Name: comparing the effects of electronic cigarette vapor and cigarette smoke in a novel in vivo exposure system
Uploaded: 2017-05-24T15:00:00.000+00:00
Duration: 10 min 44 s
Description: Watch this Scientific Journal Video about Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System at JoVE.com

This research was made possible by the Aortic Research Grant (University of Michigan) to Dr. Eliason. The authors would also like to acknowledge Nick Scott at the University of Michigan Plant Operations Sign and Graphics Department for assisting with the design and assembly of the cigarette lighting device.

The following protocol has been performed under the guidance and approval of the University of Michigan Institutional Animal Care and Use Committee (IACUC).
1. Electronic Cigarette-vapor Chamber Assembly
NOTE: The complete chamber should be placed in a fume hood during use. The chamber here was housed in a temperature controlled and filtered laboratory environment. Investigators may elect to monitor such aspects of the system to ensure consistency of the room air quality. As an option, covering the monitors with a metal cage can&#160;prevent rodent tampering while allowing the monitors to sample the interior chamber environment.&#160;
<ol>
	<li>Acquire an anesthesia chamber with air tight removable lid with volume of 20 L.</li>
	<li>Using a jig saw fitted with a blade appropriate for cutting the material, cut a 10.2 cm diameter hole in the lid of the chamber approximately 7.6 cm from the back edge of the chamber.</li>
	<li>Insert the adjustable vent into the hole and mount into place with any caulk adhesive. 
	NOTE: Ensure that the adhesive caulk is not accessible to the rodents in the chamber, as they may chew the caulk. To prevent this possible issue, apply the caulk to the outside of the chamber wall to mount the vent.</li>
	<li>Cut silicone tubing into two 15 cm segments, and attach ends to either side of a T-connector. 
	NOTE: Silicone tubing has the potential to react with some components of e-vapor or cigarette smoke. Thus, investigators may consider using non-reactive tubing.</li>
	<li>Thread both of the silicone tubes through premade holes near the front of the chamber lid so that the T-connector is inside the chamber. Ensure that the tubing is secured to the lid with either adhesive caulk or electrical tape.</li>
	<li>Connect the free ends of the silicone tubing to the output ends of two micro air pumps. Pumps should be mounted to the lid of the chamber with double-sided adhesive tape or caulk. 
	NOTE: The length of tubing connected to the output end of the pump should be short to limit the amount of vapor collection on the inside surface of the tubing during use.</li>
	<li>With a new silicone tube, attach one end to the input side of one of the air pumps (Pump A in Figure 1), and cut this tube to approximately 4 cm in length. This is where the electronic cigarette will be inserted during chamber use. Ensure that the diameter of the tube allows for a snug fit around the end of the e-cig.</li>
	<li>With a new silicone tube, attach one end to the input side of the other air pump (Pump B in Figure 1). This pump will introduce room air into the chamber. As such, the end of the tube must be placed outside the fume hood. The length of this tube is not critical, but should be as short as possible to limit airflow resistance.</li>
	<li>Inside the chamber attach two small hooks with double-sided adhesive to hold oxygen and carbon monoxide gas monitors.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55672/55672fig1.jpg" /> 
Figure 1. Schematic of Electronic Cigarette-vapor Chamber. 
Chamber is housed in fume hood (not shown). Room air pump (Pump B) introduces room air from outside the fume hood into chamber continuously at 2 L/min. E-cig pump (Pump A) puffs 133 mL of E-vapor over 4 s, with a 30 s rest interval. E-vapor and room air mix prior to being pumped into chamber. Gas monitors continuously measure carbon monoxide (CO) and oxygen (O2) concentrations inside the chamber. E-vapor is exhausted passively through vent into fume hood. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55672/55672fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Cigarette Smoke-chamber Assembly
NOTE: Virtually any brand of cigarette can be used with this system, however standardized research cigarettes such as the University of Kentucky 1R6F Research Cigarette are cost-effective, reliable, and best for this application.
<ol>
	<li>Follow steps 1.1 - 1.6, &#38; step 1.9.</li>
	<li>With a new silicone tube, attach one end to the cigarette lighting device, and the other end to the input side of the air pump (Pump A in Figure 2). The cigarette lighting device should be placed inside the fume hood and outside the chamber during use. 
	Note: Construction of the cigarette lighting device requires knowledge of metal fabrication and electrical engineering. While a step-by-step guide to construction will not be given here, see supplementary materials for plans.</li>
	<li>With a new silicone tube, attach one end to the other air pump (Pump B in Figure 2). This pump will introduce room air into the chamber, thus the end of the tube must be placed outside the fume hood.</li>
	<li>Cut several 5 mm wide vertical slits&#160;in the front wall of the chamber, and mount a computer fan on the outside of the chamber so that it covers this opening. Ensure that the front of the fan is facing towards the chamber such that the fan will blow air into the chamber through this opening.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55672/55672fig2.jpg" /> 
Figure 2. Schematic of Cigarette-smoke Chamber. 
