Tobacco Nitrate and Free Radical Levels in the Mainstream Smoke of US Cigarette Brands.

Tobacco nitrate levels have been known to impact the levels of toxicants such as polyaromatic hydrocarbons and tobacco-specific nitrosamines (TSNAs) produced during smoking. Recent work in our group showed that the intrinsic nitrate levels in individual tobacco varieties also have a large influence on the formation of gas-phase (GP) free radicals in the mainstream smoke of cigarettes produced with a single tobacco variety. As tobacco nitrate content is a potential target for future regulatory policies, we investigated whether the levels of GP free radicals in the smoke from commercially available cigarettes is also dependent on the nitrate content in the corresponding tobacco blends. GP and particulate-phase (PP) free radical yields in mainstream smoke produced from 25 popular US cigarette brands were measured by electron paramagnetic resonance (EPR) spectroscopy. For each brand, we also measured levels of the TSNAs NNN (N'-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) via HPLC-MS and the nicotine content via GC-FID. Our results show that the intrinsic nitrate levels varied >15-fold and GP radicals varied 4-fold among the 25 brands tested. The GP radicals were correlated with intrinsic nitrate levels (r = 0.87, p < 0.001). NNK and NNN levels varied >8-fold and 12-fold, respectively. We found that NNK was moderately correlated to nitrate content (r = 0.42, p = 0.03) while the NNN was strongly correlated to the nitrate content (r = 0.65, p < 0.001). Nicotine levels varied the least (<3-fold) but showed a moderate negative correlation to nitrate content (r = -0.47, p = 0.02). No statistically significant correlation was observed between nicotine and TSNA levels in mainstream smoke. Overall, this demonstrates that the nitrate content of tobacco blends used in US cigarette brands impacts toxicant output in the mainstream smoke, although other proprietary variables (total ventilation, additives, filter type, etc.) may also modulate these results.

Influence of Tobacco Variety and Curing on Free Radical Production in Cigarette Smoke.

INTRODUCTION: Cigarette smoke contains highly reactive free radicals thought to play an important role in tobacco smoke-induced harm. Previously, large variations in free radical and toxicant output have been observed in commercial cigarettes. These variations are likely because of cigarette design features (paper, filter, and additives), tobacco variety (burley, bright, oriental, etc.), and tobacco curing methods (air, sun, flue, and fire). Previous reports show that tobacco varieties and curing methods influence the production of tobacco smoke constituents like the tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK). AIMS AND METHODS: We evaluated free radical, nicotine, and NNK production in cigarette smoke from cigarettes produced with 15 different types of tobacco. Gas-phase free radicals were captured by spin trapping with N-tert-butyl-α-phenylnitrone and particulate-phase radicals were captured on a Cambridge Filter pad (CFP). Both types of radicals were analyzed using electron paramagnetic resonance spectroscopy. Nicotine and NNK were extracted from the CFP and analyzed by gas chromatography flame ionization detection and liquid chromatography-mass spectrometry, respectively. RESULTS: Gas-phase radicals varied nearly 8-fold among tobacco types with Saint James Perique tobacco producing the highest levels (42 ±â€…7 nmol/g) and Canadian Virginia tobacco-producing the lowest levels (5 ±â€…2 nmol/g). Nicotine and NNK levels in smoke varied 14-fold and 192-fold, respectively, by type. Gas-phase free radicals were highly correlated with NNK levels (r = 0.92, p < .0001) and appeared to be most impacted by tobacco curing method. CONCLUSIONS: Altogether, these data suggest that tobacco types used in cigarette production may serve as a target for regulation to reduce harm from cigarette smoking. IMPLICATIONS: Variations in cigarette free radical and NNK levels vary based on the tobacco variety and curing method. Reducing the ratio of high-producing free radical and NNK tobacco types offer a potential tool for regulators and producers looking to reduce toxicant output from cigarettes.

Effects of tobacco nitrate content on free radical levels in mainstream smoke.

