Search

PDF file - 481K, Table S-1. SRM Transitions and Collision Energies Table S-2. Equations for Mercapturic Acid Calibration Curves Table S-3. Precision and Accuracy for Mercapturic Acids in Urine, Intra-day (N=6) Table S-4. Concentrations of Mercapturic Acids in Urine of Cigarette Smokers. Comparison with Literature Values. Table S-5. Concentrations of PAH Metabolites in Urine of Smokers. Comparison with Literature Values. Figure S-1A. Chromatograms of urine extracts containing mercapturic acids: Metabolites of acrylamide (AAMA), ethylene oxide (HEMA), and acrolein (3-HPMA). Figure S-1B. Chromatograms of urine extracts containing mercapturic acids: Metabolites of 1,3-butadiene (MHBMA), acrylonitrile (CNEMA), and benzene (PMA). Figure S-1C. Chromatograms of urine extracts containing mercapturic acids: Metabolite of propylene oxide (2-HPMA) and separation from the isomer 3-HPMA. Figure S-2. Butadiene metabolite (MHBMA) calibration curves. Figure S-3. Benzene metabolite (PMA) calibration curves.

Background: Smoking tobacco preparations in a water pipe (hookah) is widespread in many places of the world and is perceived by many as relatively safe. We investigated biomarkers of toxicant exposure with water pipe compared with cigarette smoking.Methods: We conducted a crossover study to assess daily nicotine and carcinogen exposure with water pipe and cigarette smoking in 13 people who were experienced in using both products.Results: When smoking an average of 3 water pipe sessions compared with smoking 11 cigarettes per day (cpd), water pipe use was associated with a significantly lower intake of nicotine, greater exposure to carbon monoxide (CO), and a different pattern of carcinogen exposure compared with cigarette smoking, with greater exposure to benzene, and high molecular weight polycyclic aromatic hydrocarbon (PAH), but less exposure to tobacco-specific nitrosamines, 1,3-butadiene, acrolein, acrylonitrile, propylene oxide, ethylene oxide, and low molecular weight PAHs.Conclusions: A different pattern of carcinogen exposure might result in a different cancer risk profile between cigarette and water pipe smoking. Of particular concern is the risk of leukemia related to high levels of benzene exposure with water pipe use.Impact: Smoking tobacco in water pipes has gained popularity in the United States and around the world. Many believe that water pipe smoking is not addictive and less harmful than cigarette smoking. We provide data on toxicant exposure that will help guide regulation and public education regarding water pipe health risk. Cancer Epidemiol Biomarkers Prev; 22(5); 765–72. ©2013 AACR.

The most important tobacco-specific nitrosamine found in cigarette smoke and formed in ageing smoke after cigarettes are extinguished is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). It is formed from nitrosation of nicotine, under particular conditions both in indoor and outdoor environments. NNK has been classified as a potent lung carcinogen which is expected to be found primarily in the particle-phase and to be stable in particulate matter. In this study tests have been carried out to show that a bisulfate-treated filter is more efficient than an untreated filter to collect both nicotine and NNK, and that the latter is stable in outdoor particulate matter. To characterize NNK in the outdoor environment, airborne samples were collected from 11 cities in USA, UK, Hong Kong and Malta with characteristics varying from low to high population densities and from urban to suburban to rural, and with desert characteristics and distinct climates. It has been shown that airborne particle + gas phase nicotine and particle-phase NNK behave in a linearly correlated manner. A seasonal analysis was carried out on a subset of data available from five sites in California, where the load of NNK in PM10 is driven by long range transport of the air masses passing over densely populated cities. In the winter season, the load of NNK in PM is higher than in summer in a statistically significant manner. The contamination of PM with NNK shows variability, but is observed at all sites. This paper highlights the potential risk of chronic exposure to NNK in particulate matter by the inhalation pathway.

Author(s): Liu, Jia; Benowitz, Neal L.; Hatsukami, Dorothy K.; Havel, Christopher M.; Lazcano-Ponce, Eduardo; Strasser, Andrew A.; Jacob, Peyton | Abstract: In studies of tobacco toxicology, including comparisons of different tobacco products and exposure to secondhand or thirdhand smoke, exposure assessment using biomarkers is often useful. Some studies have indicated that most of the toxicity of tobacco smoke is due to gas-phase compounds. 3-Ethenylpyridine (3-EP) is a major nicotine pyrolysis product occurring in the gas phase of tobacco smoke. It has been used extensively as an environmental tracer for tobacco smoke. 3-EP would be expected to be a useful tobacco smoke biomarker as well, but nothing has been published about its metabolism and excretion in humans. In this Article we describe a solid-phase microextraction (SPME) GC-MS/MS method for determination of 3-EP in human urine and its application to the determination of 3-EP in the urine of smokers and people exposed to secondhand smoke. We conclude that 3-EP is a promising biomarker that could be useful in studies of tobacco smoke exposure and toxicology. We also point out the paucity of data on 3-EP toxicity and suggest that additional studies are needed.Graphical Abstract

