Name: A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke (Video) | JoVE Journal
Uploaded: 2012-01-02T12:00:00.000+00:00
Duration: 9 min 23 s
Description: Learn spin-trapping ESR spectroscopy to detect and quantify gas-phase free radicals in mainstream cigarette smoke. This method evaluates plant antioxidant scavenging capacity for tobacco research.

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This work was supported by the National Institute of Health, grant No. NIH/NCRR P41-RR 016292 (for ACERT).

1. Materials
All solvents used in this work were reagent grade. The spin trap, including N-tert-butyl-α-phenylnitrone (PBN) and the standard spin label 2,2,6,6-tetramethyl-1-piperinyoxyl (TEMPO) were obtained from Sigma and were used as supplied. The plant antioxidants were obtained commercially from Swanson, Inc. USA.
2. Preparation of cigarette smoke and analysis of free radicals
<ol>
	<li>To introduce antioxidants into the filter, the antioxidants pycnogenol and grape seed extract were first dissolved in 95% ethanol, while lycopene was dissolved in acetone. The solvent volumes were different depending on the antioxidant solubility.</li>
	<li>The amount of antioxidants of 0.4 mg/filter was used. They were then coated with 10 mg of activated carbon. For this purpose activated carbon was stirred for ~12 hrs with the antioxidant solution in anaerobic conditions, filtered and dried under vacuum.</li>
	<li>Antioxidants were then introduced into the conventional acetate filter (CA filter). For this purpose, the filter was cut into two pieces. The coated plant antioxidants were inserted between two pieces of the filter and wrapped with a piece of tape to form a filter - antioxidant -filter sandwich (Fig. 1A). This combined filter was subsequently attached to cigarette rods containing tobacco. The control filter was made in the same way as the antioxidant filter except no antioxidant was added.</li>
	<li>Prior to the smoking simulation, the research cigarettes were unpacked and kept in a constant humidity environment (20 °C, 60% relative humidity) using a saturated NaBr solution for a minimum of 2 days.</li>
	<li>Smoking simulation for routine analysis was performed at ambient temperature using a single-port smoking device which, as shown in Fig.1, consists of a water aspirator or GAST DOA-P104-BN Vacuum Pump/Air Compressor (Benton Harbor, Mich) connected to the spin-trapping assembly through a T-junction with one end open. Puffs were performed by plugging the open end which remained open between puffs. The gas flow was checked by flow meter and set at ~2.2 SCFH = 17.5 mL/s by adjusting a valve placed between the pump and the vent.</li>
	<li>The research cigarettes were smoked under the condition of puff volume of 35 mL for a duration of 2 seconds repeated every 60 seconds, similar to1. Smoking simulation for quantitative estimation of gas-phase free radicals was performed in conjunction with the spin trapping system as shown in Fig. 1.</li>
	<li>Ten intense puffs (35 mL/puff) were taken for each cigarette. Gas-phase free radicals were collected by passing the MCS through a Cambridge filter pad and then introduced into the spin trapping solution (0.05 M PBN in benzene, 2.0 mL).</li>
	<li>After the last puff, the bubbled trapping solution was readjusted to its initial volume (2.0 mL) with the same benzene. An aliquot was transferred into a ~250 mm long, 3 mm ID glass tube sealed at one end.</li>
	<li>The trapping solution was deoxygenated using a freeze-pump-thaw procedure. It was frozen under liquid nitrogen, and a vacuum was applied. Then it was thawed under argon atmosphere, allowing trapped gas bubbles to escape, and frozen again. After this cycle was repeated three times, the tube was flame-sealed under vacuum and used in further ESR measurements. This step is required because oxygen, which is well soluble in benzene, broadens ESR lines of organic radicals. Deoxygenation dramatically improves the signal-to-noise ratio.</li>
	<li>X-Band ESR spectra were recorded on a Bruker EMX spectrometer at a frequency of 9.34 GHz under standard conditions. The spectrometer settings used in most experiments were: center field 3312.5G, scan width 80G, modulation amplitude 0.5G, time constant 82 microseconds, scan time 40 sec.</li>
	<li>The spin trap adducts are relatively stable at the experiment conditions, which in some cases required 25 accumulations, which typically took ~20 min. However, after 12 hrs the intensity of the ESR signals in benzene solution decreased by a factor of ~5.</li>
	<li>To quantify the concentration of the trapping adduct, its initial first-derivative ESR spectrum was integrated. The resulting absorption spectrum shows a broad singlet background, most likely due to soot/tar products contained in the smoke. 
	 