Tobacco smoke free radicals play an important role in the development of smoking related adverse health effects. We previously reported that gas phase (GP) radicals vary greatly by cigarette brand and tobacco variety and are highly correlated with levels of NNK in smoke. Since NNK production in tobacco is dependent on nitrate, we proposed that GP radical production may also be associated with tobacco nitrate content. To test this, we examined the relationship between intrinsic nitrate levels in 15 individual tobacco types and the levels of free radicals delivered in mainstream smoke from cigarettes produced from these tobaccos. Intrinsic nitrate levels varied >250-fold among the tobacco types, ranging from <0.1 mg/g tobacco in the Bright Leaf types to 24.1 ±â€¯0.4 mg/g in Light Fire Cured Virginia tobacco. Among the tobacco types tested, GP radicals were highly correlated with nitrate levels (r = 0.96, p < 0.0001). To investigate nitrate-specific changes to free radical production during smoking, different concentrations of exogenous sodium nitrate were added to unsmoked shredded leaves of 4 different tobacco types (Bright Leaf Sweet Virginia, American Virginia, Semi-Oriental 456, and reconstituted). Nitrate addition resulted in dose-dependent increases in GP radicals in the corresponding smoke, supporting our hypothesis that intrinsic nitrate levels are responsible for GP radical production in cigarette smoke. We also observed increases in NNK levels as a function of added nitrate that varied significantly among the 4 tobacco types tested, implying that other tobacco-type related factors may be impacting nicotine nitrosation during pyrolysis. Altogether, these findings have identified tobacco nitrate as a key factor in the production of GP radicals, but to a lesser extent with PP radicals, as well as NNK during combustion and highlight its potential implication as a target for regulation.

Evaluating electronic cigarette cytotoxicity and inflammatory responses in vitro.

INTRODUCTION: Cigarette smoking poses many health risks and can cause chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer of the lung and other organs. Smokers can substantially reduce their risks of these diseases by quitting, but nicotine addiction makes this difficult. Alternatives, such as electronic cigarettes (e-cigarettes), may provide a similar dose of nicotine, but expose users to fewer toxic chemicals than traditional cigarettes and may still be harmful especially for dual users, therefore, we sought to develop bioassays that can assess the potential toxicity and inflammatory response induced by e-cigarette liquids (e-liquids) with and without flavors. METHODS: E-liquids with varying nicotine content and flavors were aerosolized through growth media and exposed to human bronchial epithelial cell line (BEAS-2B) and human monocyte-macrophage cell line (THP-1) in vitro. Cytotoxicity in response to e-cigarette aerosols was measured by MTT assay in BEAS-2B cells and inflammatory response was measured by TNF-α, IL-6, IL-8, and MCP-1 released from THP-1 cells. In addition, the oxidative stress marker, REDD1, and impact on phagocytosis, was assessed following exposure of BEAS-2B and THP-1 derived macrophages, respectively. Cigarette smoke extract was used as a positive control with known cytotoxicity and impairment of inflammatory response. RESULTS: E-cigarette aerosols induced moderate cellular toxicity in bronchial epithelial cells. Our data also show that low nicotine levels are less damaging to the bronchial epithelial cells, and flavors in e-liquids influence the combined inflammatory response markers, phagocytosis, and REDD1 when examined in vitro. CONCLUSIONS: Our in vitro bioassays can be utilized to effectively measure flavor and nicotine-induced effects of e-cigarettes on combined inflammatory response and cytotoxicity in human macrophages and human bronchial epithelial cells, respectively.

Evidence from an fMRI study that dessert-flavored e-cigarettes engage taste-related, but not smoking-related, brain circuitry for female daily smokers.

Regulations limiting the sale of flavored e-cigarette products are controversial for their potential to interfere with e-cigarette use as a cessation aid in addition to curbing youth use. Limited research suggests that flavor might enhance the addictive potential of e-cigarettes; however, the acute effects of flavored aerosols on brain function among humans have not been assessed. The present study aimed to isolate and compare the neural substrates of flavored and unflavored e-cigarette aerosols on brain function among nine female daily smokers. Participants inhaled aerosolized e-liquid with 36 mg/mL of nicotine with and without a strawberry-vanilla flavor while undergoing functional magnetic resonance imaging. We used general linear modeling to compare whole-brain mean neural activation and seed-to-voxel task-based functional connectivity between the flavored and unflavored inhalation runs. Contrary to our hypothesis, the flavored aerosol was associated with weaker activation than the unflavored aerosol in the brain stem and bilateral parietal-temporal-occipital region of the cortex. Instead, the flavor engaged taste-related brain regions while suppressing activation of the neural circuits typically engaged during smoking and nicotine administration. Alternatively, functional connectivity between subcortical dopaminergic brain seeds and cortical brain regions involved in motivation and reward salience were stronger during the flavored compared to unflavored aerosol run. The findings suggest that fruity and dessert-flavored e-cigarettes may dampen the reward experience of aerosol inhalation for smokers who initiate e-cigarette use by inhibiting activation of dopaminergic brain circuits. These preliminary findings may have implications for understanding how regulations on flavored e-cigarettes might impact their use as cessation aids. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Free Radical and Nicotine Yields in Mainstream Smoke of Chinese Marketed Cigarettes: Variation with Smoking Regimens and Cigarette Brands.

