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1.
Sci Rep ; 11(1): 4747, 2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33637800

RESUMEN

Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0-1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.

2.
BMC Res Notes ; 13(1): 492, 2020 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-33087173

RESUMEN

OBJECTIVES: Cigarette smoke aqueous aerosol extracts (AqE) have been used for assessing tobacco products, particularly with in vitro models such as oxidative stress and inflammation. These test articles can be generated easily, but there are no standardised methods for the generation and characterisation or stability. We investigated the effects of pro-oxidant smoke-derived chemicals by using 3R4F AqE generated under standardised conditioning and smoking regimes and assessed the stability over 31-week timeframe. Twenty batches generated from ten puffs per cigarette bubbled through 20 ml cell culture media were used fresh and thawed from frozen aliquots stored at - 80 ºC. RESULTS: Nicotine levels quantified by gas chromatography/mass spectrometry and optical density at 260 nm showed chemical and physical stability from week 0 (fresh sample) to weeks 1, 4, 8 and 31 (frozen samples). No significant change in H292 human bronchial epithelial cell viability or oxidative stress were observed between fresh AqE at week 0 and frozen AqE at 31 weeks. AqEs generated by our protocol were stable for up to 31 weeks for all tested end points, suggesting that it may not be necessary to use freshly generated AqE for each study, thus reducing batch-to-batch variability.

3.
Toxicol Lett ; 335: 51-63, 2020 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-33091563

RESUMEN

Electronic cigarettes (e-cigarettes) and tobacco heating products (THPs) have reduced yields of toxicants and have recently emerged as a potentially safer alternative to combustible cigarettes. To understand if reduced toxicant exposure is associated with reductions in biological responses, there is a need for high-quality pre-clinical in vitro studies. Here, we investigated the cytotoxic response of human umbilical vein endothelial cells to conventional cigarette aqueous aerosol extracts (AqE) and highly concentrated AqEs from e-cigarettes (two generations of atomisers) and THPs (two variants). All AqE samples were generated by a standardized methodology and characterized for nicotine, propylene glycol and vegetable glycerol. The cigarette AqE caused a maximum 100 ± 0.00 % reduction in cell viability at 35 % dose (2.80 puffs) as opposed to 96.63 ± 2.73 % at 50 % (20 puffs) and 99.85 ± 0.23 % at 75 % (30 puffs) for the two THP variants (glo Bright Tobacco, glo Rich Tobacco), and 99.07 ± 1.61 % at the neat ePen2.0 e-cigarette (200 puffs). The AqE of the remaining e-cigarettes either resulted in an incomplete dose-response or did not elicit any response. The methods utilized were suitably sensitive to not only differentiate between cigarette, THP and e-cigarette aerosols but also to distinguish between products within each product category.

4.
Toxicol Rep ; 7: 1010-1019, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32874925

RESUMEN

In vitro studies have supported the toxicological evaluation of chemicals and complex mixtures including cigarette smoke and novel tobacco and nicotine products which include tobacco heating products (THP). This new environment requires faster testing, higher throughput and appropriate in vitro studies, to support product innovation and development. In this study, total particulate matter (TPM) from a commercially available THP and a reference cigarette (3R4F) were assessed up to 500 µg/mL using two in vitro micronucleus techniques. V79 and TK6 cells were assessed using conventional OECD 487 manual scoring techniques, whereas, CHO cells were assessed using contemporary, automated high content screening approaches (Cellomics ArrayScan® VTI). V79 cells gave the most consistent response with all three treatment conditions producing a clear positive genotoxic response. Human TK6 cells only produced dose-dependent response, indicative of a weak-positive response. CHO cells demonstrated a positive response with TPM using long (24 h) -S9 conditions. All three cell lines equally demonstrated a negative response with THP TPM up to 500 µg/mL. In conclusion, THP TPM did not increase micronuclei formation above control levels even at doses far exceeding that tested with reference cigarette smoke, in most cases up to 10x the dose delivered compared to that of cigarette smoke. This study supports the growing belief that THPs are less risky than conventional cigarettes and that 21st century screening techniques can be employed to support product design and decision making, as a potential 1st screen prior to more traditional assessments.

