Nicotine Delivery of a Menthol-Flavored Heat-not-Burn Tobacco Product During Directed Use.

INTRODUCTION: IQOS was authorized by the U.S. Food and Drug Administration (FDA) as a modified-risk tobacco product. We conducted a pharmacokinetic study evaluating the nicotine delivery and subjective effects of IQOS use among current menthol cigarette smokers to better understand if IQOS is an acceptable cigarette alternative in light of the proposed menthol cigarette ban. AIMS AND METHODS: Participants were adult smokers of >4 menthol cigarettes per day. After 14-hour nicotine abstinence, participants were provided an IQOS device and menthol heatstick to puff every 20 seconds for a total of 14 puffs. Blood samples were collected at baseline and during active use to calculate nicotine boost from baseline to peak concentration. Nicotine withdrawal symptoms were collected before and after IQOS use. In addition, a modified Product Evaluation Scale for IQOS was collected after use. RESULTS: Participants (n = 8) were a mean age of 43.9 years, 63% were female, 88% identified as White, and they smoked a mean of 17.1 menthol cigarettes per day. After IQOS use, the mean nicotine boost obtained was 15.96 ng/mL (SD = 6.91) (range 9.31 to 30.55 ng/mL). Most (75%) participants reported enjoying use of the product "a lot" or greater and more than half (62.5%) reported reduced cigarette cravings. Most participants reported no side effects after use; however, two experienced dry mouth, three experienced dizziness, one experienced throat irritation, and one experienced headache. CONCLUSION: We found that directed use (14 puffs) of menthol IQOS delivered a mean nicotine boost of 15.96 ng/mL which reduced craving for a cigarette. The majority of participants enjoyed use of IQOS and reported mild side effects. IMPLICATIONS: Menthol IQOS delivered a sufficient dose of nicotine perceived as satisfying by menthol cigarette smokers and it reduced craving with mild side effects. Menthol IQOS has potential to serve as a less harmful alternative for menthol cigarette smokers. The availability of modified risk products like IQOS should be considered by FDA's Comprehensive Plan for Tobacco and Nicotine Regulation.

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.

Switching to Progressively Reduced Nicotine Content Cigarettes in Smokers With Low Socioeconomic Status: A Double-Blind Randomized Clinical Trial.

INTRODUCTION: The Food and Drug Administration issued an advanced notice of proposed rulemaking for setting a product standard for nicotine levels in cigarettes, with an emphasis on minimally or non-addicting very low nicotine content (VLNC). METHODS: A 33 week, two-arm, double-blind randomized trial conducted in Hershey, Pennsylvania, USA and Washington, DC, USA included adult daily cigarette smokers (≥5 cigarettes per day) with less than a college degree, and who had no plans to quit within the next six months. Participants were randomized to either reduced nicotine content (RNC) study cigarettes tapered every three weeks to a final VLNC (0.2 mg/cigarette) for six weeks or to usual nicotine content (UNC) study cigarettes (11.6 mg/cigarette). Outcomes included acceptability of study cigarettes measured by attrition (primary outcome), compliance, reduction in cigarette dependence and tobacco biomarkers, and post-intervention cessation. RESULTS: The RNC (n = 122) versus UNC (n = 123) group had higher attrition (adjusted Hazard Ratio 3.4; 95% confidence interval [CI] 1.99 to 5.81). At the end of the intervention, cotinine levels were 50% lower in the RNC group (mean group difference -137 ng/mL; 95% CI -172, -102). The RNC group smoked fewer CPD (-4.1; 95% CI -6.44, -1.75) and had lower carbon monoxide levels (-4.0 ppm; 95% CI -7.7, -0.4). Forty seven percent (29/62) of the RNC group were biochemically-confirmed compliant with smoking VLNC cigarettes (mean cotinine = 8.9 ng/ml). At three month follow-up, only compliant VLNC smokers quit with an assisted quit attempt (N = 6/22, 27%). CONCLUSIONS: This study supports a VLNC standard in cigarettes. IMPLICATIONS: Differential dropout and noncompliance indicate some smokers had difficulty transitioning to cigarettes with reduced nicotine. These smokers will benefit from supplemental nicotine in medicinal or noncombustible tobacco products if a nicotine reduction standard is established. Other smokers successfully transitioned to very low nicotine content cigarettes exclusively and substantially reduced their exposure to nicotine.

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.

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.