Free radicals and nicotine are components of cigarette smoke that are thought to contribute to the development of smoking-induced diseases. China has the largest number of smokers in the world, yet little is known about the yields of tobacco smoke constituents in different Chinese brands of cigarettes. In this study, gas-phase and particulate-phase free radicals as well as nicotine yields were quantified in mainstream cigarette smoke from five popular Chinese brands and two research cigarettes (3R4F and 1R6F). Mainstream smoke was generated under International Organization of Standardization (ISO) and Canadian Intense (CI) smoking regimens using a linear smoking machine. Levels of free radicals and nicotine were measured by electron paramagnetic resonance spectroscopy (EPR) and gas chromatography with flame-ionization detection, respectively. Under the ISO puffing regimen, Chinese brand cigarettes produced an average of 3.0 ± 1.2 nmol/cig gas-phase radicals, 118 ± 44.7 pmol/cig particulate-phase radicals, and 0.6 ± 0.2 mg/cig nicotine. Under the CI puffing regimen, Chinese brand cigarettes produced an average of 5.6 ± 1.2 nmol/cig gas-phase radicals, 282 ± 92.1 pmol/cig particulate-phase radicals, and 2.1 ± 0.4 mg/cig nicotine. Overall, both gas- and particulate-phase free radicals were substantially lower compared to the research cigarettes under both regimens, whereas no significant differences were observed for nicotine levels. When Chinese brands were compared, the highest free radical and nicotine yields were found in "LL" and "BS" brands, while lowest levels were found in "YY". These results suggested that the lower radical delivery by Chinese cigarettes compared to United States reference cigarettes may be associated with reductions in oxidant-related harm.

Free Radical Production and Characterization of Heat-Not-Burn Cigarettes in Comparison to Conventional and Electronic Cigarettes.

With conventional cigarettes, the burning cone reaches temperatures of >900 °C, resulting in the production of numerous toxicants and significant levels of highly reactive free radicals. In attempts to eliminate combustion while still delivering nicotine and flavorings, a newer alternative tobacco product has emerged known as "heat-not-burn" (HnB). These products heat tobacco to temperatures of 250-350 °C depending on the device allowing for the volatilization of nicotine and flavorants while potentially limiting the production of combustion-related toxicants. To better understand how the designs of these new products compare to conventional cigarettes and different styles of electronic cigarettes (e-cigs), we measured and partially characterized their production of free radicals. Smoke or aerosols were trapped by a spin trap phenyl-N-tert-butylnitrone (PBN) and analyzed for free radicals using electron paramagnetic resonance (EPR). Free radical polarity was assessed by passing the aerosol or smoke through either a polar or nonpolar trap prior to being spin trapped with PBN. Particulate-phase radicals were detected only for conventional cigarettes. Gas-phase free radicals were detected in smoke/aerosol from all products with levels for HnB (IQOS, Glo) (12 pmol/puff) being similar to e-cigs (Juul, SREC, box mod e-cig) and hybrid devices (Ploom) (5-40 pmol/puff) but 50-fold lower than conventional cigarettes (1R6F). Gas phase radicals differed in polarity with HnB products and conventional cigarettes producing more polar radicals compared to those produced from e-cigs. Free radical production should be considered in evaluating the toxicological profile of nicotine delivery products and identification of the radicals is of paramount importance.

An Electronic Aerosol Delivery System for Functional Magnetic Resonance Imaging.