5.
Toxicol Lett ; 334: 110-116, 2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-32707277

RESUMEN

Endothelial cell migration is a critical process in the maintenance of healthy blood vessels. Impaired endothelial migration is reportedly associated with the development of cardiovascular diseases. Here, we report on the development of a 96-well in vitro endothelial migration assay for the purpose of comparative toxicological assessment of a novel THP relative to cigarette smoke, to be able to rapidly inform regulatory decision making. Uniform scratches were induced in confluent human umbilical vein endothelial cells using the 96-pin wound maker and exposed to 3R4F cigarette or THP aqueous extracts (AqE). Endothelial migration was recorded over 24 h, and the rate of wound closure calculated using mean relative wound density rather than migration rate as previously reported. This self-normalising parameter accounts for starting wound size, by comparing the density of the scratch to the outer region at each time-point. Furthermore, wound width acceptance criteria was defined to further increase the sensitivity of the assay. 3R4F and THP AqE samples were tested at comparable nicotine concentrations. 3R4F showed significant cytotoxicity and inhibition of wound healing whereas THP AqE did not show any response in either endpoint. This 96-well endothelial migration assay was suitably sensitive to distinguish combustible cigarette and THP test articles.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Sistemas Electrónicos de Liberación de Nicotina , Nicotina/toxicidad , Material Particulado/toxicidad , Productos de Tabaco/toxicidad , Tabaco/toxicidad , Aerosoles , Ensayos de Migración Celular , Endotelio Vascular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Células Endoteliales de la Vena Umbilical Humana , Humanos
6.
Toxicol Lett ; 315: 14-22, 2019 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-31400404

RESUMEN

In vitro testing can facilitate the rapid assessment of next generation nicotine delivery products (NGPs) with comparisons to combustible tobacco products. In vitro assays for cytotoxicity and oxidative stress were employed at BAT (UK) and JT (Japan) to test total particulate matter (TPM) of a scientific reference cigarette and aerosol collected mass (ACM) of a commercially available E-cigarette and two tobacco heating products (THP). 3R4F TPMs were generated using the Health Canada intense (HCI) regimen, a modified regime (mHCI) for the THP ACMs and the CORESTA recommended method no. 81 for the E-cigarette ACM. Human lung cells were exposed to the test product TPM/ACMs at concentrations between 0-200 µg/ml followed by the employment of commercially available assays for endpoint analysis that included reactive oxygen species (ROS) generation, the glutathione ratio (GSH:GSSG), activation of the antioxidant response elements (ARE) and cellular viability. TPM/ACM nicotine concentrations were quantified using a UPLC-PDA technique. At both laboratories the 3R4F TPM induced significant and dose-dependent responses in all in vitro assays, whereas no significant responses could be measured for the NGP ACMs. In conclusion, both laboratories obtained comparable results across all endpoints therefore demonstrating the utility of the in vitro techniques combined with standardised test products to support the assessment of NGPs.


Asunto(s)
Aerosoles/análisis , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Pulmón/efectos de los fármacos , Nicotina/análisis , Material Particulado/análisis , Productos de Tabaco/análisis , Sistemas Electrónicos de Liberación de Nicotina , Humanos , Japón , Reino Unido
7.
Food Chem Toxicol ; 132: 110546, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31163219

RESUMEN

In this study, a variety of test matrices from tobacco and nicotine delivery products were assessed against a 3R4F Kentucky reference cigarette using the in vitro micronucleus assay. Testing was conducted using two Chinese hamster cell lines (CHO and V79), and a human lymphoblastoid cell line (TK6), in accordance with established guidelines. Total particulate matter (TPM) from a 3R4F Reference cigarette was compared to an electronic cigarette e-liquid, electronic cigarette TPM and TPM from a commercial tobacco heating product using a standard and an extended treatment condition with recovery period. Cells were assessed with 3R4F TPM prior to assessment of the other tobacco and nicotine product test matrices. These cell lines gave varied responses to 3R4F TPM with the most robust response using V79 cells. The use of an extended exposure/recovery period was seen to increase assay sensitivity for CHO and V79 cell lines but was less clear for TK6 cells. Negative responses were observed for all products except 3R4F across all treatment conditions in V79 cells. The most potent response to cigarette smoke was following extended treatment with recovery, suggesting this may be a more appropriate treatment for the future assessment of tobacco and nicotine product test matrices.


Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Material Particulado/toxicidad , Productos de Tabaco/toxicidad , Contaminación por Humo de Tabaco/efectos adversos , Animales , Células CHO , Cricetulus , Humanos , Masculino , Pruebas de Micronúcleos , Mitocondrias/efectos de los fármacos , Material Particulado/análisis , Ratas Sprague-Dawley , Productos de Tabaco/análisis , Contaminación por Humo de Tabaco/análisis
8.
Food Chem Toxicol ; 132: 110584, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31228600

RESUMEN

Conduct of the mouse lymphoma assay (MLA) is underpinned by Organisation for Economic Co-operation and Development (OECD) Test Guideline 490 and International Conference on Harmonisation S2(R1) guidance and is a recognised in vitro genotoxicity test battery assay. It has been used on a limited number of occasions for the assessment of some tobacco and nicotine products, such as e-cigarettes and tobacco heating products (THP). The aim of this study was to assess the suitability of the MLA for genotoxicity testing with a variety of tobacco and nicotine products. Total particulate matter (TPM) from a 3R4F cigarette was compared against a commercial electronic cigarette liquid (e-liquid), electronic cigarette (e-cigarette) aerosol matter captured from the same e-liquid, and TPM from a commercial THP. Treatment conditions included 3 h exposures with and without metabolic activation and a longer 24 h exposure without metabolic activation (-S9) at concentrations up to 500 µg/mL. Under all treatment conditions, 3R4F produced a clear positive response with regard to induction of mutation. In contrast, no marked induction of mutation was observed for the e-liquid, e-cigarette aerosol or THP. Additionally, data are presented as a function of nicotine equivalents for comparisons between these different tobacco products and test matrices.


Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Material Particulado/toxicidad , Productos de Tabaco/toxicidad , Contaminación por Humo de Tabaco/efectos adversos , Animales , Línea Celular Tumoral , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Pruebas de Mutagenicidad , Nicotina/toxicidad , Ratas Sprague-Dawley
9.
Regul Toxicol Pharmacol ; 93: 62-70, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29080849

RESUMEN

Cigarette smoking is a major risk factor for many adverse health conditions. Novel tobacco heating products (THPs) heat tobacco, reducing exposure to many of the harmful combustion toxicants in conventional cigarette emissions. In vitro studies have been employed to support the toxicological evaluation of chemicals and complex mixtures, including cigarette smoke. The use of automated robotics platforms for in vitro toxicological screening complements traditional testing approaches. Multiparametric toxicity and oxidative stress endpoints were used to assess in vitro biological responses elicited after exposure to total particulate matter (TPM) from two commercially available THPs, and the reference tobacco product 3R4F, in human bronchial epithelial cells. A luciferase-based reporter gene assay was used to assess antioxidant response element (ARE) transcriptional activation in stably transfected H292 cells after 6 and 24 h exposures. High-content screening was used to assess 10 endpoints normal human bronchial epithelial cells after 4 or 24 h exposures. 3R4F TPM stimulated significant increases in ARE activation (p < 0.005) and moderate activity in HCS cell-based assays compared to THP at comparable doses. THPs showed little or no activity in all assays. HCS techniques can extend safety assessments providing information quickly in the early stages of product innovation and development.


Asunto(s)
Aerosoles/análisis , Sistemas Electrónicos de Liberación de Nicotina/métodos , Calefacción/métodos , Mucosa Respiratoria/efectos de los fármacos , Productos de Tabaco/análisis , Aerosoles/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Material Particulado/análisis , Material Particulado/farmacología , Mucosa Respiratoria/fisiología
10.
Regul Toxicol Pharmacol ; 93: 71-83, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29080850