Pharmacokinetic Profile of Spectrum Reduced Nicotine Cigarettes.

INTRODUCTION: Spectrum research cigarettes have been developed with varying nicotine content for use in studies evaluating the effects of a regulatory policy reducing the permissible nicotine content in cigarettes. This study aimed to characterize the nicotine pharmacokinetic profile of Spectrum cigarettes. METHODS: Twelve daily smokers attended four sessions and had blood nicotine, exhaled carbon monoxide, and subjective effects measured before and after smoking either a single cigarette of their preferred brand or high (10.9 mg/cigarette), medium (3.2 mg/cigarette), or low (0.2 mg/cigarette) nicotine content Spectrum research cigarettes, in a double-blind design with order counterbalanced. RESULTS: The boost in blood nicotine concentration was dose-dependent, with a boost of 0.3, 3.9, and 17.3 ng/mL for low-, medium-, and high-nicotine content Spectrum cigarettes. The high dose Spectrum had a similar nicotine boost to the "preferred brand" cigarettes (19 ng/mL). Subjects took longer puffs on the low nicotine cigarettes, but smoked these cigarettes faster than other cigarette types. High nicotine Spectrum cigarettes reduced the urge to smoke more than other cigarette types. CONCLUSIONS: This study shows that Spectrum research cigarettes produce blood nicotine absorption in a dose-dependent manner, and therefore, are appropriate for use in studies of nicotine reduction in cigarettes. IMPLICATIONS: This is the first study to determine the pharmacokinetic profile of Spectrum reduced nicotine content research cigarettes following an overnight abstinence. These data could provide evidence to regulatory agencies about the effects of reduced nicotine cigarettes when considering regulations on tobacco reduction.

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.

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.

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.

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.

Highly reactive free radicals in electronic cigarette aerosols.

Electronic cigarette (EC) usage has increased exponentially, but limited data are available on its potential harmful effects. We tested for the presence of reactive, short-lived free radicals in EC aerosols by electron paramagnetic resonance spectroscopy (EPR) using the spin-trap phenyl-N-tert-butylnitrone (PBN). Radicals were detected in aerosols from all ECs and eliquids tested (2.5 × 10(13) to 10.3 × 10(13) radicals per puff at 3.3 V) and from eliquid solvents propylene glycol and glycerol and from "dry puffing". These results demonstrate, for the first time, the production of highly oxidizing free radicals from ECs which may present a potential toxicological risk to EC users.

Influence of omega-3 fatty acids on Tamoxifen-induced suppression of rat mammary carcinogenesis.

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague-Dawley rats were injected ip with 1-methyl-1-nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO-rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n-3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n-3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n-3FA.

H2S concentrations in the arterial blood during H2S administration in relation to its toxicity and effects on breathing.

Our aim was to establish in spontaneously breathing urethane-anesthetized rats, the relationship between the concentrations of H2S transported in the blood and the corresponding clinical manifestations, i.e., breathing stimulation and inhibition, during and following infusion of NaHS at increasing rates. The gaseous concentration of H2S (CgH2S, one-third of the total soluble form) was computed from the continuous determination of H2S partial pressure in the alveolar gas, while H2S, both dissolved and combined to hemoglobin, was measured at specific time points by sulfide complexation with monobromobimane (CMBBH2S). We found that using a potent reducing agent in vitro, H2S added to the whole blood had little interaction with the plasma proteins, as sulfide appeared to be primarily combined and then oxidized by hemoglobin. In vivo, H2S was undetectable in the blood in its soluble form in baseline conditions, while CMBBH2S averaged 0.7 ± 0.5 µM. During NaHS infusion, H2S was primarily present in nonsoluble form in the arterial blood: CMBBH2S was about 50 times higher than CgH2S at the lowest levels of exposure and 5 or 6 times at the levels wherein fatal apnea occurred. CgH2S averaged only 1.1 ± 0.7 µM when breathing increased, corresponding to a CMBBH2S of 11.1 ± 5.4 µM. Apnea occurred at CgH2S above 5.1 µM and CMBBH2S above 25.4 µM. At the cessation of exposure, CMBBH2S remained elevated, at about 3 times above baseline for at least 15 min. These data provide a frame of reference for studying the putative effects of endogenous H2S and for testing antidotes against its deadly effects.

Comparative metabolism of benzo[a]pyrene by human keratinocytes infected with high-risk human papillomavirus types 16 and 18 as episomal or integrated genomes.