BACKGROUND: Public health concerns over the addictive potential of electronic cigarettes (e-cigs) have heightened in recent years. Brain function during e-cig use could provide an objective measure of the addictive potential of new vaping products to facilitate research; however, there are limited methods for delivering e-cig aerosols during functional magnetic resonance imaging (fMRI). The current study describes the development and feasibility testing of a prototype to deliver up to four different e-cig aerosols during fMRI. METHODS: Standardized methods were used to test the devices' air flow variability, nicotine yield, and free radical production. MRI scans were run with and without the device present to assess its safety and effects on MRI data quality. Five daily smokers were recruited to assess plasma nicotine absorption from e-liquids containing nicotine concentrations of 8, 11, 16, 24, and 36 mg/ml. Feedback was collected from participants through a semi-structured interview and computerized questionnaire to assess comfort and subjective experiences of inhaling aerosol from the device. RESULTS: Nicotine yield captured from the aerosol produced by the device was highly correlated with the nicotine concentration of the e-liquids used (R2 = 0.965). Nicotine yield was reduced by a mean of 48% and free radical production by 17% after traveling through the device. The e-liquid containing the highest nicotine concentration tested (36 mg/ml) resulted in the highest plasma nicotine boost (6.6 ng/ml). Overall, participants reported that the device was comfortable to use and inhaling the e-cig aerosols was tolerable. The device was determined to be safe for use during fMRI and had insignificant effects on scan quality. CONCLUSIONS: With the current project, we were able to design a working prototype that safely and effectively delivers e-cig aerosols during fMRI. The device has the potential to be used to assess brain activation during e-cig use and to compare brain reactivity to varying flavors, nicotine concentrations, and other e-cig characteristics.

Emissions of Free Radicals, Carbonyls, and Nicotine from the NIDA Standardized Research Electronic Cigarette and Comparison to Similar Commercial Devices.

E-cigarettes (e-cigs) are a diverse and continuously evolving group of products with four generations currently in the market. The National Institute on Drug Abuse (NIDA) standardized research e-cigarette (SREC) is intended to provide researchers with a consistent e-cig device with known characteristics. Thus, we conducted laboratory-based characterizations of oxidants and nicotine in aerosols produced from SREC and other closed-system, breath-activated, commercially available e-cigs (Blu and Vuse). We hypothesized that oxidant and nicotine production will be significantly affected in all devices by changes in puffing parameters. All e-cigs were machine vaped and the aerosols generated were examined for nicotine, carbonyls, and free-radicals while varying the puff-volumes and puff-durations to reflect typical human usage. The data were normalized on a per puff, per gram aerosol, and per milligram nicotine basis. We found that aerosol production generally increased with increasing puff-duration and puff-volume in all e-cigs tested. Increased puff-duration and puff-volume increased nicotine delivery for Blu and Vuse but not the SREC. We report, for the first time, reactive free-radicals in aerosols from all closed-system e-cigs tested, albeit at levels lower than cigarette smoke. Formaldehyde, acetaldehyde, acetone, and propionaldehyde were detected in the aerosols of all tested e-cigs. Carbonyl and free radical production is affected by puff-duration and puff volume. Overall, SREC was more efficient at aerosol and nicotine production than both Blu and Vuse. In terms of carbonyl and free radical levels, SREC delivered lower or similar levels to both other devices.

Free Radical, Carbonyl, and Nicotine Levels Produced by Juul Electronic Cigarettes.

INTRODUCTION: Free radicals and carbonyls produced by electronic cigarettes (e-cigs) have the potential to inflict oxidative stress. Recently, Juul e-cigs have risen drastically in popularity; however, there is no data on nicotine and oxidant yields from this new e-cig design. METHODS: Aerosol generated from four different Juul flavors was analyzed for carbonyls, nicotine, and free radicals. The e-liquids were analyzed for propylene glycol (PG) and glycerol (GLY) concentrations. To determine the effects of e-liquid on oxidant production, Juul pods were refilled with nicotine-free 30:70 or 60:40 PG:GLY with or without citral. RESULTS: No significant differences were found in nicotine (164 ± 41 µg/puff), free radical (5.85 ± 1.20 pmol/puff), formaldehyde (0.20 ± 0.10 µg/puff), and acetone (0.20 ± 0.05 µg/puff) levels between flavors. The PG:GLY ratio in e-liquids was ~30:70 across all flavors with GLY being slightly higher in tobacco and mint flavors. In general, when Juul e-liquids were replaced with nicotine-free 60:40 PG:GLY, oxidant production increased up to 190% and, with addition of citral, increased even further. CONCLUSIONS: Juul devices produce free radicals and carbonyls, albeit, at levels substantially lower than those observed in other e-cig products, an effect only partially because of a low PG:GLY ratio. Nicotine delivery by these devices was as high as or higher than the levels previously reported from cigarettes. IMPLICATIONS: These findings suggest that oxidative stress and/or damage resulting from Juul use may be lower than that from cigarettes or other e-cig devices; however, the high nicotine levels are suggestive of a greater addiction potential.