Room air pump (Pump B) introduces room air from outside the fume hood into chamber continuously at 2 L/min. Pump A draws on lit cigarette for 40 s with rate of 2 L/min, and 20 s later the computer fan evacuates the chamber over 3 min. Smoke and room air mix prior to being pumped into chamber. Gas monitors continuously measure carbon monoxide (CO) and oxygen (O2) chamber concentrations. Smoke is exhausted through vent into fume hood. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55672/55672fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Microcontroller Assembly and Software
<ol>
	<li>Control the pumping system of the E-cigarette&#160;vapor chamber and cigarette smoke chamber by separate microcontrollers. Download the microcontroller software and upload the operating codes provided in the <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55672/supplementary_materials.zip" target="_blank">Supplementary Materials</a>. The electronic cigarette vapor code will run the room air pump continuously and activate the E-cigarette&#160;pump for a 4 s duration every 30 s. The cigarette smoke code will run the room air pump continuously, activate the cigarette pump for 40 s, and activate the computer fan 20 s after the cigarette pump has stopped. The fan will shut off after running for 3 min. 
	Note: The timing of the pumps and fan can be adjusted as needed. Reference manufacturer instructions on how to upload relevant codes to the microcontroller.</li>
	<li>To assemble the microcontroller, connect zip wires, diodes, resisters, and capacitors to the bread board as depicted in Figure 3 and attach alligator-clip wires to the corresponding air pumps (and computer fan for cigarette smoke chamber). House the microcontroller outside of the fume hood if possible.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55672/55672fig3.jpg" /> 
Figure 3. Schematic of Microcontroller. 
Schematic of microcontroller and bread board to operate timing of air pumps and fan. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55672/55672fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Animals
<ol>
	<li>Use adult rats weighing 450 - 520 g.</li>
	<li>Group rats according to exposure type (e.g., electronic cigarette, cigarette smoke, room air pumped into chamber).</li>
	<li>At this time prior to exposure, collect 500 &#181;L of blood in EDTA coated tubes from tail vein using syringe to measure baseline serum cotinine concentration.</li>
	<li>Spin down blood samples at 20,000 x g for 30 min at 4 &#730;C and collect serum. Ensure that samples are chilled on ice throughout this process.</li>
	<li>Following the manufacturer protocol, assay for cotinine in the serum samples collected. Samples may also be stored at -80 &#730;C for later use.</li>
</ol>
5. Operating the Electronic cigarette Chamber
<ol>
	<li>Clean the inside of the chamber with 70% ethanol, then with deionized water, and let air dry until the chamber is completely dry (or about 30 min).</li>
	<li>Calibrate gas monitors and mount entire monitor on the wall inside the chamber.</li>
	<li>Place rats inside the chamber. Note that up to three animals can be exposed simultaneously.</li>
	<li>Ensure the E-cigarette&#160;is fully charged with adequate E-liquid and insert the E-cigarette&#160; into input tube. It is critical to ensure that the E-cigarette&#160;battery and E-liquid levels are adequate throughout the duration of the 90-min exposure.</li>
	<li>Turn the air pumps on, and start the timer.</li>
	<li>During exposure observe the gas monitors to ensure chamber contains &#62;20% O2 and 0 ppm CO. 
	Note: If oxygen levels fall, the chamber may not be well ventilated or the oxygen monitor may not be appropriately calibrated.</li>
	<li>Once the exposure time has reached 90 min, remove the E-cigarette&#160;and continue to run the gas pumps to vent the remaining vapor. Additionally, the top of the chamber can be lifted to hasten ventilation.</li>
	<li>Once the vapor has cleared, remove the rats and clean the chamber.</li>
	<li>Collect 500 &#181;L blood from tail vein from each rat approximately 1 h after exposure at the conclusion of the experimental protocol.</li>
	<li>Follow steps 4.4 -&#160;4.5 to isolate serum and assay for cotinine.</li>
</ol>
6. Operating the Tobacco Cigarette Chamber
<ol>
	<li>Follow steps 5.1 - 5.3.</li>
	<li>Insert cigarette into cigarette lighting device, with the end of the cigarette against the heating elements.</li>
	<li>Turn on the cigarette lighting device until cigarette butt begins to smolder (about 5 s).</li>
	<li>Once the cigarette is lit, turn on the pumping system, start the timer, and observe the cigarette burn to completion (about 40 s).</li>
	<li>Once the cigarette has burned, carefully remove the spent cigarette from the cigarette lighting device with forceps.</li>
	<li>Ensure that CO levels do not rise above 1000 ppm and O2 levels do not fall below 20%. Computer fan timing and duration is critical in preventing carbon monoxide accumulation.</li>
	<li>After 4 min turn the pumping system off and return to step 6.2 until rats are exposed to tobacco smoke for 90 min (or approximately 23 cigarettes). 
	NOTE: Carbon monoxide levels 4 min following cigarette burning should fall below 400 ppm, otherwise carbon monoxide may begin to accumulate in chamber.</li>
	<li>When exposure is complete, continue to leave the pumping system on to ventilate residual smoke. When carbon monoxide falls below 100 ppm remove rats from chamber. This should take 5 - 10 min.</li>
	<li>Remove rats and clean the chamber.</li>
	<li>Collect 500 &#181;L blood from tail vein approximately 1 h after exposure at the conclusion of the experimental protocol as with the E-cigarettes.</li>
	<li>Follow steps 4.4 - 4.5 to isolate serum and assay for cotinine.</li>
</ol>