RESUMEN

In vitro studies have been widely used to support the toxicological evaluation of chemicals and complex mixtures including cigarette smoke. In this study, the total particulate matter and whole aerosol from a Kentucky reference 3R4F cigarette and two commercially available tobacco heating products (THPs) were assessed using in vitro mutagenicity, cytotoxicity and tumour-promoting activity assays. The Ames assay assessed mutagenicity using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA102 ± metabolic activation (S9). The mouse lymphoma assay was used with short 3 h and longer 24 h exposures. The Bhas 42 cell transformation assay was incorporated as an in vitro alternative for detecting tumour promoters, and the neutral red uptake cell viability assay provided an acute measure of cytotoxicity. To complement the approach, the Ames assay was also employed with S. typhimurium tester strains TA98, TA100, TA1535, TA97 and TA102 using a scaled down methodology for the assessment of aerosols. All the in vitro techniques employed produced a clear positive response with cigarette smoke and in contrast, a negative response to THPs at doses equivalent to or higher than a cigarette smoke test matrix. The data show little difference between the THPs assessed suggesting parity between products.


Asunto(s)
Aerosoles/toxicidad , Sistemas Electrónicos de Liberación de Nicotina/métodos , Calefacción/métodos , Mutágenos/toxicidad , Aerosoles/análisis , Animales , Células 3T3 BALB , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Ratones , Ratones Endogámicos BALB C , Pruebas de Mutagenicidad/métodos , Mutágenos/análisis , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Pruebas de Toxicidad/métodos
11.
Regul Toxicol Pharmacol ; 93: 52-61, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28987911

RESUMEN

Tobacco heating products (THPs) represent a subset of the next-generation nicotine and tobacco product category, in which tobacco is typically heated at temperatures of 250-350 °C, thereby avoiding many of the harmful combustion-related toxicant emissions of conventional cigarettes. In this study, we have assessed aerosol generation and cytotoxicity from two commercially available THPs, THP1.0 and THS, relative to tobacco smoke from 3R4F reference cigarettes, using an adapted Borgwaldt RM20S Smoking Machine. Quantification of nicotine in the exposed cell-culture media showed greater delivery of nicotine from both THPs than from the cigarette. Using Neutral Red Uptake assay, THPs demonstrated reduced in vitro cytotoxicity in H292 human bronchial epithelial cells as compared with 3R4F cigarette exposure at the air-liquid interface (p < 0.0001). Both THPs demonstrated a statistically similar reduction in biological response, with >87% viability relative to 3R4F at a common aerosol dilution (1:40, aerosol:air). A similar response was observed when plotted against nicotine; a statistical difference between 3R4F and THPs (p < 0.0001) and no difference between the THPs (p = 0.0186). This pre-clinical in vitro biological testing forms part of a larger package of data to help assess the safety and risk reduction potential of next-generation tobacco products relative to cigarettes, using a weight of evidence approach.


Asunto(s)
Citotoxinas/análisis , Sistemas Electrónicos de Liberación de Nicotina/métodos , Calefacción/métodos , Nicotina/análisis , Productos de Tabaco/análisis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Citotoxinas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Nicotina/farmacología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/fisiología
12.
Regul Toxicol Pharmacol ; 90: 342-357, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28954704

RESUMEN

Cigarette smoking causes many human diseases including cardiovascular disease, lung disease and cancer. Novel tobacco products with reduced yields of toxicants compared to cigarettes, such as tobacco-heating products, snus and electronic cigarettes, hold great potential for reducing the harms associated with tobacco use. In the UK several public health agencies have advocated a potential role for novel products in tobacco harm reduction. Public Health England has stated that "The current best estimate is that e-cigarettes are around 95% less harmful than smoking" and the Royal College of Physicians has urged public health to "Promote e-cigarettes widely as substitute for smoking". Health related claims on novel products such as 'reduced exposure' and 'reduced risk' should be substantiated using a weight of evidence approach based on a comprehensive scientific assessment. The US FDA, has provided draft guidance outlining a framework to assess novel products as Modified Risk Tobacco Products (MRTP). Based on this, we now propose a framework comprising pre-clinical, clinical, and population studies to assess the risk profile of novel tobacco products. Additionally, the utility of this framework is assessed through the pre-clinical and part of the clinical comparison of a commercial e-cigarette (Vype ePen) with a scientific reference cigarette (3R4F) and the results of these studies suggest that ePen has the potential to be a reduced risk product.


Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina/métodos , Reducción del Daño , Nicotina/toxicidad , Productos de Tabaco/toxicidad , Dispositivos para Dejar de Fumar Tabaco/efectos adversos , Tabaco/toxicidad , Aerosoles , Guías como Asunto , Humanos , Salud Pública , Medición de Riesgo/métodos , Medición de Riesgo/normas , Fumar/efectos adversos , Prevención del Hábito de Fumar/métodos , Tabaco/química , Estados Unidos , United States Food and Drug Administration
13.
Toxicol Lett ; 277: 123-128, 2017 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-28658606

RESUMEN

Cigarette smoking is a risk factor for several diseases. There has been a steep increase in the use of e-cigarettes that may offer a safer alternative to cigarette smoking. In vitro models of smoking-related diseases may provide valuable insights into disease mechanisms associated with tobacco use and could be used to assess e-cigarettes. We previously reported the application of a 'scratch wound' assay, measuring endothelial cell migration rate following artificial wounding, in the presence or absence of cigarette smoke extracts. This study reports the comparative effects of two commercial e-cigarette products (Vype ePen and Vype eStick) and a scientific reference cigarette (3R4F) on endothelial migration in vitro. Puff-matched extracts were generated using the Health Canada Intense (HCI) regime for cigarettes and a modified HCI for e-cigarettes. Exposure to 3R4F extract (20h) induced concentration-dependent inhibition of endothelial cell migration, with complete inhibition at concentrations >20%. E-cigarette extracts did not inhibit migration, even at double the 3R4F extract nicotine concentration, allowing cells to migrate into the wounded area. Our data demonstrate that e-cigarettes do not induce the inhibition of endothelial cell migration in vitro when compared to 3R4F. The scratch wound assay enables the comparative assessment between tobacco and nicotine products in vitro.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Sistemas Electrónicos de Liberación de Nicotina/efectos adversos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humo/efectos adversos , Fumar/efectos adversos , Aerosoles , Células Cultivadas , Seguridad de Productos para el Consumidor , Relación Dosis-Respuesta a Droga , Humanos , Exposición por Inhalación/efectos adversos , Nicotina/administración & dosificación , Nicotina/toxicidad , Agonistas Nicotínicos/administración & dosificación , Agonistas Nicotínicos/toxicidad , Medición de Riesgo , Factores de Tiempo
14.
Food Chem Toxicol ; 106(Pt A): 533-546, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28595930

RESUMEN

This study assessed the toxicological and biological responses of aerosols from a novel hybrid tobacco product. Toxicological responses from the hybrid tobacco product were compared to those from a commercially available Tobacco Heating Product (c-THP), a prototype THP (p-THP) and a 3R4F reference cigarette, using in vitro test methods which were outlined as part of a framework to substantiate the risk reduction potential of novel tobacco and nicotine products. Exposure matrices used included total particulate matter (TPM), whole aerosol (WA), and aqueous aerosol extracts (AqE) obtained after machine-puffing the test products under the Health Canada Intense smoking regime. Levels of carbonyls and nicotine in these matrices were measured to understand the aerosol dosimetry of the products. The hybrid tobacco product tested negative across the in vitro assays including mutagenicity, genotoxicity, cytotoxicity, tumour promotion, oxidative stress and endothelial dysfunction. All the THPs tested demonstrated significantly reduced responses in these in vitro assays when compared to 3R4F. The findings suggest these products have the potential for reduced health risks. Further pre-clinical and clinical assessments are required to substantiate the risk reduction of these novel products at individual and population levels.