Second Hand Smoke (SHS) has always been primarily linked with indoor pollution. To date nicotine was the favoured marker for SHS alongside measurements of particulate matter (PM) levels. As nicotine is mainly found in the gas-phase and reactive in the outdoor environment it is not ideal as a marker for the SHS-driven particulate component in PM. Nicotelline, a minor tobacco alkaloid that is stable, found almost exclusively in the particle phase and easy to quantify even at low concentrations, is being proposed as a better marker. It is the first study using bisulfate-treated quartz fiber filters to show that airborne nicotine (gas+particle phase) is directly proportional to airborne nicotelline in countries that have different climates. The analytical method developed has been validated to show that the use of untreated filters is suitable for the quantification of nicotelline even at low concentrations. Although nicotelline exhibits a seasonal and geographical variation, this is the first comprehensive study which demonstrates the ubiquitous presence of nicotelline in PM from outdoor air samples collected in the USA (0.1-285.6 pgm-3), UK (2.3-9.1 pgm-3), Hong Kong (3.8-109.3 pgm-3) and Malta (4.2-280.8 pgm-3). From the nicotelline apportionment factor of 1589 ng/mg of tobacco smoke PM we estimate the fraction of outdoor airborne PM derived from SHS to be in the range of 0.03-0.08%. While it is unlikely for tobacco smoke-related toxics in outdoor PM to be considered a major health hazard, in heavily polluted microenvironments this marker would be useful in tracing the presence of SHS and emerging Third Hand Smoke components that form or are found in airborne and settled PM that could induce serious health effects.

Synthetic cannabinoid receptor agonists (SCRAs) are a dynamic class of new psychoactive substances (NPS), with novel chemotypes emerging each year. Following the putative detection of 5F-CUMYL-P7AICA in Australia in 2016, the scaffold-hopping SCRAs 5F-CUMYL-PICA, 5F-CUMYL-PINACA, and 5F-CUMYL-P7AICA were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-quadrupole time-of-flight-MS (LC-QTOF-MS). Since little is known of the pharmacology of 7-azaindole SCRAs like 5F-CUMYL-P7AICA, the binding affinities and functional activities of all compounds at cannabinoid type 1 and type 2 receptors (CB1 and CB2 , respectively) were assessed using tritiated radioligand competition experiments and fluorescence-based plate reader membrane potential assays. Despite CB1 binding affinities differing by over two orders of magnitude (Ki = 2.95-174 nM), all compounds were potent and efficacious CB1 agonists (EC50 = 0.43-4.7 nM), with consistent rank order for binding and functional activity (5F-CUMYL-PINACA >5F-CUMYL-PICA >5F-CUMYL-P7AICA). Additionally, 5F-CUMYL-P7AICA was found to exert potent cannabimimetic effects in mice, inducing hypothermia (6°C, 3 mg/kg) through a CB1 -dependent mechanism.

To describe the effect of e-liquid flavors on nicotine intake and pharmacology of e-cigarettes.11 males and 3 females participated in a 3-day inpatient crossover study with strawberry, tobacco, and their usual flavor e-liquid. Nicotine levels were nominally 18mg/mL in the strawberry (pH 8.29) and tobacco (pH 9.10) e-liquids and ranged between 3-18mg/mL in the usual brands (mean pH 6.80). Each day consisted of a 15-puff session followed by 4h of abstinence, then 90min of ad libitum use. Subjects used a KangerTech mini ProTank 3.After 15 puffs, the amount of nicotine inhaled and systemically retained were not significantly different between the strawberry and tobacco e-liquids but plasma AUC(0→180) was significantly higher with the strawberry e-liquid. While not significantly different, Cmax was 22% higher and various early time point AUCs to measure rate of rise of nicotine in blood ranged between 17 and 23% higher with the strawberry e-liquid compared to the tobacco e-liquid. During ad libitum use, systemic exposure to nicotine (AUC(0→90)) was the same for the tobacco and usual brand e-liquids but were both significantly lower than after using the strawberry e-liquid. The usual flavors were more liked and satisfying than the strawberry and tobacco e-liquids.Flavors influence nicotine exposure through flavor liking, may affect rate of nicotine absorption possibly through pH effects, and contribute to heart rate acceleration and subjective effects of e-cigarettes. E-cigarette users titrate their nicotine exposure but the extent of titration may vary across flavors.

Introduction Many electronic cigarette manufacturers have begun offering liquids containing “nicotine salts,” which are formed when an acid is mixed in a solution with free-base nicotine. Type of salt could play a significant role in the abuse liability of electronic cigarette liquids. As a first step to understanding nicotine salts, this study sought to identify the types of acids present in 23 commercially available electronic cigarette liquids. Aims and Methods Twenty-three electronic cigarette liquids advertised as containing nicotine salts were purchased for analysis. These liquids were tested for the presence of 11 different organic acids that were deemed likely to be used in a nicotine salt formulation. Liquids were analyzed using a combination of liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry methods, then compared to authentic acid standards for identification. Results Six of the 11 possible acids were identified in the liquids, from most to least common: lactic, benzoic, levulinic, salicyclic, malic, and tartaric acid. Acid(s) could not be identified in one of the liquids. Though most liquids contained only one type, three of the liquids contained multiple acids. Conclusions These data demonstrate that several types of salts/acids are currently being used in electronic cigarette liquids. The type and concentration of salt(s) used in these liquids may differentially alter sensations in the throat and upper airway, and overall pharmacology of the aerosols by altering liquid pH and from flavor and sensory characteristics of the acids themselves. Implications This study demonstrates that at least six different types of acids are being used to create the nicotine salts in electronic cigarette liquids, with the acids lactic, benzoic, and levulinic being the most frequently identified. Identification of these acids can serve as the foundation for future research to determine if type of nicotine salt alters pharmacological and toxicological effects of electronic cigarettes.