Effects of Charcoal on Carbonyl Delivery from Commercial, Research, and Make-Your-Own Cigarettes.

Previous literature has shown that adding charcoal to cigarette filters can have varying effects on the delivery of toxic carbonyls depending on filter design, amount of charcoal, and puffing profiles. However, these studies have relied on either comparisons between commercially available charcoal and noncharcoal filtered cigarettes or experimental modification of filters to insert a charcoal plug into existing cellulose acetate filters. Make-your-own (MYO) cigarettes can help obviate many of the potential pitfalls of previous studies; thus, we conducted studies using commercial charcoal cigarettes and MYO cigarettes to determine the effects of charcoal on carbonyl delivery. To do this, we analyzed carbonyls in mainstream smoke by HPLC-UV after derivatization with 2,4-dinitrophenylhydrazine (DNPH). Charcoal was added in-line after the cigarettes or through the use of MYO charcoal cigarette tubes. MYO cigarettes had carbonyl deliveries similar to that of 3R4F research cigarette, regardless of tobacco type. The greatest effect on carbonyl delivery was observed with 200 mg of charcoal, significantly reducing all carbonyls under both methods tested. However, "on-tow" design charcoal filters, available on many commercially available charcoal brands, appeared to have a minimal effect on carbonyl delivery under intense smoking methods. Overall, we found that charcoal, when added in sufficient quantity (200 mg) as a plug, can substantially reduce carbonyl delivery for both MYO and conventional cigarettes. As carbonyls are related to negative health outcomes, such reductions may be associated with reductions in carbonyl-related harm in smokers.

Little Cigars, Filtered Cigars, and their Carbonyl Delivery Relative to Cigarettes.

Introduction: Little cigars and filtered cigars are currently growing in popularity due to their low cost and wide variety of flavors while retaining an appearance similar to cigarettes. Given the health consequences associated with cigarette use, it is important to understand the potential harm associated with these similar products. This includes the potential harm associated with carbonyls (eg, acetaldehyde, acrolein, formaldehyde, etc.), an important class of toxicants and carcinogens in tobacco smoke. Our objective was to determine the carbonyl levels in mainstream smoke from little and filtered cigars compared to cigarettes. Methods: We examined two brands each of little cigars and filtered cigars, as well as two research cigarettes for carbonyl delivery using the International Organization of Standards (ISO) and the Health Canada Intense (HCI) machine-smoking protocols. Results: On a per puff basis, the levels of five of the seven carbonyls were higher from little cigars than filtered cigars and cigarettes (ISO: 56-116%; HCI: 39-85%; p < .05). On a per unit basis, most carbonyl levels were higher from both cigar types than cigarettes using the ISO method (ISO: 51-313%; p < .05) whereas only filtered cigars were higher using the HCI method (HCI: 53-99%; p < .05). Conclusion: These findings suggest that cigar smokers can be exposed to higher levels of carbonyls per cigar than cigarette smokers per cigarette. Implications: These data will increase our understanding of the relative harm from carbonyl exposure from little and filtered cigars both for cigar-only smokers and the cumulative harm among the growing population of cigarette-cigar multi-product smokers.

Effect of Charcoal in Cigarette Filters on Free Radicals in Mainstream Smoke.