<ol>
	<li>Schoenborn, C. A., Gindi, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+Cigarette+Use+Among+Adults:+United+States,+2014+Key+findings.">Electronic Cigarette Use Among Adults: United States, 2014 Key findings.</a> NCHS. , (2014).</li><li>Singh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+Use+Among+Middle+and+High+School+Students+-+United+States,+2011-2015.">Tobacco Use Among Middle and High School Students - United States, 2011-2015.</a> MMWR. 65 (14), 361-367 (2016).</li><li>Flora, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+potential+impurities+and+degradation+products+in+electronic+cigarette+formulations+and+aerosols.">Characterization of potential impurities and degradation products in electronic cigarette formulations and aerosols.</a> Regul. Toxicol. Pharmacol: RTP. 74, 1-11 (2016).</li><li>Tierney, P. A., Karpinski, C. D., Brown, J. E., Luo, W., Pankow, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavour+chemicals+in+electronic+cigarette+fluids.">Flavour chemicals in electronic cigarette fluids.</a> Tob. Control. , (2015).</li><li>Sleiman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emissions+from+Electronic+Cigarettes:+Key+Parameters+Affecting+the+Release+of+Harmful+Chemicals.">Emissions from Electronic Cigarettes: Key Parameters Affecting the Release of Harmful Chemicals.</a> Environ. Sci. Technol. 50 (17), 9644-9651 (2016).</li><li>Hwang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+cigarette+inhalation+alters+innate+immunity+and+airway+cytokines+while+increasing+the+virulence+of+colonizing+bacteria.">Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria.</a> Int J Mol Med (Berlin, Germany). 94 (6), 667-679 (2016).</li><li>Cheng, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+evaluation+of+electronic+cigarettes.">Chemical evaluation of electronic cigarettes.</a> Tob Control. 23, 11-18 (2014).</li><li>Etter, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+longitudinal+study+of+cotinine+in+long-term+daily+users+of+e-cigarettes.">A longitudinal study of cotinine in long-term daily users of e-cigarettes.</a> Drug Alcohol Depend. 160, 218-221 (2016).</li><li>Etter, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levels+of+saliva+cotinine+in+electronic+cigarette+users.">Levels of saliva cotinine in electronic cigarette users.</a> Addiction. 109 (5), 825-829 (2014).</li><li>Marsot, A., Simon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+and+Cotinine+Levels+With+Electronic+Cigarette:+A+Review.">Nicotine and Cotinine Levels With Electronic Cigarette: A Review.</a> Int. J. Toxicol. 35 (2), 179-185 (2016).</li><li>Flouris, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+impact+of+active+and+passive+electronic+cigarette+smoking+on+serum+cotinine+and+lung+function.">Acute impact of active and passive electronic cigarette smoking on serum cotinine and lung function.</a> Inhal. Toxicol. 25 (2), 91-101 (2013).</li><li>Bot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cotinine+levels+in+cigarette+smokers:+impact+of+mental+health+and+other+correlates.">Plasma cotinine levels in cigarette smokers: impact of mental health and other correlates.</a> Eur Addict Res. 20 (4), 183-191 (2014).</li><li>Zhu, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+hundred+and+sixty+brands+of+e-cigarettes+and+counting:+implications+for+product+regulation.">Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation.</a> Tob. Control. 23, 3-9 (2014).</li></ol>

The authors have nothing to disclose.