Introduction Dual use of electronic cigarettes (e-cigarettes) and combustible cigarettes is a major public health issue. It is generally accepted that exclusive e-cigarette use is less harmful than exclusive combustible cigarette use, but most e-cigarette users continue to smoke combustible cigarettes as well. To what extent the use of e-cigarettes reduces harm in people who continue to smoke combustible cigarettes has been debated. The aim of this study was to explore the utility of biomarkers as measures of dual use. Methods In two human studies of participants who used e-cigarettes only or both combustible cigarettes and e-cigarettes, we measured urine concentrations of the metabolites of nicotine (total nicotine equivalents) as well as two biomarkers of tobacco exposure: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific carcinogen metabolite, and nicotelline, a tobacco alkaloid not found in significant concentrations in e-cigarette products. Results The presence of nicotine metabolites indicates either e-cigarette or combustible cigarette use. Nicotelline (half-life of 2–3 hours) indicates recent combustible cigarette use and NNAL (half-life of 10 days or more), indicates combustible cigarette use occurring within several weeks prior to sample collection. Conclusions Nicotelline and NNAL are useful biomarkers for combustible tobacco use in users e-cigarettes. The application of these biomarkers provides a tool to help assess whether, or to what extent, dual use of e-cigarettes and combustible cigarettes reduces harm compared to sole use of combustible cigarettes. These biomarkers can also verify exclusive use of e-cigarettes over short (24 hour) or long (several week) time periods. Implications: To what extent dual use of e-cigarettes and combustible cigarettes reduce harm compared to smoking combustible cigarettes only is of considerable public health interest. We show that the levels of the minor tobacco alkaloid nicotelline and the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are extremely low in electronic cigarette fluids. The urine biomarkers nicotelline and the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are indicative of cigarette smoking and can be used to assess recent and past smoking in dual users.

Author(s): St Helen, Gideon; Ross, Kathryn C; Dempsey, Delia A; Havel, Christopher M; Jacob, Peyton; Benowitz, Neal L | Abstract: ObjectiveTo characterize vaping behavior and nicotine intake during ad libitum e-cigarette access.MethodsThirteen adult e-cigarette users had 90 minutes of videotaped ad libitum access to their usual e-cigarette. Plasma nicotine was measured before and every 15 minutes after the first puff; subjective effects were measured before and after the session.ResultsAverage puff duration and interpuff interval were 3.5±1.4 seconds (±SD) and 118±141 seconds, respectively. 12% of puffs were unclustered puffs while 43%, 28%, and 17% were clustered in groups of 2-5, 6-10, and g10 puffs, respectively. On average, 4.0±3.3 mg of nicotine was inhaled; the maximum plasma nicotine concentration (Cmax) was 12.8±8.5 ng/mL. Among the 8 tank users, number of puffs was positively correlated with amount of nicotine inhaled, Cmax, and area under the plasma nicotine concentration-time curve (AUC0→90min) while interpuff interval was negatively correlated with Cmax and AUC0→90.ConclusionVaping patterns differ from cigarette smoking. Plasma nicotine levels were consistent with intermittent dosing of nicotine from e-cigarettes compared to the more bolus dosing from cigarettes. Differences in delivery patterns and peak levels of nicotine achieved could influence the addictiveness of e-cigarettes compared to conventional cigarettes.

Author(s): St Helen, Gideon; Nardone, Natalie; Addo, Newton; Dempsey, Delia; Havel, Christopher; Jacob, Peyton; Benowitz, Neal L | Abstract: AimTo describe systemic nicotine exposure and subjective effects of electronic cigarettes (e-cigarettes) in people who use both e-cigarettes and cigarettes (dual users), including within-subject comparisons of e-cigarette and cigarette use.DesignTwo-arm, counterbalanced cross-over study. Participants used their usual brand of e-cigarette or cigarette during a standardized session in a 2-week study.SettingHospital research ward, San Francisco, CA, USA.ParticipantsThirty-six healthy (eight women, 28 men) participants.MeasurementsPlasma nicotine was analyzed by gas chromatography-tandem mass spectrometry; nicotine withdrawal, urge to smoke and vape, affective states, craving, satisfaction and psychological reward were measured by standardized questionnaires.FindingsCompared with cigarettes, average maximum plasma nicotine concentration (Cmax ) was lower with e-cigarettes [6.1 ± 5.5 ng/ml, mean ± standard deviation (SD) versus 20.2 ± 11.1 ng/ml, P l 0.001] and time of maximal concentration (Tmax ) was longer (6.5 ± 5.4 versus 2.7 ± 2.4 minutes, P l 0.001). Use of both products resulted in a reduction in the severity of withdrawal symptoms, negative affect and urge to use either product. E-cigarettes were less rewarding and satisfying and reduced craving to a lesser degree than cigarettes. We were not able to detect any differences in withdrawal symptoms, affective states and urge to smoke cigarettes between e-cigarette and cigarette use.ConclusionSystemic nicotine exposure was, on average, lower with single use of e-cigarettes compared with cigarettes, and e-cigarettes were judged to be less satisfying and rewarding and reduced craving less than cigarettes.