	After subtraction of this background the separated triplet of the trapping adduct was integrated one more time (Fig. 2).</li>
</ol>
3. Representative Results
Most of the free radicals in burning cigarette-produced smoke (gas phase) are instantaneous and unstable. In order to observe these radicals a spin trap technique is employed. It captures gas-phase free radicals by transforming them into a spin adduct which is more stable and can be detected by ESR (Fig.1). In the present study, the spin-trap solution of 0.05 M PBN was used to collect smoke gas phase free radicals, which are a mixture of oxygen- and carbon- centered radicals which are difficult to separate15. In our case, however, the observed hyperfine splitting constants aN = 13.7G and aH = 1.95G are very similar to the corresponding values for trapping adducts of alkoxyl free radicals (RO.)2, suggesting that they are the major product. We showed that weak ESR signals and low reproducibility observed in our initial measurements (Fig. 2) were due to the moisture in the smoke flow. To resolve this problem, we added a liquid nitrogen (LN2) trap between the Cambridge filter and the spin trap solution. The LN2 trap removed water from the MCS flow by quick freezing and capturing it on the inner wall of the glass tube. This greatly improved ESR signals and allowed for highly reproducible results (Fig. 3).
The amount of trapped free radicals was determined using a reference sample. For control samples without antioxidants, the typical adduct concentration in benzene estimated by comparison of its double integral spectrum with a double integral spectrum for known concentration of TEMPO was 1.24 μM (Fig.2). Since the amount of air passed through each cigarette during smoking was ~350 ml, this gives an estimate for the free radical concentration in the gas phase of MCS of ~7.1X10-9M, and a total number of radicals trapped from the gas phase of a whole cigarette of ~1.5X1015. An estimate for the total amount of free radicals in the smoke of a whole cigarette, including both gas and particulate phase, was ~1016 free radicals9.
Different levels of the scavenging effect of plant antioxidants on gas-phase free radicals in main stream cigarette smoke were observed. Their scavenging rates were presented in Fig. 4. Lycopene and grape seed extract showed the highest rates while lower rate was observed for pycnogenol (Fig.4).
<img alt="Cigarette smoke analysis setup diagram with spin trap, Cambridge filter, and liquid nitrogen process." src="/files/ftp_upload/3406/3406fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. A diagram of improved smoking simulation design for collecting gas-phase free radicals in mainstream cigarette smoke (MCS) using spin trap. MCS was drawn by a water aspirator through CA filter, then Cambridge filter (yellow filter) and passed through a liquid nitrogen trap to remove H2O. The gas-phase products finally went to spin trap and bubbled through the spin trap solution. Plant antioxidant was placed between two pieces of conventional acetate filters (Enlarged in circle A) attached to cigarette to scavenge free radicals in MCS.
<img alt="Derivative, integral graph set; EPR spectra; analysis: baseline subtraction, double integration." src="/files/ftp_upload/3406/3406fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. A quantitative estimate for the spin-trapping adduct concentration in benzene requires subtracting a broad background signal from the first integral spectrum. The hyperfine splitting parameters for the spin-trapping adduct are aH = 1.95 G, aN = 13.7G.
<img alt="Electron spin resonance spectra comparison with and without liquid nitrogen; graph analysis." src="/files/ftp_upload/3406/3406fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3. Passing through LN2 trap substantially improves the quality of ESR signals obtained by spin-trapping of MCS.
<img alt="Spectral analysis graph; EPR spectra of antioxidants: pycnogenol, grape seed, lycopene, control." src="/files/ftp_upload/3406/3406fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4. Effect of natural antioxidants on the concentration of free radicals in MCS. The relative signal intensities are: control - 100%, pycnogenol - 55%, grape seed extract - 12%, lycopene - 10%.

