RESUMO
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.
Assuntos
Catepsina B , Lipopolissacarídeos , Masculino , Humanos , Camundongos , Animais , Catepsina B/metabolismo , Catepsina B/farmacologia , Lipopolissacarídeos/farmacologia , Ensaios de Triagem em Larga Escala , Inflamação/induzido quimicamente , Inflamação/metabolismo , Macrófagos , Citocinas/metabolismo , Interleucina-1beta/metabolismoRESUMO
OBJECTIVES: A cure for cancer is out of reach for most patients due to chemoresistance. Cancer-associated fibroblasts (CAFs) play a vital role in cancer chemoresistance, but detailed understanding of the process particularly in chemoresistant lung cancer is lacking. In this study, we investigated programmed death-ligand 1 (PDL-1) as a potential biomarker for CAF-induced chemoresistance and evaluated its role and the underlying mechanisms of chemoresistance in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: A systemic search of gene expression profiles of multiple tissues in NSCLC was carried out to determine the expression intensities of traditional fibroblast biomarkers and CAF-secreted protumorigenic cytokines. PDL-1 expression in CAFs was analyzed by ELISA, Western blotting, and flow cytometry. Human cytokine array was used to identify specific cytokines secreted from CAFs. Role of PDL-1 in NSCLC chemoresistance was assessed using CRISPR/Cas9 knockdown and various functional assays including MTT, cell invasion, sphere formation, and cell apoptosis. In vivo experiments were conducted using a co-implantation xenograft mouse model with live cell imaging and immunohistochemistry. RESULTS: We demonstrated that chemotherapy-stimulated CAFs promoted tumorigenic and stem cell-like properties of NSCLC cells, which contribute to their chemoresistance. Subsequently, we revealed that PDL-1 expression is upregulated in chemotherapy-treated CAFs and is associated with poor prognosis. Silencing PDL-1 expression suppressed CAFs' ability to promote stem cell-like properties and invasiveness of lung cancer cells, favoring chemoresistance. Mechanistically, an upregulation of PDL-1 in chemotherapy-treated CAFs led to an increase in hepatocyte growth factor (HGF) secretion, which stimulates cancer progression, cell invasion, and stemness of lung cancer cells, while inhibiting apoptosis. CONCLUSION: Our results show that PDL-1-positive CAFs modulate stem cell-like properties of NSCLC cells by secreting elevated HGF, thereby promoting chemoresistance. Our finding supports PDL-1 in CAFs as a chemotherapy response biomarker and as a drug delivery and therapeutic target for chemoresistant NSCLC.
Assuntos
Antineoplásicos , Fibroblastos Associados a Câncer , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Fibroblastos Associados a Câncer/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fibroblastos , Citocinas/metabolismo , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
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.
Assuntos
6-Aminonicotinamida , Via de Pentose FosfatoRESUMO
This study investigated the inhalation toxicity of the emissions from 3-D printing with acrylonitrile butadiene styrene (ABS) filament using an air-liquid interface (ALI) in vitro model. Primary normal human-derived bronchial epithelial cells (NHBEs) were exposed to ABS filament emissions in an ALI for 4 hours. The mean and mode diameters of ABS emitted particles in the medium were 175 ± 24 and 153 ± 15 nm, respectively. The average particle deposition per surface area of the epithelium was 2.29 × 107 ± 1.47 × 107 particle/cm2, equivalent to an estimated average particle mass of 0.144 ± 0.042 µg/cm2. Results showed exposure of NHBEs to ABS emissions did not significantly affect epithelium integrity, ciliation, mucus production, nor induce cytotoxicity. At 24 hours after the exposure, significant increases in the pro-inflammatory markers IL-12p70, IL-13, IL-15, IFN-γ, TNF-α, IL-17A, VEGF, MCP-1, and MIP-1α were noted in the basolateral cell culture medium of ABS-exposed cells compared to non-exposed chamber control cells. Results obtained from this study correspond with those from our previous in vivo studies, indicating that the increase in inflammatory mediators occur without associated membrane damage. The combination of the exposure chamber and the ALI-based model is promising for assessing 3-D printer emission-induced toxicity.
Assuntos
Acrilonitrila , Poluição do Ar em Ambientes Fechados , Acrilonitrila/toxicidade , Poluição do Ar em Ambientes Fechados/análise , Butadienos/toxicidade , Células Epiteliais , Humanos , Tamanho da Partícula , Material Particulado , Impressão Tridimensional , Estireno/análise , Estireno/toxicidadeRESUMO
The lung is an organ that is directly exposed to the external environment. Given the large surface area and extensive ventilation of the lung, it is prone to exposure to airborne substances, such as pathogens, allergens, chemicals, and particulate matter. Highly elaborate and effective mechanisms have evolved to protect and maintain homeostasis in the lung. Despite these sophisticated defense mechanisms, the respiratory system remains highly susceptible to environmental challenges. Because of the impact of respiratory exposure on human health and disease, there has been considerable interest in developing reliable and predictive in vitro model systems for respiratory toxicology and basic research. Human air-liquid-interface (ALI) organotypic airway tissue models derived from primary tracheobronchial epithelial cells have in vivo-like structure and functions when they are fully differentiated. The presence of the air-facing surface allows conducting in vitro exposures that mimic human respiratory exposures. Exposures can be conducted using particulates, aerosols, gases, vapors generated from volatile and semi-volatile substances, and respiratory pathogens. Toxicity data have been generated using nanomaterials, cigarette smoke, e-cigarette vapors, environmental airborne chemicals, drugs given by inhalation, and respiratory viruses and bacteria. Although toxicity evaluations using human airway ALI models require further standardization and validation, this approach shows promise in supplementing or replacing in vivo animal models for conducting research on respiratory toxicants and pathogens.