1,4-Butanediol (BDO) - used as solvent, and abused for its euphoric effects - is converted to gamma-hydroxybutyrate (GHB) by the enzyme alcohol dehydrogenase (ADH). This double-blind, placebo-controlled crossover study with six healthy volunteers is the first to date investigating the role of the ADH inhibitor fomepizole (4MP) in moderating this conversion in humans. Participants received on two different days either intravenous placebo or 15 mg/kg 4MP, followed by oral administration of 25 mg/kg BDO. Pretreatment with 4MP resulted in significantly higher BDO maximal plasma concentration (p=0.001) and AUC (p=0.028), confirming that ADH is the primary pathway for the conversion of BDO to GHB in humans. With 4MP, the mean arterial pressure was significantly lower at 105 minutes compared to baseline (p=0.003), indicating that blood pressure lowering, observed not with a temporal relationship to 4MP administration but after the maximum BDO concentration was reached, may be an intrinsic effect of BDO. This article is protected by copyright. All rights reserved.

BackgroundHeated tobacco products (also called ‘heat-not-burn’ products) heat tobacco at temperatures below that of combustion, causing nicotine and other compounds to aerosolise. One such product, IQOS from Philip Morris International, is being marketed internationally with claims of harm reduction. We sought to determine whether exposure to IQOS aerosol impairs arterial flow-mediated dilation (FMD), a measure of vascular endothelial function that is impaired by tobacco smoke.MethodsWe exposed anaesthetised rats (n=8/group) via nose cone to IQOS aerosol from single HeatSticks, mainstream smoke from single Marlboro Red cigarettes or clean air for a series of consecutive 30 s cycles over 1.5–5 min. Each cycle consisted of 15 or 5 s of exposure followed by removal from the nose cone. We measured pre-exposure and postexposure FMD, and postexposure serum nicotine and cotinine.ResultsFMD was impaired comparably by ten 15 s exposures and ten 5 s exposures to IQOS aerosol and to cigarette smoke, but not by clean air. Serum nicotine levels were similar to plasma levels after humans have smoked one cigarette, confirming that exposure conditions had real-world relevance. Postexposure nicotine levels were ~4.5-fold higher in rats exposed to IQOS than to cigarettes, despite nicotine being measured in the IQOS aerosol at ~63% the amount measured in smoke. When IQOS exposure was briefer, leading to comparable serum nicotine levels to the cigarette group, FMD was still comparably impaired.ConclusionsAcute exposures to IQOS aerosol impairs FMD in rats. IQOS use does not necessarily avoid the adverse cardiovascular effects of smoking cigarettes.

RATIONALE: Gamma- hydroxybutyrate acid (GHB), a GABA(B) receptor agonist approved for treatment of narcolepsy, impairs driving ability, but little is known about doses and plasma concentrations associated with impairment and time course of recovery. OBJECTIVE: To assess effects of oral GHB (Xyrem®) upon driving as measured by a driving simulator, and to determine plasma concentrations associated with impairment and the time course of recovery. METHODS: Randomized, double-blind, two-arm crossover study, during which sixteen participants received GHB 50 mg/kg orally or placebo. GHB blood samples were collected prior to and at one, three and six hours post dosing. Driving simulator sessions occurred immediately after blood sampling. RESULTS: Plasma GHB was not detectable at baseline or six hours post dosing. Median GHB concentrations at one and three hours were 83.1 mg/L (range 54–110) and 24.4 mg/L (range 7.2–49.7), respectively. Compared to placebo, at one hour post GHB dosing, significant differences were seen for the life-threatening outcomes collisions (p

Author(s): St Helen, Gideon; Havel, Christopher; Dempsey, Delia A; Jacob, Peyton; Benowitz, Neal L | Abstract: AimsTo measure the systemic retention of nicotine, propylene glycol (PG) and vegetable glycerin (VG) in electronic cigarette (e-cigarette) users, and assess the abuse liability of e-cigarettes by characterizing nicotine pharmacokinetics.DesignE-cigarette users recruited over the internet participated in a 1-day research ward study. Subjects took 15 puffs from their usual brand of e-cigarette. Exhaled breath was trapped in gas-washing bottles and blood was sampled before and several times after use.SettingSan Francisco, California, USA.ParticipantsThirteen healthy, experienced adult e-cigarette users (six females and seven males).MeasurementsPlasma nicotine was analyzed by gas chromatography-mass spectrometry (GC-MS/MS) and nicotine, VG and PG in e-liquids and gas traps were analyzed by LC-MS/MS. Heart rate changes and subjective effects were assessed.FindingsE-cigarettes delivered an average of 1.33 (0.87-1.79) mg [mean and 95% confidence interval (CI)] of nicotine, and 93.8% of the inhaled dose, 1.22 (0.80-1.66) was systemically retained. Average maximum plasma nicotine concentration (Cmax ) was 8.4 (5.4-11.5) ng/ml and time of maximal concentration (Tmax ) was 2-5 minutes. One participant had Tmax of 30 minutes. 84.4% and 91.7% of VG and PG, respectively, was systemically retained. Heart rate increased by an average of 8.0 beats per minute after 5 minutes. Withdrawal and urge to smoke decreased and the e-cigarettes were described as satisfying.ConclusionsE-cigarettes can deliver levels of nicotine that are comparable to or higher than typical tobacco cigarettes, with similar systemic retention. Although the average maximum plasma nicotine concentration in experienced e-cigarette users appears to be generally lower than what has been reported from tobacco cigarette use, the shape of the pharmacokinetic curve is similar, suggesting addictive potential.