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<li>Church, D. F., Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Free-radical+chemistry+of+cigarette+smoke+and+its+toxicological+implications%5BTitle%5D)+AND+%22Environ.+Health.+Perspect%22%5BJournal%5D)">Free-radical chemistry of cigarette smoke and its toxicological implications.</a> Environ. Health. Perspect. 64, 111-126 (1985).</li>
<li>Deliconstantinos, G., Villiotou, V., Stavrides, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scavenging+effects+of+hemoglobin+and+related+heme+containing+compounds+on+nitric+oxide%2C+reactive+oxidants+and+carcinogenic+volatile+nitrosocompounds+of+cigarette+smoke.+a+new+method+for+protection+against+the+dangerous+cigarette+constituents%5BTitle%5D)+AND+%22Anticancer.+Res%22%5BJournal%5D)">Scavenging effects of hemoglobin and related heme containing compounds on nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke. a new method for protection against the dangerous cigarette constituents.</a> Anticancer. Res. 14, 2717-2726 (1994).</li>
<li>Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco+smoke+carcinogens+and+lung+cancer%5BTitle%5D)+AND+%22Journal+of+the+National+Cancer+Institute%22%5BJournal%5D)">Tobacco smoke carcinogens and lung cancer.</a> Journal of the National Cancer Institute. 91, 1194-1210 (1999).</li>
<li>Kodama, M., Kaneko, M., Aida, M., Inoue, F., Nakayama, T., Akimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Free+radical+chemistry+of+cigarette+smoke+and+its+implication+in+human+cancer%5BTitle%5D)+AND+%22Anticancer.+Res%22%5BJournal%5D)">Free radical chemistry of cigarette smoke and its implication in human cancer.</a> Anticancer. Res. 17, 433-437 (1997).</li>
<li>Nair, A. K., Brandt, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+smoking+on+health+care+costs%5BTitle%5D)+AND+%22Journal+of+Oklahoma+State+Medical+Association%22%5BJournal%5D)">Effects of smoking on health care costs.</a> Journal of Oklahoma State Medical Association. 93, 245-250 (2000).</li>
<li>Nishizawa, M., Kohno, M., Nishimura, M., Kitagawa, A., Niwano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presence+of+Peroxyradicals+in+Cigarette+Smoke+and+the+Scavenging+Effect+of+Shikonin%2C+a+Naphthoquinone+Pigment%5BTitle%5D)+AND+%22Chem.+Pharm.+Bull%22%5BJournal%5D)">Presence of Peroxyradicals in Cigarette Smoke and the Scavenging Effect of Shikonin, a Naphthoquinone Pigment.</a> Chem. Pharm. Bull. 53, 796-799 (2005).</li>
<li>Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cigarette+smoke+and+the+involvement+of+free+radical+reactions+in+chemical+carcinogenesis%5BTitle%5D)+AND+%22Br.+J.+Cancer%22%5BJournal%5D)">Cigarette smoke and the involvement of free radical reactions in chemical carcinogenesis.</a> Br. J. Cancer. 8, 19-23 (1987).</li>
<li>Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cigarette+smoke+radicals+and+the+role+of+free+radicals+in+chemical+carcinogenicity%5BTitle%5D)+AND+%22Environ.+Health.+Perspect%22%5BJournal%5D)">Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity.</a> Environ. Health. Perspect. 105, 875-882 (1997).</li>
<li>Rozzi, N. I., Singh, R. K., Vierling, R. A., Watkins, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Supercritical+fluid+extraction+of+lycopene+from+tomato+processing+byproducts%5BTitle%5D)+AND+%22J.+Agric.+Food.+Chem%22%5BJournal%5D)">Supercritical fluid extraction of lycopene from tomato processing byproducts.</a> J. Agric. Food. Chem. 50, 2638-2643 (2002).</li>
<li>Shopland, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco+use+and+its+contribution+to+early+cancer+mortality+with+a+special+emphesis+on+cigarette+smoking%5BTitle%5D)+AND+%22Environmental.+Health.+Perspectives%22%5BJournal%5D)">Tobacco use and its contribution to early cancer mortality with a special emphesis on cigarette smoking.</a> Environmental. Health. Perspectives. 103, 131-142 (1995).</li>
<li>Shrikhandle, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wine+products+with+health+benefits%5BTitle%5D)+AND+%22Food+Res.+Int%22%5BJournal%5D)">Wine products with health benefits.</a> Food Res. Int. 33, 469-474 (2000).</li>
<li>Valavanidis, A., Haralambous, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparative+study+by+electron+paramagnetic+resonance+of+free+radical+species+in+the+mainstream+and+sidestream+smoke+of+cigarettes+with+conventional+acetate+filters+and+%27bio-filters%27%5BTitle%5D)+AND+%22Redox.+Rep%22%5BJournal%5D)">A comparative study by electron paramagnetic resonance of free radical species in the mainstream and sidestream smoke of cigarettes with conventional acetate filters and 'bio-filters'.</a> Redox. Rep. 6, 161-171 (2001).</li>
<li>Valavanidis, A., Vlachogianni, T., Fiotakis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco+smoke%3A+involvement+of+reactive+oxygen+species+and+stable+free+radicals+in+mechanisms+of+oxidative+damage%2C+carcinogenesis+and+synergistic+effects+with+other+respirable+particles%5BTitle%5D)+AND+%22International+Journal+of+Environmental+Research+and+Public+Health%22%5BJournal%5D)">Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles.</a> International Journal of Environmental Research and Public Health. 6, 445-462 (2009).</li>
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No conflicts of interest declared.