The addition of charcoal in cigarette filters may be an effective means of reducing many toxicants from tobacco smoke. Free radicals are a highly reactive class of oxidants abundant in cigarette smoke, and here we evaluated the effectiveness of charcoal to reduce free radical delivery by comparing radical yields from commercially available cigarettes with charcoal-infused filters to those without and by examining the effects of incorporating charcoal into conventional cigarette filters on radical production. Commercial cigarettes containing charcoal filters produced 40% fewer gas-phase radicals than did regular cellulose acetate filter cigarettes when smoked using the International Organization of Standardization (ISO, p = 0.07) and Canadian Intense (CI, p < 0.01) smoking protocols. While mean-particulate-phase radicals were 25-27% lower in charcoal cigarettes, differences from noncharcoal products were not significant ( p = 0.06-0.22). When cellulose acetate cigarette filters were modified to incorporate different types and amounts of activated charcoal, reductions in gas-phase (>70%), but not particulate-phase, radicals were observed. The reductions in gas-phase radicals were similar for the three types of charcoal. Decreases in radical production were dose-responsive with increasing amounts of charcoal (25-300 mg) with as little as 25 mg of activated charcoal reducing gas-phase radicals by 41%. In all studies, charcoal had less of an effect on nicotine delivery, which was decreased 33% at the maximal amount of charcoal tested (300 mg). Overall, these results support the potential consideration of charcoal in cigarette filters as a means to reduce exposure to toxic free radicals from cigarettes and other combustible tobacco products.

Influence of Smoking Puff Parameters and Tobacco Varieties on Free Radicals Yields in Cigarette Mainstream Smoke.

Cigarette smoke is a major exogenous source of free radicals, and the resulting oxidative stress is one of the major causes of smoking-caused diseases. Yet, many of the factors that impact free radical delivery from cigarettes remain unclear. In this study, we machine-smoked cigarettes and measured the levels of gas- and particulate-phase radicals by electron paramagnetic resonance (EPR) spectroscopy using standardized smoking regimens (International Organization of Standardization (ISO) and Canadian Intense (CI)), puffing parameters, and tobacco blends. Radical delivery per cigarette was significantly greater in both gas (4-fold) and particulate (6-fold) phases when cigarettes were smoked under the CI protocol compared to the ISO protocol. Total puff volume per cigarette was the major factor with radical production being proportional to total volume, regardless of whether volume differences were achieved by changes in individual puff volume or puff frequency. Changing puff shape (bell vs sharp vs square) or puff duration (1-5 s), without changing volume, had no effect on radical yields. Tobacco variety did have a significant impact on free radical production, with gas-phase radicals highest in reconstituted > burley > oriental > bright tobacco and particulate-phase radicals highest in burley > bright > oriental > reconstituted tobacco. Our findings show that modifiable cigarette design features and measurable user smoking behaviors are key factors determining free radical exposure in smokers.

Effect of flavoring chemicals on free radical formation in electronic cigarette aerosols.

BACKGROUND: Flavoring chemicals, or flavorants, have been used in electronic cigarettes (e-cigarettes) since their inception; however, little is known about their toxicological effects. Free radicals present in e-cigarette aerosols have been shown to induce oxidative stress resulting in damage to proliferation, survival, and inflammation pathways in the cell. Aerosols generated from e-liquid solvents alone contain high levels of free radicals but few studies have looked at how these toxins are modulated by flavorants. OBJECTIVES: We investigated the effects of different flavorants on free radical production in e-cigarette aerosols. METHODS: Free radicals generated from 49 commercially available e-liquid flavors were captured and analyzed using electron paramagnetic resonance (EPR). The flavorant composition of each e-liquid was analyzed by gas chromatography mass spectroscopy (GCMS). Radical production was correlated with flavorant abundance. Ten compounds were identified and analyzed for their impact on free radical generation. RESULTS: Nearly half of the flavors modulated free radical generation. Flavorants with strong correlations included ß-damascone, δ-tetradecalactone, γ-decalactone, citral, dipentene, ethyl maltol, ethyl vanillin, ethyl vanillin PG acetal, linalool, and piperonal. Dipentene, ethyl maltol, citral, linalool, and piperonal promoted radical formation in a concentration-dependent manner. Ethyl vanillin inhibited the radical formation in a concentration dependent manner. Free radical production was closely linked with the capacity to oxidize biologically-relevant lipids. CONCLUSIONS: Our results suggest that flavoring agents play an important role in either enhancing or inhibiting the production of free radicals in flavored e-cigarette aerosols. This information is important for developing regulatory strategies aimed at reducing potential harm from e-cigarettes.