© 2015 American Chemical Society. Smokeless tobacco products, such as moist snuff or chewing tobacco, contain many of the same carcinogens as tobacco smoke; however, the impact on children of indirect exposure to tobacco constituents via parental smokeless tobacco use is unknown. As part of the California Childhood Leukemia Study, dust samples were collected from 6 homes occupied by smokeless tobacco users, 6 homes occupied by active smokers, and 20 tobacco-free homes. To assess children's potential for exposure to tobacco constituents, vacuum-dust concentrations of five tobacco-specific nitrosamines, including N′-nitrosonornicotine [NNN] and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [NNK], as well as six tobacco alkaloids, including nicotine and myosmine, were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We used generalized estimating equations derived from a multivariable marginal model to compare levels of tobacco constituents between groups, after adjusting for a history of parental smoking, income, home construction date, and mother's age and race/ethnicity. The ratio of myosmine/nicotine was used as a novel indicator of the source of tobacco contamination, distinguishing between smokeless tobacco products and tobacco smoke. Median dust concentrations of NNN and NNK were significantly greater in homes with smokeless tobacco users compared to tobacco-free homes. In multivariable models, concentrations of NNN and NNK were 4.8- and 6.9-fold higher, respectively, in homes with smokeless tobacco users compared to tobacco-free homes. Median myosmine/nicotine ratios were lower in homes with smokeless tobacco users (1.8%) compared to homes of active smokers (7.7%), confirming that cigarette smoke was not the predominant source of tobacco constituents in homes with smokeless tobacco users. Children living with smokeless tobacco users may be exposed to carcinogenic tobacco-specific nitrosamines via contact with contaminated dust and household surfaces. (Chemical Presented).

Exposure to thirdhand smoke (THS) is a recently described health concern that arises in many indoor environments. However, the carcinogenic potential of THS, a critical consideration in risk assessment, remains untested. Here we investigated the effects of short-term early exposure to THS on lung carcinogenesis in A/J mice. Forty weeks after THS exposure from 4 to 7 weeks of age, the mice had increased incidence of lung adenocarcinoma, tumor size and, multiplicity, compared with controls. In vitro studies using cultured human lung cancer cells showed that THS exposure induced DNA double-strand breaks and increased cell proliferation and colony formation. RNA sequencing analysis revealed that THS exposure induced endoplasmic reticulum stress and activated p53 signaling. Activation of the p53 pathway was confirmed by an increase in its targets p21 and BAX. These data indicate that early exposure to THS is associated with increased lung cancer risk.

Thirdhand smoke (THS) is the fraction of cigarette smoke that persists in indoor environments after smoking. We investigated the effects of neonatal and adult THS exposure on bodyweight and blood cell populations in C57BL/6 J mice. At the end of neonatal exposure, THS-treated male and female mice had significantly lower bodyweight than their respective control mice. However, five weeks after neonatal exposure ended, THS-treated mice weighed the same as controls. In contrast, adult THS exposure did not change bodyweight of mice. On the other hand, both neonatal and adult THS exposure had profound effects on the hematopoietic system. Fourteen weeks after neonatal THS exposure ended, eosinophil number and platelet volume were significantly higher, while hematocrit, mean cell volume, and platelet counts were significantly lower compared to control. Similarly, adult THS exposure also decreased platelet counts and increased neutrophil counts. Moreover, both neonatal and adult THS exposure caused a significant increase in percentage of B-cells and significantly decreased percentage of myeloid cells. Our results demonstrate that neonatal THS exposure decreases bodyweight and that THS exposure induces persistent changes in the hematopoietic system independent of age at exposure. These results also suggest that THS exposure may have adverse effects on human health.

Significance Electronic cigarettes, also known as e-cigarettes, are devices designed to imitate regular cigarettes and deliver nicotine via inhalation without combusting tobacco. They are purported to deliver nicotine without other toxicants and to be a safer alternative to regular cigarettes. However, little toxicity testing has been performed to evaluate the chemical nature of vapour generated from e–cigarettes. The aim of this study was to screen e-cigarette vapours for content of four groups of potentially toxic and carcinogenic compounds: carbonyls, volatile organic compounds, nitrosamines and heavy metals. Materials and methods Vapours were generated from 12 brands of e-cigarettes and the reference product, the medicinal nicotine inhaler, in controlled conditions using a modified smoking machine. The selected toxic compounds were extracted from vapours into a solid or liquid phase and analysed with chromatographic and spectroscopy methods. Results We found that the e-cigarette vapours contained some toxic substances. The levels of the toxicants were 9–450 times lower than in cigarette smoke and were, in many cases, comparable with trace amounts found in the reference product. Conclusions Our findings are consistent with the idea that substituting tobacco cigarettes with e-cigarettes may substantially reduce exposure to selected tobacco-specific toxicants. E-cigarettes as a harm reduction strategy among smokers unwilling to quit, warrants further study. (To view this abstract in Polish and German, please see the supplementary files online.)