Here we describe a method for constructing chambers that expose rodents to E-vapor and cigarette smoke in a controlled fashion (Figure 6). Construction of the E-cigarette chamber is relatively simple and inexpensive compared to commercial exposure systems14,15,16. The parts and tools required to build the chamber are readily available from commercial suppliers online. Similarly, constructing the cigarette smoke chamber is relatively simple with the exception of the cigarette lighting device which must be fabricated (see <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55672/supplementary_materials.zip" target="_blank">Supplementary Materials</a> for plans).
Once the chambers are constructed, a critical step of the exposure system is calibrating the chambers to expose the rodents to the desired amount of nicotine. In both the E-cigarette chamber and the cigarette chamber, adjusting the amount of total exposure time is perhaps the easiest method for increasing or decreasing the amount of nicotine exposure. Increasing the puff time in the E-cigarette exposure system may increase the nicotine dose, however drawing vapor from the E-cigarette for an extended length of time has been shown to increase the levels of ROS, aldehydes, and other hazardous compounds, and may or may not reflect the typical habits of an E-cigarette user5. The puff duration and total exposure time can be adjusted by modifying the code uploaded to the microcontroller. Also, it should be noted that the concentration of nicotine in E-cigarette solutions, as well as the voltage of the E-cigarette heating element, can vary significantly and should be taken into account when calibrating the system.
One of the greatest advantages of this exposure system is its versatility. Virtually any brand of e-cig or e-cig solution can be used with this system. This is an especially useful feature given that the E-cigarette&#160;market now includes over 400 brands and thousands of E-cigarette solutions13. Additionally, the exposure system is compatible with multiple experimental endpoints allowing for the effects of E-cigarette on various organ systems and disease processes to be studied. We also acknowledge that there are several limitations to this exposure paradigm, such as the method by which the animals are exposed to the vapor. E-cigarette users directly inhale E-vapor while in this paradigm the rodents inhale E-vapor passively. Additionally, the rodents will also likely absorb compounds within the vapor or smoke by other routes (i.e.&#160;direct dermal absorption, and ingestion while preening). However, we think that the benefits of the exposure system far outweigh the limitations.
Overall, this exposure paradigm delivers consistent and clinically relevant E-vapor and cigarette smoke exposure, and may help aid the research effort in determining the adverse health effects of E-cigarette and cigarette smoke.

Since entering the US market in 2004, electronic cigarettes (E-cigarettes) have expanded into a billion-dollar industry, and it is estimated that nearly 9 million adults use them regularly1. In 2014 and 2015, more high school students had used E-cigarettes than conventional cigarettes2. The growing number of E-cigarettes users has spawned a research effort to evaluate their potential adverse health effects.
E-cigarettes generate a vapor (dubbed &#34;E-vapor&#34;) by heating a viscous solution that typically contains a mixture of water, polyethylene glycol or vegetable glycerin, nicotine, and flavorings3,4. It has been shown that E-vapor contains several harmful compounds including Reactive Oxygen Species (ROS), nicotine, various aldehydes, and polycyclic aromatic hydrocarbons5,6. Many of these compounds are formed during the vaporization process of E-liquid prior to inhalation7. Notably, several of these harmful compounds are also present in cigarette smoke, raising concern that E-cigarettes use may have similar adverse health consequences7.
There is little consensus on the health effects of E-cigarettes. To address this, several animal models of E-vapor exposure have been developed (Table 1). These models employ a variety of methods such as whole-body E-vapor exposure and mechanical ventilation. While current models have provided insightful data, few make direct comparisons to cigarette smoke within the same exposure system (Table 1). Additionally, while several human studies have shown E-cigarettes&#160;users and cigarette smokers to have serum cotinine levels between 30-200 ng/mL, many models of E-vapor and cigarette smoke exposure fall outside this range8,9,10,11,12.
Herein we present a method for comparing the effects of cigarette smoke and E-vapor exposure in vivo that yields serum cotinine levels similar to human studies.