Effects of Solvent and Temperature on Free Radical Formation in Electronic Cigarette Aerosols.

The ever-evolving market of electronic cigarettes (e-cigarettes) presents a challenge for analyzing and characterizing the harmful products they can produce. Earlier we reported that e-cigarette aerosols can deliver high levels of reactive free radicals; however, there are few data characterizing the production of these potentially harmful oxidants. Thus, we have performed a detailed analysis of the different parameters affecting the production of free radical by e-cigarettes. Using a temperature-controlled e-cigarette device and a novel mechanism for reliably simulating e-cigarette usage conditions, including coil activation and puff flow, we analyzed the effects of temperature, wattage, and e-liquid solvent composition of propylene glycol (PG) and glycerol (GLY) on radical production. Free radicals in e-cigarette aerosols were spin-trapped and analyzed using electron paramagnetic resonance. Free radical production increased in a temperature-dependent manner, showing a nearly 2-fold increase between 100 and 300 °C under constant-temperature conditions. Free radical production under constant wattage showed an even greater increase when going from 10 to 50 W due, in part, to higher coil temperatures compared to constant-temperature conditions. The e-liquid PG content also heavily influenced free radical production, showing a nearly 3-fold increase upon comparison of ratios of 0:100 (PG:GLY) and 100:0 (PG:GLY). Increases in PG content were also associated with increases in aerosol-induced oxidation of biologically relevant lipids. These results demonstrate that the production of reactive free radicals in e-cigarette aerosols is highly solvent dependent and increases with an increase in temperature. Radical production was somewhat dependent on aerosol production at higher temperatures; however, disproportionately high levels of free radicals were observed at ≥100 °C despite limited aerosol production. Overall, these findings suggest that e-cigarettes can be designed to minimize exposure to these potentially harmful products.

A Survey of Nicotine Yields in Small Cigar Smoke: Influence of Cigar Design and Smoking Regimens.

Introduction: Although the popularity of small cigar brands that resemble cigarettes, including both little cigars (LC) and filtered cigars (FC), has been on the rise, little is known about the delivery of nicotine from these products. Our objective was to determine the nicotine yields of small cigars in comparison to cigarettes. Methods: Nicotine yields from LC, FC, and 3R4F and 1R6F research cigarettes were determined from mainstream smoke generated on a smoking machine under the International Organization of Standardization (ISO) and Canadian Intense (CI) methods. Market characteristics (price and package label) and physical features (filter ventilation, product weight and filter weight, product length, and diameter) were also determined for eight brands of small cigars. Results: Nicotine yields in small cigars averaged 1.24 and 3.49 mg/unit on ISO and CI regimens, respectively, compared with 0.73 and 2.35 mg/unit, respectively, for the research cigarettes. Nicotine yields per puff were similar between small cigars and cigarettes. We also found that FC did not differ from LC in nicotine yields. FC and LC differ from each other in many physical design features (unit weight, filter weight, and filter length), but are similar in others (unit length, diameter, and filter ventilation). Conclusions: Nicotine delivery from small cigars is similar to or greater than that from cigarettes. Thus, for future research and regulatory purposes, standard definitions need to be developed for small cigars, and FC and LC should be evaluated as separate entities. Implications: Small cigars are similar to cigarettes in their design and use. Although nicotine yields per puff were similar between products, small cigars delivered substantially higher amounts of nicotine per unit than cigarettes. These findings support the growing body of evidence to justify regulating all small cigars, including LC and FC in a similar fashion as cigarettes.

Effects of Topography-Related Puff Parameters on Carbonyl Delivery in Mainstream Cigarette Smoke.