Exposure to thirdhand smoke (THS) is a newly described health risk. Evidence supports its widespread presence in indoor environments. However, its genotoxic potential, a critical aspect in risk assessment, is virtually untested. An important characteristic of THS is its ability to undergo chemical transformations during aging periods, as demonstrated in a recent study showing that sorbed nicotine reacts with the indoor pollutant nitrous acid (HONO) to form tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-4-(3-pyridyl)butanal (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The goal of this study was to assess the genotoxicity of THS in human cell lines using two in vitro assays. THS was generated in laboratory systems that simulated short (acute)- and long (chronic)-term exposures. Analysis by liquid chromatography-tandem mass spectrometry quantified TSNAs and common tobacco alkaloids in extracts of THS that had sorbed onto cellulose substrates. Exposure of human HepG2 cells to either acute or chronic THS for 24h resulted in significant increases in DNA strand breaks in the alkaline Comet assay. Cell cultures exposed to NNA alone showed significantly higher levels of DNA damage in the same assay. NNA is absent in freshly emitted secondhand smoke, but it is the main TSNA formed in THS when nicotine reacts with HONO long after smoking takes place. The long amplicon-quantitative PCR assay quantified significantly higher levels of oxidative DNA damage in hypoxanthine phosphoribosyltransferase 1 (HPRT) and polymerase β (POLB) genes of cultured human cells exposed to chronic THS for 24h compared with untreated cells, suggesting that THS exposure is related to increased oxidative stress and could be an important contributing factor in THS-mediated toxicity. The findings of this study demonstrate for the first time that exposure to THS is genotoxic in human cell lines.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is tobacco specific and has a longer half-life than other tobacco biomarkers studied thus far. An accurate measurement of the NNAL half-life is important for optimal use to assess exposure to tobacco smoke. We determined the half-life of NNAL in urine in eight daily smokers on a clinical research ward and in five occasional smokers in a real-life environment. Total NNAL in urine was monitored for 14 days in daily smokers after stopping smoking and for up to 60 days in occasional smokers. The average half-life for the terminal phase in the daily smoker group using a two-compartmental body model was 10.3 days (beta phase), and using a noncompartmental model, it was 9.1 days. In the occasional group, these values were 17.6 and 16.0 days, respectively. The alpha-phase half-lives were 14.3 and 27.8 hours for the two groups, respectively. The inter-subject coefficient of variation of the NNAL terminal half-life ranged from 14% to 30%, and the intra-subject coefficient of variation ranged from 3% to 18%. There was very good agreement between the plasma and urinary half-lives in two subjects with plasma analyses: 7.4 versus 7.9 days and 9.2 versus 10.7 days. Mean renal clearance of NNAL was 13 ± 2.3 mL/min. The terminal half-life of NNAL of 10 to 18 days indicates that this biomarker can be used to detect tobacco smoke exposure for 6 to 12 weeks after cessation of exposure and requires a similar time to assess the steady levels of NNAL after switching from one tobacco product to another. (Cancer Epidemiol Biomarkers Prev 2009;18(12):3421–5)

© 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. The association between cigarette smoke exposure and the acute respiratory distress syndrome in patients with the most common acute respiratory distress syndrome risk factors of sepsis, pneumonia, and aspiration has not been well studied. The goal of this study was to test the association between biomarker-confirmed cigarette smoking and acute respiratory distress syndrome in a diverse cohort. Design: Prospective cohort. Setting: Tertiary care center. Patients: Four hundred twenty-six critically ill patients with acute respiratory distress syndrome risk factors (excluding trauma and transfusion) Interventions: None. Measurements and Main Results: We obtained smoking histories and measured urine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (a biomarker of cigarette smoke exposure) on urine samples obtained at the time of study enrollment. The association between cigarette smoke exposure and acute respiratory distress syndrome differed based on acute respiratory distress syndrome risk factor (p < 0.02 for interaction). In patients with nonpulmonary sepsis as the primary acute respiratory distress syndrome risk factor (n = 212), 39% of those with acute respiratory distress syndrome were current smokers by history compared with 22% of those without acute respiratory distress syndrome (odds ratio, 2.28; 95% CI, 1.24-4.19; p = 0.008). Likewise, cigarette smoke exposure as measured by urine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol was significantly associated with acute respiratory distress syndrome in this group. The increased risk of acute respiratory distress syndrome in nonpulmonary sepsis was restricted to patients with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol levels consistent with active smoking and was robust to adjustment for other acute respiratory distress syndrome predictors. Cigarette smoke exposure as measured by history or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol was not associated with acute respiratory distress syndrome in patients with other risk factors (e.g., pneumonia and aspiration). Conclusions: Cigarette smoking measured both by history and biomarker is associated with an increased risk of acute respiratory distress syndrome in patients with nonpulmonary sepsis. This finding has important implications for tobacco product regulation and for understanding the pathogenesis of acute respiratory distress syndrome.