Asunto(s)
Aerosoles/química , Sistemas Electrónicos de Liberación de Nicotina/instrumentación , Aromatizantes/química , Tabaco/química , Adulto , Seguridad de Productos para el Consumidor , Sistemas Electrónicos de Liberación de Nicotina/métodos , Sistemas Electrónicos de Liberación de Nicotina/normas , Femenino , Calor , Humanos , Masculino , Mutagénesis , Material Particulado , Fumar
15.
Environ Mol Mutagen ; 58(4): 190-198, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28444993

RESUMEN

In vitro cell transformation assays (CTA) are used to assess the carcinogenic potential of chemicals and complex mixtures and can detect nongenotoxic as well as genotoxic carcinogens. The Bhas 42 CTA has been developed with both initiation and promotion protocols to distinguish between these two carcinogen classes. Cigarette smoke is known to be carcinogenic and is positive in in vitro genotoxicity assays. Cigarette smoke also contains nongenotoxic carcinogens and is a tumour promoter and cocarcinogen in vivo. We have combined a suite of in vitro assays to compare the relative biological effects of new categories of tobacco and nicotine products with traditional cigarettes. The Bhas promotion assay has been included in this test battery to provide an in vitro surrogate for detecting tumor promoters. The activity of an electronic cigarette (e-cigarette; Vype ePen) was compared to that of a reference cigarette (3R4F) in the promotion assay, using total particulate matter (TPM)/aerosol collected matter (ACM) and aqueous extracts (AqE) of product aerosol emissions. 3R4F TPM was positive in this assay at concentrations ≥6 µg/mL, while e-cigarette ACM did not have any promoter activity. AqE was found to be a lesssuitable test matrix in this assay due to high cytotoxicity. This is the first study to use the Bhas assay to compare tobacco and nicotine products and demonstrates the potential for its future application as part of a product assessment framework. These data add to growing evidence suggesting that e-cigarettes may provide a safer alternative to traditional cigarettes. Environ. Mol. Mutagen. 58:190-198, 2017. © 2017 Wiley Periodicals, Inc.


Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina/efectos adversos , Neoplasias/patología , Animales , Carcinógenos/toxicidad , Transformación Celular Neoplásica , Técnicas In Vitro , Ratones
16.
Toxicol Mech Methods ; 26(6): 465-476, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27690198

RESUMEN

Tobacco smoking is a risk factor for various diseases. The underlying cellular mechanisms are not fully characterized, but include oxidative stress, apoptosis, and necrosis. Electronic-cigarettes (e-cigarettes) have emerged as an alternative to and a possible means to reduce harm from tobacco smoking. E-cigarette vapor contains significantly lower levels of toxicants than cigarette smoke, but standardized methods to assess cellular responses to exposure are not well established. We investigated whether an in vitro model of the airway epithelium (human bronchial epithelial cells) and commercially available assays could differentiate cellular stress responses to aqueous aerosol extracts (AqE) generated from cigarette smoke and e-cigarette aerosols. After exposure to AqE concentrations of 0.063-0.500 puffs/mL, we measured the intracellular glutathione ratio (GSH:GSSG), intracellular generation of oxidant species, and activation of the nuclear factor erythroid-related factor 2 (Nrf2)-controlled antioxidant response elements (ARE) to characterize oxidative stress. Apoptotic and necrotic responses were characterized by increases in caspase 3/7 activity and reductions in viable cell protease activities. Concentration-dependent responses indicative of oxidative stress were obtained for all endpoints following exposure to cigarette smoke AqE: intracellular generation of oxidant species increased by up to 83%, GSH:GSSG reduced by 98.6% and transcriptional activation of ARE increased by up to 335%. Caspase 3/7 activity was increased by up to 37% and the viable cell population declined by up to 76%. No cellular stress responses were detected following exposure to e-cigarette AqE. The methods used were suitably sensitive to be employed for comparative studies of tobacco and nicotine products.


Asunto(s)
Aerosoles/toxicidad , Sistemas Electrónicos de Liberación de Nicotina/efectos adversos , Células Epiteliales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Humo/efectos adversos , Aerosoles/química , Bronquios/citología , Bronquios/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Células Epiteliales/metabolismo , Humanos , Nicotina/química , Nicotina/toxicidad , Tabaco/toxicidad
17.
Environ Mol Mutagen ; 55(8): 662-72, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24889675