Smoking topography parameters differ substantially between individual smokers and may lead to significant variation in tobacco smoke exposure and risk for tobacco-caused diseases. However, to date, little is known regarding the impact of individual puff parameters on the delivery of many harmful smoke constituents including carbonyls. To examine this, we determined the effect of altering individual puff parameters on mainstream smoke carbonyl levels in machine-smoked reference cigarettes. Carbonyls including formaldehyde, acetaldehyde, crotonaldehyde, propionaldehyde, methyl ethyl ketone (MEK), acrolein, and acetone were determined in cigarette smoke by HPLC after derivatization with 2,4-dinitrophenylhydrazine (DNPH). Deliveries of all carbonyls were nearly two-fold greater when cigarettes were smoked according to the more intense Health Canada Intense (HCI) protocol compared to the International Organization of Standardization (ISO) method, consistent with the two-fold difference in total puff volume between methods (ISO: 280-315 mL; CI: 495-605 mL). When individual topography parameters were assessed, changes in puff volume alone had the greatest effect on carbonyl delivery as predicted with total carbonyls being strongly correlated with overall puff volume (r2: 0.52-0.99) regardless of how the differences in volume were achieved. All seven of the carbonyls examined showed a similar relationship with puff volume. Minor effects on carbonyl levels were observed from vent blocking and changing the interpuff interval, while effects of changing puff duration and peak flow rate were minimal. Overall, these results highlight the importance of considering topography, especially puff volume, when the toxicant delivery and potential exposure smokers receive are assessed. The lack of an impact of other behaviors, including puff intensity and duration independent of volume, indicate that factors such as temperature and peak flow rate may have minimal overall effects on carbonyl production and delivery.

Variation in Free Radical Yields from U.S. Marketed Cigarettes.

Free radicals in tobacco smoke are thought to be an important cause of smoking-induced diseases, yet the variation in free radical exposure to smokers from different brands of commercially available cigarettes is unknown. We measured the levels of highly reactive gas-phase and stable particulate-phase radicals in mainstream cigarette smoke by electron paramagnetic resonance (EPR) spectroscopy with and without the spin-trapping agent phenyl-N-tert-butylnitrone (PBN), respectively, in 27 popular US cigarettes and the 3R4F research cigarette, machine-smoked according to the FTC protocol. We find a 12-fold variation in the levels of gas-phase radicals (1.2 to 14 nmol per cigarette) and a 2-fold variation in the amounts of particulate-phase radicals (44 to 96 pmol per cigarette) across the range of cigarette brands. Gas and particulate-phase radicals were highly correlated across brands (ρ = 0.62, p < 0.001). Both radicals were correlated with TPM (gas-phase: ρ = 0.38, p = 0.04; particulate-phase: ρ = 0.44, p = 0.02) and ventilation (gas- and tar-phase: ρ = -0.58, p = 0.001), with ventilation explaining nearly 30% of the variation in radical levels across brands. Overall, our findings of significant brand variation in free radical delivery under standardized machine-smoked conditions suggest that the use of certain brands of cigarettes may be associated with greater levels of oxidative stress in smokers.

Soy protein concentrate mitigates markers of colonic inflammation and loss of gut barrier function in vitro and in vivo.

Whereas a number of studies have examined the effects of soy isoflavones and tocopherols on colonic inflammation, few have examined soy protein. We determined the radical scavenging and cytoprotective effects of soy protein concentrate (SPC) in vitro and its anti-inflammatory effects in dextran sulfate sodium (DSS)-treated mice. Cotreatment with SPC protected Caco-2 human colon cells from H2O2-induced cell death and mitigated intracellular oxidative stress. Treatment of differentiated Caco-2 cells with SPC blunted DSS-induced increases in monolayer permeability. Pepsin/pancreatin-digested SPC had reduced radical scavenging activity, but retained the monolayer protective effects of SPC. In vivo, 1.5% DSS caused body weight loss, colon shortening, and splenomegaly in CF-1 mice. Co-treatment with 12% SPC mitigated DSS-induced body weight loss and splenomegaly. DSS increased colonic interleukin (IL)-1ß, IL-6, and monocyte chemotactic protein-1 expression. The levels of these markers were significantly lower in mice co-treated with SPC. SPC prevented DSS-mediated reductions in colonic glucagon-like peptide 2 levels, suggesting that SPC can prevent loss of gut barrier function, but no significant effect on claudin 1 and occludin mRNA levels of was observed. SPC-treated mice had lower colonic mRNA expression of toll-like receptor 4 and nucleotide-binding oligomerization domain-containing protein-like receptor family, pyrin domain containing protein 3 (NLRP3), and lower caspase-1 enzyme activity than DSS-treated mice. In summary, SPC exerted antioxidant and cytoprotective effects in vitro and moderated the severity of DSS-induced inflammation and loss of gut barrier function in vivo. These effects appear to be mediated in part through reduced NLRP3 expression and caspase 1 activity.