A reliable estimate of the effect of different free-radical scavengers in tobacco smoke requires a reproducible technique for quantitative detection of free radicals. Previously1, it has been shown that small volumes of higher concentration spin trap solutions in non-polar solvents are most effective at trapping free radicals from tobacco smoke. Cigarette smoke always contains water vapor from combustion of organic compounds and the residual moisture in tobacco which can end up in the trapping solvent. This admixture of water in the PBN spin trap solution substantially decreases the lifetime of the spin trapping adducts and the intensity of their ESR signals. Removing this moisture by simple passing MCS through a U-shaped tube cooled with liquid nitrogen dramatically improved the quality of ESR spectra in our experiments, even though some fraction of gas-phase radicals might be also trapped on the frozen surface. 
Using this technique we compared relative efficiency of different radical-scavenging natural compounds introduced into cigarette filters. We found that lycopene and grape seed extract immediately after incorporating into cigarette filters are able to scavenge up to 90% of free radicals from the gas phase MSC. Such high scavenging capacity ranks these cheap readily available natural compounds among the most efficient reported free radical protectors like haemoglobin and shikonine8,14. Yet, in our experiments cigarette filters loaded with studied natural antioxidants lost a noticeable part of their scavenging capacity after a week of storage at room temperature. Solving this problem can stimulate future application of lycopene and grape seed extract in commercial cigarette filters.