<table><tbody><tr><td>blu PLUS Rechargeable Kit</td><td>blu eCigs</td><td>N/A</td><td></td></tr><tr><td>1R6F Reference Cigarettes</td><td>Center for Tob Ref&amp;nbsp; Prod UK</td><td>N/A</td><td></td></tr><tr><td>Lexan Anesthesia Chamber 20 L</td><td>Jorgensen Laboratories</td><td>JOR265</td><td></td></tr><tr><td>Arduino UNO</td><td>Arduino</td><td>2877</td><td></td></tr><tr><td>Diode Rectifier - 1 A; 50 V</td><td>Spark Fun</td><td>COM-08589</td><td></td></tr><tr><td>Resistor 10 KOhm 1/6th W PTH - 20 pack</td><td>Spark Fun</td><td>COM-11508</td><td></td></tr><tr><td>Electrolytic Decoupling Capacitors - 100 uF/25 V</td><td>Spark Fun</td><td>COM-00096</td><td></td></tr><tr><td>Solderless Plug-in BreadBoard&amp;nbsp;</td><td>BusBoard Prototype Systems</td><td>BB400</td><td></td></tr><tr><td>Alligator-Clip Wires</td><td>BusBoard Prototype Systems</td><td>CA-M-20</td><td></td></tr><tr><td>ZipWire</td><td>BusBoard Prototype Systems</td><td>ZW-MM-10</td><td></td></tr><tr><td>Standard Fan 80 ST2</td><td>Cooler Master</td><td>R4-S8R-20AK-GP</td><td></td></tr><tr><td>ARIC 4&quot; adjustable vent</td><td>Bestlouver</td><td>N/A</td><td></td></tr><tr><td>ToxiPro&amp;nbsp; Carbon Monoxide (CO) Monitor</td><td>Honeywell Analytics</td><td>54-00-10316</td><td></td></tr><tr><td>ToxiPro Oxygen (O&lt;sub&gt;2&lt;/sub&gt;) Monitor</td><td>Honeywell Analytics</td><td>54-45-90-VD</td><td></td></tr><tr><td>ToxiPro IQ Express Docking Station</td><td>Honeywell/Sperian Biosystems&amp;nbsp;</td><td>54-46-9100</td><td></td></tr><tr><td>Command Wall Hook Small Wire 6-Pack</td><td>3M</td><td>N/A</td><td></td></tr><tr><td>Micro Water/Air Pump</td><td>Xiamen Conjoin Electronics</td><td>CJWP40-A12A1</td><td></td></tr><tr><td>1/4&quot; Silicon Tubing</td><td>NewAge</td><td>2801470-100</td><td></td></tr><tr><td>T Connector</td><td>Bel-Art Scienceware</td><td>F196060000</td><td></td></tr><tr><td>Plastic Whole Blood tube with spray-coated K2EDTA</td><td>Becton, Dickinson and Company</td><td>367841</td><td></td></tr><tr><td>Cotinine ELISA kit</td><td>Calbiotech</td><td>CO096D</td><td></td></tr></tbody></table>

comparing the effects of electronic cigarette vapor and cigarette smoke in a novel in vivo exposure system