Assuntos
Ar , Brônquios/citologia , Células Epiteliais/citologia , Modelos Biológicos , Traqueia/citologia , Técnicas de Cultura de Células , Humanos , Testes de ToxicidadeRESUMO
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.
Assuntos
Poluentes Atmosféricos/toxicidade , Incineração , Nanotubos de Carbono/química , Material Particulado/toxicidade , Plásticos/toxicidade , Brônquios , Linhagem Celular , Dano ao DNA , Células Epiteliais , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
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 (â¼0.58 µg/cm2 delivered dose), SiO2-nFe2O3 (â¼0.55 µg/cm2 delivered dose), or gas metal arc mild steel welding fumes (GMA-MS, â¼0.58 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.
Assuntos
Carcinógenos/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Compostos Férricos/toxicidade , Nanopartículas Metálicas/toxicidade , Dióxido de Silício/química , Carcinógenos/química , Proliferação de Células/efeitos dos fármacos , DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Compostos Férricos/química , Humanos , Nanopartículas Metálicas/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Three independently conducted asbestos exposure evaluations were conducted using wire gauze pads similar to standard practice in the laboratory setting. All testing occurred in a controlled atmosphere inside an enclosed chamber simulating a laboratory setting. Separate teams consisting of a laboratory technician, or technician and assistant simulated common tasks involving wire gauze pads, including heating and direct wire gauze manipulation. Area and personal air samples were collected and evaluated for asbestos consistent with the National Institute of Occupational Safety Health method 7400 and 7402, and the Asbestos Hazard Emergency Response Act (AHERA) method. Bulk gauze pad samples were analyzed by Polarized Light Microscopy and Transmission Electron Microscopy to determine asbestos content. Among air samples, chrysotile asbestos was the only fiber found in the first and third experiments, and tremolite asbestos for the second experiment. None of the air samples contained asbestos in concentrations above the current permissible regulatory levels promulgated by OSHA. These findings indicate that the level of asbestos exposure when working with wire gauze pads in the laboratory setting is much lower than levels associated with asbestosis or asbestos-related lung cancer and mesothelioma.
Assuntos
Absorventes Higiênicos , Poluição do Ar em Ambientes Fechados/análise , Amianto/análise , Monitoramento Ambiental , Laboratórios , Pesquisa , Amianto/administração & dosagem , HumanosRESUMO
Acrolein is a highly reactive unsaturated aldehyde that is formed during the burning of gasoline and diesel fuels, cigarettes, woods and plastics. In addition, acrolein is generated during the cooking or frying of food with fats or oils. Acrolein is also used in the synthesis of many organic chemicals and as a biocide in agricultural and industrial water supply systems. The total emissions of acrolein in the United States from all sources are estimated to be 62,660 tons/year. Acrolein is classified by the Environmental Protection Agency as a high-priority air and water toxicant. Acrolein can exert toxic effects following inhalation, ingestion, and dermal exposures that are dose dependent. Cardiovascular tissues are particularly sensitive to the toxic effects of acrolein based primarily on in vitro and in vivo studies. Acrolein can generate free oxygen radical stress in the heart, decrease endothelial nitric oxide synthase phosphorylation and nitric oxide formation, form cytoplasmic and nuclear protein adducts with myocyte and vascular endothelial cell proteins and cause vasospasm. In this manner, chronic exposure to acrolein can cause myocyte dysfunction, myocyte necrosis and apoptosis and ultimately lead to cardiomyopathy and cardiac failure. Epidemiological studies of acrolein exposure and toxicity should be developed and treatment strategies devised that prevent or significantly limit acrolein cardiovascular toxicity.
Assuntos
Acroleína/efeitos adversos , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/epidemiologia , Poluentes Ambientais/efeitos adversos , Animais , Doenças Cardiovasculares/metabolismo , Fumar Cigarros/efeitos adversos , Fumar Cigarros/epidemiologia , Humanos , Estresse Oxidativo/fisiologiaRESUMO
This investigation aims to assess whether the hepatocellular carcinoma cell line, HepG2, is an appropriate model to assess the role of poly (ADP-ribose) polymerase (PARP) during acute ethanol toxicosis. HepG2 cells were dosed with graded concentrations of ethanol, ranging from 100 mM to 800 mM, for 6 hours to assess PARP activity induction, while another parallel experiment examined cellular damage via medium aspartate aminotransferase activity and cellular viability via MTT reduction. Aspartate aminotransferase activity was significantly elevated at 600 mM ethanol (FOLD; P < 0.01), with further increases at the 800 mM dose (1.43 fold; P < 0.001), compared to controls. Cellular viability was not significantly decreased compared to controls among all dose groups. PARP activity measured in total cell lysates showed a significant decreasing trend with respect to ethanol dose, reaching statistical significance at the 100 mM dose group (P < 0.05). Paradoxically, exposure to 50 µM etoposide (Positive apoptosis-inducing control) did not demonstrate significant PARP activity ablation. When analyzing PARP activity observation temporally, a significant correlation (R(2) =0.5314) was observed between activity and assay sequence. Overall, a clear HepG2 insensitivity to ethanol was observed.