a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

The overall goal of this procedure is to measure the amount of free radicals in mainstream tobacco smoke and to assess the scavenging capacity of several antioxidants. This is accomplished by first preparing filters containing several different scavenger compounds as well as controls without scavenger filter. Preparation is followed by smoking simulation with the smoke of research cigarettes, passing through a spin trapping solution.Electron spin resonance, abbreviated ESR is then used to record spectra of trapped radicals as well as of reference samples. Next obtained ESR Spectra are subjected to routine analysis in order to separate the signal of the trapped gas phase radicals from the background and to quantify the radical concentration using a reference sample. Ultimately, results show quantitative information on the content of free radicals in mainstream cigarette smoke and estimates of scavenging capacity of natural antioxidants through spin trapping ESR spectroscopy.The main advantage of this technique over the existing methods of tobacco smoke analysis like gas chromatography, is that ESR is specifically sensitive to to free radicals. The implications of this technique can help in reducing hazardous effects of tobacco smoke because theoreticals is a major group of carcinogen. This method can provide insight in the studying free radical in the gasket smoke, and it can also be applied to other systems such as industrial smoke exhaust and smacked The National Biomedical Research Center for electron spin resonance technology, also known as a cert, is an outgrowth of our extensive experience in developing modern ESR technology.For researchers engaged in electrons spin resonance, we offer a wide variety of service for them and for the biomedical community. We make our facilities regularly available To introduce antioxidants into the cigarette filter. First, dissolve the antioxidants, pycnogenol, and grapee extract in 95%ethanol and dissolve lycopene and acetone.Adjust the solvent volumes depending on the antioxidants solubility. Next, combine the dissolved antioxidants with activated carbon by stirring in anaerobic conditions for approximately 12 hours. Anaerobic conditions are achieved by bubbling Argonne through the mixture while stirring.Following incubation, filter the solution and dry under a vacuum. Then introduce the antioxidants and carbon into a conventional acetate filter by first cutting the filter into two pieces. Insert the coated plant antioxidants between two pieces of the filter to form a filter antioxidant filter sandwich, and reinsert it into the cigarettes paper cylinder.Prepare the control filter in the same way as the antioxidant filter, except not to add the antioxidant prior to experimental setup. The research cigarettes should be unpacked and kept in a constant humidity environment using a saturated sodium bromide solution for a minimum of two days. Retrieve the cigarette rods containing tobacco and attach to the combined filter.Perform smoking simulation for routine analysis at ambient temperature using a single port smoking device, which consists of a water aspirator or vacuum pump slash air compressor connected to the spin trapping assembly through a T junction with one end open. Use a flow meter to check the gas flow and set at approximately 2.2 standard cubic feet per hour by adjusting a valve placed between the pump and the vent. Smoke the research cigarettes using an intense puff volume of 35 milliliters for a duration of two seconds and repeat every 60 seconds.The open end of the smoking device should be plugged to perform puffs and left open between puffs. While the end is unplugged, there is little draft through the cigarette, which corresponds to the conditions between puffs. Take 10 intense puffs for each cigarette collecting gas phase free radicals by passing the mainstream cigarette smoke through a Cambridge filter pad, and then introducing into the spin trapping solution.The liquid nitrogen trap removes water vapor from the mainstream smoke, but does not substantially affect the gas phase radicals. After the last puff transfer an aliquot of the bubbled trapping solution into an approximately 10 inch long, three millimeter internal diameter glass sample tube. Since oxygen broadens ESR spectra and decreases the method sensitivity, deoxy oxygenate the solution using the freeze pump thaw method by first dipping the tube into liquid nitrogen, then evacuating the solution with an oil plump, and then thawing the solution under Argonne.Repeat this freeze pump thaw method several times. Next, seal the tube under vacuum. The tube is now ready.For further ESR measurements, record X-band ESR spectra on a Bruker EMX spectrometer at a frequency of 9.34 gigahertz. Under standard conditions, consult the written protocol for typical spectrometer settings used. For these experiments perform 25 ESR accumulations, which typically take approximately 20 minutes.Although the spin trap attics are relatively stable at the experimental conditions after 12 hours, the intensity of the ESR signals in benzene solution decreased by a factor of five. To quantify the concentration of the trapping addict, integrate the initial first derivative ESR spectrum. The resulting absorption spectrum shows a broad singlet background, most likely due to soot and tar products contained in the smoke.After subtraction of this background, integrate the separated triplet of the trapping addict. One more time to get the free radical concentration. The values of double integrals for the spin trapping solution are compared with those for a solution of a nitrox radical tempo with known concentration from the experiment demonstrated here, as well as additional calculations found in the written protocol.A free radical concentration in the gas phase of mainstream cigarette smoke of approximately 7.1 times 10 to the minus nine molar was obtained. From this, it was determined that the total number of radicals trapped from the gas phase of a whole cigarette is around 1.5 times 10 to the 15th. During this experiment, initial measurements led to weak ESR signals and low reproducibility due to the moisture in the smoke flow.To resolve this problem, a liquid nitrogen trap was added to the smoking simulation device between the Cambridge filter and the spin trap solution. The liquid nitrogen trap removed water from the mainstream cigarette smoke flow by quick freezing and capturing it on the inner wall of the glass tube. This greatly improved ESR signals and allowed for highly reproducible results.Different levels of the scavenging effect of plant antioxidants on gas phase, free radicals in mainstream cigarette smoke were observed and their scavenging rates are presented. Lycopene and grape seed extract showed the highest rates while lower rate was observed for pycnogenol. Once master, this technique can be done in less than one hour if performed properly.Once attempting the procedure, it's important to pass the smoke through a liquid nitrogen trap. It improves the quality of a SR spectra and the reproducibility once developed. The technique helped the researchers in health and environment to explore and to elevate the presence of health threatening free radicals in various volatile compounds.After watching the video, you should have a good understanding of how to quantify free radicals in the mainstream tobacco smoke in order to reduce hazardous effects of smoking.

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Bioengineering

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium.  A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Source: Li, T., et al. Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells. J. Vis. Exp. (2020).This video demonstrates how cigarette smoke extract weakens antimicrobial defense in lung epithelial cells by reducing AMP production, leading to increased bacterial invasion. The procedure measures intracellular bacterial load to assess the impact of smoke exposure on host-pathogen interactions.

Analyzing the Effects of Cigarette Smoke on Antimicrobial Defense in Lung Epithelial Cells

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems.

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2'-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2'-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

Learn spin-trapping ESR spectroscopy to detect and quantify gas-phase free radicals in mainstream cigarette smoke. This method evaluates plant antioxidant scavenging capacity for tobacco research.

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

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