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Carbon monoxide and oxygen monitoring
Oxygen concentrations did not fall below 20% during e-vapor exposure and CO concentrations remained undetectable throughout exposure. Gas monitors during cigarette smoke exposure indicated that oxygen concentration remained above 20%. Carbon monoxide concentrations did not exceed 1,000 ppm (Figure 4).
<img alt="Figure 4" src="/files/ftp_upload/55672/55672fig4.jpg" /> 
Figure 4: Carbon Monoxide&#160;Concentrations in cigarette-smoke Chamber. Carbon monoxide concentration was recorded every 30 s over the course of introducing smoke from 1R6F cigarettes. Results shown are averages from 3 consecutive 4 min cycles. Carbon monoxide concentrations did not exceed 1,000 ppm. The cigarette is burned to completion over 40 s, and the fan is activated 20 s later (i.e.&#160;fan is activated 1 min following cigarette ignition).
Pre- and Postexposure Serum Cotinine
Pre-exposure and 1 h postexposure serum cotinine for E-vapor group (n = 3) was 4.2 &#177;0.4 ng/mL and 171.6 &#177;20.5 ng/mL, respectively. Pre-exposure and 1 h postexposure serum cotinine for cigarette smoke group (n = 3) was 3.9 &#177;0.3 ng/mL and 98.8 &#177;2.1 ng/mL, respectively (Figure 5).
<img alt="Figure 5" src="/files/ftp_upload/55672/55672fig5.jpg" /> 
Figure 5: Serum Cotinine Levels Following Cigarette-smoke or Electronic Cigarette-vapor Exposure. Serum cotinine was measured prior to protocol initiation, and 1 h after a 90 min exposure session. Pre-exposure and 1 h post-exposure serum cotinine for e-vapor group was 4.2 &#177;0.4 ng/mL and 171.6 &#177;20.5 ng/mL, respectively. Pre-exposure and 1 h postexposure serum cotinine for cigarette smoke group was 3.9 &#177;0.3 ng/mL and 98.8 &#177;2.1 ng/mL, respectively. Difference between pre- and postexposure serum cotinine concentrations were statistically significant. *P &#60;0.05.
<img alt="Figure 6" src="/files/ftp_upload/55672/55672fig6.jpg" /> 
Figure 6: Electronic Cigarette-vapor Chamber and Cigarette Smoke-chamber. Image of the electronic cigarette vapor (right) and cigarette smoke chamber (left) within the fume hood. Red boxes contain microcontrollers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55672/55672fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Animal model</td>
			<td colspan="2">Electronic cigarette</td>
			<td colspan="2">Tobacco cigarette</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Exposure method</td>
			<td>Model organism</td>
			<td>Brand (nicotine)</td>
			<td>Cotinine ng/mL (serum, [urine])</td>
			<td>Brand</td>
			<td>Cotinine ng/mL (serum, [urine])</td>
			<td></td>
		</tr>
		<tr>
			<td>whole-body exposure</td>
			<td>C57BL/6J mice</td>
			<td>Joytech 510-T (1.8%)</td>
			<td>62.3 &#177;3.3, [892.5 &#177;234]</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>McGrath-Morrow</td>
		</tr>
		<tr>
			<td>mechanical ventilator</td>
			<td>BALB/cJ mice</td>
			<td>not reported</td>
			<td>[400 - 500]</td>
			<td>not reported</td>
			<td>[500 - 800]</td>
			<td>Ponzoni</td>
		</tr>
		<tr>
			<td>whole-body exposure</td>
			<td>CD-1 mice</td>
			<td>multiple* (0.6 - 24%)</td>
			<td>not reported</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Hwang</td>
		</tr>
		<tr>
			<td>whole-body exposure</td>
			<td>Wistar albino rats</td>
			<td>Ego T (0.9%)</td>
			<td>not reported</td>
			<td>not reported</td>
			<td>not reported</td>
			<td>Salturk</td>
		</tr>
		<tr>
			<td>whole-body exposure</td>
			<td>C57BL/6 mice</td>
			<td>NJOY (1.8%)</td>
			<td>267 &#177;17</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Sussan</td>
		</tr>
		<tr>
			<td>whole-body exposure</td>
			<td>C57BL/6J mice</td>
			<td>CoolCart, Vapor Titan</td>
			<td>500 &#177;10</td>
			<td>3R4F Reference Cigarettes</td>
			<td>76 &#177;7.6</td>
			<td>Husari</td>
		</tr>
		<tr>
			<td colspan="5">*Xtreme Vaping, Vapure, Vape Addict Juice, Grimm Creations, Green Smart Living, Free Masons Elixer</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Characteristics of Electronic Cigarette Exposure Models.

Watch this Scientific Journal Video about Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System at JoVE.com

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

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

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

11.4K Views.  University of Michigan Medical School.  Dit protocol beschrijft een methode om knaagdieren bloot te stellen aan elektronische sigarettendamp (E-damp) en sigarettenrook. Blootstellingskamers worden geconstrueerd door het aanpassen van verdovingskamers met een geautomatiseerd pompsysteem dat E-damp of sigarettenrook aan knaagdieren levert. Dit systeem kan gemakkelijk worden aangepast om veel experimentele eindpunten in te vullen. 

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

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

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

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

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

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.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

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

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

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.

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Bioengineering

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

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

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.

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and ...

Chemistry

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

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

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.

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Immunology and Infection

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

In Vitro Model of Murine Airway Epithelial Cells and Chronic Smoke Exposure

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.

Author Spotlight: Development and Characterization of an In Vitro Model to Study Chronic Cigarette Smoke Exposure and Its Impact on Airway Epithelial Cells in COPD Research

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

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

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