To measure the systemic retention of nicotine, propylene glycol (PG) and vegetable glycerin (VG) in electronic cigarette (e-cigarette) users, and assess the abuse liability of e-cigarettes by characterizing nicotine pharmacokinetics.E-cigarette users recruited over the internet participated in a 1-day research ward study. Subjects took 15 puffs from their usual brand of e-cigarette. Exhaled breath was trapped in gas-washing bottles and blood was sampled before and several times after use.San Francisco, California, USA.Thirteen healthy, experienced adult e-cigarette users (six females and seven males).Plasma nicotine was analyzed by gas chromatography-mass spectrometry (GC-MS/MS) and nicotine, VG and PG in e-liquids and gas traps were analyzed by LC-MS/MS. Heart rate changes and subjective effects were assessed.E-cigarettes delivered an average of 1.33 (0.87-1.79) mg [mean and 95% confidence interval (CI)] of nicotine, and 93.8% of the inhaled dose, 1.22 (0.80-1.66) was systemically retained. Average maximum plasma nicotine concentration (Cmax ) was 8.4 (5.4-11.5) ng/ml and time of maximal concentration (Tmax ) was 2-5 minutes. One participant had Tmax of 30 minutes. 84.4% and 91.7% of VG and PG, respectively, was systemically retained. Heart rate increased by an average of 8.0 beats per minute after 5 minutes. Withdrawal and urge to smoke decreased and the e-cigarettes were described as satisfying.E-cigarettes can deliver levels of nicotine that are comparable to or higher than typical tobacco cigarettes, with similar systemic retention. Although the average maximum plasma nicotine concentration in experienced e-cigarette users appears to be generally lower than what has been reported from tobacco cigarette use, the shape of the pharmacokinetic curve is similar, suggesting addictive potential.

Background: Water pipe tobacco smoking is spreading globally and is increasingly becoming popular in the United States, particularly among young people. Although many perceive water pipe smoking to be relatively safe, clinical experimental studies indicate significant exposures to tobacco smoke carcinogens following water pipe use. We investigated biomarkers of nicotine intake and carcinogen exposure from water pipe smoking in the naturalistic setting of hookah bars. Methods: Fifty-five experienced water pipe users were studied before and after smoking water pipe in their customary way in a hookah bar. Urine samples were analyzed for nicotine, cotinine, the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and mercapturic acid metabolites of volatile organic compounds (VOC). Results: We found an average 73-fold increase in nicotine, 4-fold increase in cotinine, 2-fold increase in NNAL, and 14% to 91% increase in VOC mercapturic acid metabolites immediately following water pipe smoking. We saw moderate to high correlations between changes in tobacco-specific biomarkers (nicotine, cotinine, and NNAL) and several mercapturic acid metabolites of VOCs. Conclusion: Water pipe smoking in a hookah bar is associated with significant nicotine intake and carcinogen exposure. Impact: Given the significant intake of nicotine and carcinogens, chronic water pipe use could place users at increased risk of cancer and other chronic diseases. Cancer Epidemiol Biomarkers Prev; 23(6); 1055–66. ©2014 AACR.

Thirdhand smoke (THS) refers to components of secondhand smoke that stick to indoor surfaces and persist in the environment. Little is known about exposure levels and possible remediation measures to reduce potential exposure in contaminated areas. This study deals with the effect of aging on THS components and evaluates possible exposure levels and remediation measures. We investigated the concentration of nicotine, five nicotine related alkaloids, and three tobacco specific nitrosamines (TSNAs) in smoke exposed fabrics. Two different extraction methods were used. Cotton terry cloth and polyester fleece were exposed to smoke in controlled laboratory conditions and aged before extraction. Liquid chromatography-tandem mass spectrometry was used for chemical analysis. Fabrics aged for 19 months after smoke exposure retained significant amounts of THS chemicals. During aqueous extraction, cotton cloth released about 41 times as much nicotine and about 78 times the amount of tobacco specific nitrosamines (TSNAs) as polyester after one hour of aqueous extraction. Concentrations of nicotine and TSNAs in extracts of terry cloth exposed to smoke were used to estimate infant/toddler oral exposure and adult dermal exposure to THS. Nicotine exposure from THS residue can be 6.8 times higher in toddlers and 24 times higher in adults and TSNA exposure can be 16 times higher in toddlers and 56 times higher in adults than what would be inhaled by a passive smoker. In addition to providing exposure estimates, our data could be useful in developing remediation strategies and in framing public health policies for indoor environments with THS.

Secondhand smoke (SHS) is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS), the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS) in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR) assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF) and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer. © 2014 Sarker et al.

Particulate matter (PM) derived from tobacco smoke contains numerous toxic substances. Since the PM and gas phase of tobacco smoke may distribute differently in the environment and substances in them may have different human bioavailability, multiple tracers and biomarkers for tobacco smoke constituents are desirable. Nicotelline is a relatively nonvolatile alkaloid present in tobacco smoke, and therefore, it has the potential to be a suitable tracer and biomarker for tobacco smoke-derived PM. We describe experiments demonstrating that nicotelline is present almost entirely in the PM, in both freshly generated cigarette smoke and aged cigarette smoke. An excellent correlation between the mass of nicotelline and the mass of the PM in aged cigarette smoke was found. We also describe experiments suggesting that the main source of nicotelline in tobacco smoke is dehydrogenation of another little-studied tobacco alkaloid, anatalline, during the burning process. We show that nicotelline metabolites can be measured in the urine of smokers and that nicotelline can be measured in house dust from homes of smokers and nonsmokers. We conclude that nicotelline should be useful as a tracer and biomarker for PM derived from tobacco smoke. © 2013 American Chemical Society.