RESUMEN

Tobacco smoke is a complex mixture of over 6,000 individual chemical constituents. Approximately 150 of these have been identified as 'tobacco smoke toxicants' due to their known toxicological effects. A number of these toxicants are present in the gaseous phase of tobacco smoke. This presents a technical challenge when assessing the toxicological effects of these chemicals in vitro. We have adapted a commercially available tobacco smoke exposure system to enable the assessment of the contribution of individual smoke toxicants to the overall toxicological effects of whole mainstream cigarette smoke (WS). Here we present a description of the exposure system and the methodology used. We use the example of a gaseous tobacco smoke toxicant, ethylene oxide (EtO), a Group 1 IARC carcinogen and known mutagen, to illustrate how this methodology can be applied to the assessment of genotoxicity of gaseous chemicals in the context of WS. In the present study we found that EtO was positive in Salmonella typhimurium strain YG1042, a strain that is sensitive to tobacco smoke. However, EtO did not increase the mutagenicity of the WS mixture when it was added at greatly higher concentrations than those found typically in WS. The findings presented here demonstrate the suitability of this exposure system for the assessment of the mutagenic potential of gases in vitro. Whilst we have focused on tobacco smoke toxicants, this system has broad application potential in studying the biological effects of exposure to a wide range of gaseous compounds that are present within complex aerosol mixtures.


Asunto(s)
Pruebas de Mutagenicidad/métodos , Humo/análisis , Contaminación por Humo de Tabaco/efectos adversos , Tabaco , Óxido de Etileno/toxicidad , Técnicas In Vitro , Pruebas de Mutagenicidad/instrumentación , Mutágenos , Salmonella typhimurium/efectos de los fármacos , Tabaco/química
18.
Altern Lab Anim ; 39(3): 233-55, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21777038

RESUMEN

Carcinogenesis is a highly complex, multi-stage process that can occur over a relatively long period before its clinical manifestation. While the sequence in which a cancer cell acquires the necessary traits for tumour formation can vary, there are a number of mechanisms that are common to most, if not all, cancers across the spectrum of possible causes. Many aspects of carcinogenesis can be modelled in vitro. This has led to the development of a number of mechanistically driven, cell-based assays to assess the pro-carcinogenic and anti-carcinogenic potential of chemicals. A review is presented of the current in vitro models that can be used to study carcinogenesis, with examples of cigarette smoke testing in some of these models, in order to illustrate their potential applications. We present an overview of the assays used in regulatory genotoxicity testing, as well as those designed to model other aspects that are considered to be hallmarks of cancer. The latter assays are described with a view to demonstrating the recent advances in these areas, to a point where they should now be considered for inclusion in an overall testing strategy for chemical carcinogens.


Asunto(s)
Pruebas de Carcinogenicidad/métodos , Animales , Apoptosis , Ciclo Celular , Línea Celular Transformada , Línea Celular Tumoral , Transformación Celular Neoplásica , Humanos , Modelos Animales , Pruebas de Mutagenicidad , Neovascularización Patológica , Humo/efectos adversos , Tabaco
19.
Biomarkers ; 14 Suppl 1: 90-6, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19604067

RESUMEN

The mechanism(s) by which cigarette smoke contributes to lung diseases, such as cancer, remains unclear. Recent developments in our knowledge of cell signalling events suggest that cigarette smoke causes oxidative stress and proinflammatory responses in cells of the lung. Cigarette smoke is a complex mixture of over 4000 compounds and high levels of oxidants and reactive oxygen species (ROS) have been detected in both mainstream and sidestream smoke. Oxidative stress that ensues, when the antioxidant defences are depleted, is accompanied by increases in ROS production in lung epithelial cells. Cigarette smoke-mediated oxidative stress produces DNA damage and activates survival signalling cascades resulting in uncontrolled cell proliferation and transformation. Intervention studies using antioxidants have provided compelling evidence that oxidative stress plays a critical role in the aetiology of smoking-related disorders.


Asunto(s)
Células Epiteliales/efectos de los fármacos , Pulmón/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Humo/efectos adversos , Fumar/efectos adversos , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Daño del ADN , Células Epiteliales/metabolismo , Células Epiteliales/patología , Medicina Basada en la Evidencia , Glutatión/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Pulmón/metabolismo , Pulmón/patología , Especies Reactivas de Oxígeno/metabolismo , Medición de Riesgo , Transducción de Señal/efectos de los fármacos
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