectal cancer patients where these food additives may unknowingly and unnecessarily hasten the progression of their cancers.

Assessing electronic cigarette emissions: linking physico-chemical properties to product brand, e-liquid flavoring additives, operational voltage and user puffing patterns.

Users of electronic cigarettes (e-cigs) are exposed to particles and other gaseous pollutants. However, major knowledge gaps on the physico-chemical properties of such exposures and contradictory data in published literature prohibit health risk assessment. Here, the effects of product brand, type, e-liquid flavoring additives, operational voltage, and user puffing patterns on emissions were systematically assessed using a recently developed, versatile, e-cig exposure generation platform and state-of-the-art analytical methods. Parameters of interest in this systematic evaluation included two brands (A and B), three flavors (tobacco, menthol, and fruit), three types of e-cigs (disposable, pre-filled, and refillable tanks), two puffing protocols (4 and 2 s/puff), and four operational voltages (2.2-5.7 V). Particles were generated at a high number concentration (106-107 particles/cm3). The particle size distribution was bi-modal (∼200 nm and 1 µm). Furthermore, organic species (humectants propylene glycol and glycerin, nicotine) that were present in e-liquid and trace metals (potassium and sodium) that were present on e-cig heating coil were also released into the emission. In addition, combustion-related byproducts, such as benzene and toluene, were also detected in the range of 100-38,000 ppbv/puff. Parametric analyzes performed in this study show the importance of e-cig brand, type, flavor additives, user puffing pattern (duration and frequency), and voltage on physico-chemical properties of emissions. This observed influence is indicative of the complexity associated with the toxicological screening of emissions from e-cigs and needs to be taken into consideration.

Assessment of reactive oxygen species generated by electronic cigarettes using acellular and cellular approaches.

Electronic cigarettes (e-cigs) have fast increased in popularity but the physico-chemical properties and toxicity of the generated emission remain unclear. Reactive oxygen species (ROS) are likely present in e-cig emission and can play an important role in e-cig toxicity. However, e-cig ROS generation is poorly documented. Here, we generated e-cig exposures using a recently developed versatile exposure platform and performed systematic ROS characterization on e-cig emissions using complementary acellular and cellular techniques: 1) a novel acellular Trolox-based mass spectrometry method for total ROS and hydrogen peroxide (H2O2) detection, 2) electron spin resonance (ESR) for hydroxyl radical detection in an acellular and cellular systems and 3) in vitro ROS detection in small airway epithelial cells (SAEC) using the dihydroethidium (DHE) assay. Findings confirm ROS generation in cellular and acellular systems and is highly dependent on the e-cig brand, flavor, puffing pattern and voltage. Trolox method detected a total of 1.2-8.9nmol H2O2eq./puff; H2O2 accounted for 12-68% of total ROS. SAEC cells exposed to e-cig emissions generated up to eight times more ROS compared to control. The dependency of e-cig emission profile on e-cig features and operational parameters should be taken into consideration in toxicological studies.

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.