BACKGROUND: Infection with human papillomavirus (HPV) is a critical factor in the development of cervical cancer. Smoking is an additional risk factor. Tobacco smoke carcinogens, such as benzo[a]pyrene (B[a]P), and their cytochrome P450-related metabolites are present in significantly higher levels in the cervical mucus of women smokers than in nonsmokers. We determined the metabolism and P450 expression of B[a]P-treated human keratinocytes infected with HPV-16 or -18. MATERIALS AND METHODS: Monolayer cultures of uninfected primary human foreskin keratinocytes, human vaginal and cervical keratinocytes carrying episomal genomes of HPV-16 and -18, respectively, and invasive cervical carcinoma cell lines carrying either HPV-16 or -18 genomes integrated into the host DNA, were incubated with 0.1 µM [(3)H]B[a]P. The resulting oxidative metabolites were analyzed and quantified by radioflow high-performance liquid chromatography. Additionally, all cell lines were incubated with unlabeled 0.1 µM B[a]P for Western blot analysis of cytochrome P450 1A1 and 1B1. RESULTS: Significant enhancement in levels of both detoxification and activation metabolites was found in incubations with all types of HPV-infected cells compared with control incubations (P < 0.05). The highest capacity to metabolize B[a]P was observed with cells containing integrated HPV-18 genomes. Induction of cytochrome 1B1 was observed in HPV-16 and -18 integrated, and in HPV-16 episomal cell types. CONCLUSIONS: Both viral genotype and genomic status in the host cell affect B[a]P metabolism and cytochrome P450 1B1 expression. An increase of DNA-damaging metabolites might result from exposure of HPV-infected women to cigarette smoke carcinogens.

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.

The effects of reduced nicotine content cigarettes on biomarkers of nicotine and toxicant exposure, smoking behavior and psychiatric symptoms in smokers with mood or anxiety disorders: A double-blind randomized trial.

BACKGROUND: The U.S. Food and Drug Administration and the government of New Zealand have proposed a reduction of the nicotine content in cigarettes to very low levels. This study examined the potential effects of this regulation in smokers with affective disorders. METHODS: In a randomized controlled parallel group trial conducted at two sites in the USA (Penn State University, Hershey, PA and Massachusetts General Hospital, Boston, MA) 188 adult smokers with a current (n = 118) or lifetime (n = 70) anxiety or unipolar mood disorder, not planning to quit in the next 6 months, were randomly assigned (1:1) to smoke either Usual Nicotine Content (UNC) (11.6 mg nicotine/cigarette) research cigarettes, or Reduced Nicotine Content (RNC) research cigarettes where the nicotine content per cigarette was progressively reduced to 0.2 mg in five steps over 18 weeks. Participants were then offered the choice to either receive assistance to quit smoking, receive free research cigarettes, or resume using their own cigarette brand during a 12-week follow-up period. Main outcomes were biomarkers of nicotine and toxicant exposure, smoking behavior and dependence and severity of psychiatric symptoms. The pre-registered primary outcome was plasma cotinine. RESULTS: A total of 143 (76.1%) randomized participants completed the randomized phase of the trial, 69 (73.4%) in the RNC group and 74 (78.8%) in the UNC group. After switching to the lowest nicotine content cigarettes, compared to smokers in the UNC group, at the last randomized visit the RNC group had significantly lower plasma cotinine (metabolite of nicotine): difference between groups, -175.7, 95% CI [-218.3, -133.1] ng/ml. Urine NNAL (metabolite of NNK, a lung carcinogen), exhaled carbon-monoxide, cigarette consumption, and cigarette dependence were also significantly lower in the RNC group than the UNC group. No between-group differences were found on a range of other biomarkers (e.g. 8-isoprostanes) or health indicators (e.g. blood pressure), or on 5 different psychiatric questionnaires, including the Kessler K6 measure of psychological distress. At the end of the subsequent 12-week treatment choice phase, those randomized to the RNC group were more likely to have quit smoking, based on initial intent-to-treat sample, n = 188 (18.1% RNC v 4.3% UNC, p = 0.004). CONCLUSION: Reducing nicotine content in cigarettes to very low levels reduces some toxicant exposures and cigarette addiction and increases smoking cessation in smokers with mood and/or anxiety disorders, without worsening mental health. TRIAL REGISTRATION: TRN: NCT01928758, registered August 21, 2013.