The precise quantitation of smoking during pregnancy is difficult in retrospective studies. Routinely collected blood specimens from newborns, stored as dried blood spots, may provide a low-cost method to objectively measure maternal smoking close to the time of delivery. This article compares cotinine levels in dried blood spots to those in umbilical cord blood to assess cotinine in dried blood spots as a biomarker of maternal smoking close to the time of delivery. The California Genetic Disease Screening Program provided dried blood spots from 428 newborns delivered in 2001-2003 with known umbilical cord blood cotinine levels. Cotinine in dried blood spots was measured in 6.35-mm punches by using liquid chromatography-tandem mass spectrometry (quantitation limit, 3.1 ng/mL). Repeated measures of cotinine in dried blood spots were highly correlated (R2= 0.99, P < 0.001) among 100 dried blood spots with cotinine quantitated in 2 separate punches. Linear regression revealed that cotinine levels in dried blood spots were slightly lower than those in umbilical cord blood and predicted umbilical cord blood cotinine levels well (β = 0.95, R2= 0.80, and P < 0.001 for both cotinine levels in log10 scale). When defining active smoking as a cotinine level of 10 ng/mL or more and using umbilical cord blood cotinine as the criterion standard, we found that measurements of cotinine in dried blood spots had high sensitivity (92.3%) and specificity (99.7%) in the prediction of maternal active smoking. Cotinine levels in dried blood spots are an accurate biomarker of maternal smoking close to the time of delivery. © 2013 The Author. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved.

Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered – Thirdhand smoke (THS) – the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed. The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS.

Background: Smoking tobacco preparations in a water pipe (hookah) is widespread in many places of the world and is perceived by many as relatively safe. We investigated biomarkers of toxicant exposure with water pipe compared with cigarette smoking. Methods: We conducted a crossover study to assess daily nicotine and carcinogen exposure with water pipe and cigarette smoking in 13 people who were experienced in using both products. Results: When smoking an average of 3 water pipe sessions compared with smoking 11 cigarettes per day (cpd), water pipe use was associated with a significantly lower intake of nicotine, greater exposure to carbon monoxide (CO), and a different pattern of carcinogen exposure compared with cigarette smoking, with greater exposure to benzene, and high molecular weight polycyclic aromatic hydrocarbon (PAH), but less exposure to tobacco-specific nitrosamines, 1,3-butadiene, acrolein, acrylonitrile, propylene oxide, ethylene oxide, and low molecular weight PAHs. Conclusions: A different pattern of carcinogen exposure might result in a different cancer risk profile between cigarette and water pipe smoking. Of particular concern is the risk of leukemia related to high levels of benzene exposure with water pipe use. Impact: Smoking tobacco in water pipes has gained popularity in the United States and around the world. Many believe that water pipe smoking is not addictive and less harmful than cigarette smoking. We provide data on toxicant exposure that will help guide regulation and public education regarding water pipe health risk. Cancer Epidemiol Biomarkers Prev; 22(5); 765–72. ©2013 AACR.

Objectives The objectives of this study were: (1) to characterise the exposure of non-smokers exposed to secondhand smoke (SHS) in a vehicle using biomarkers, (2) to describe the time course of the biomarkers over 24 h, and (3) to examine the relationship between tobacco biomarkers and airborne concentrations of SHS markers. Methods Eight non-smokers were individually exposed to SHS in cars with fully open front windows and closed back windows over an hour from a smoker who smoked three cigarettes at 20 min intervals. The non-smokers sat in the back seat on the passenger side, while the smoker sat in the driver9s seat. Plasma cotinine and urine cotinine, 3-hydroxycotinine (3HC) and 4-(methylnitrosoamino)-(3-pyridyl)-1-butanol (NNAL) were compared in samples taken at baseline (BL) and several time-points after exposure. Nicotine, particulate matter (PM 2.5 ) and carbon monoxide (CO) were measured inside and outside the vehicle and ventilation rates in the cars were measured. Results Average plasma cotinine and the molar sum of urine cotinine and 3HC (COT+3HC) increased four-fold, urine cotinine increased six-fold and urine NNAL increased ∼27 times compared to BL biomarker levels. Plasma cotinine, urine COT+3HC and NNAL peaked at 4–8 h post-exposure while urine cotinine peaked within 4 h. Plasma cotinine was significantly correlated to PM 2.5 (Spearman correlation r s =0.94) and CO (r s =0.76) but not to air nicotine. The correlations between urine biomarkers, cotinine, COT+3HC and NNAL, and air nicotine, PM 2.5 and CO were moderate but non-significant (r s range = 0.31–0.60). Conclusions Brief SHS exposure in cars resulted in substantial increases in levels of tobacco biomarkers in non-smokers. For optimal characterisation of SHS exposure, tobacco biomarkers should be measured within 4–8 h post-exposure. Additional studies are needed to better describe the relationship between tobacco biomarkers and environmental markers of SHS.

Author(s): Havel, Christopher M; Benowitz, Neal L; Jacob, Peyton; St Helen, Gideon | Abstract: IntroductionCharacterization of aerosols generated by electronic cigarettes (e-cigarettes) is one method used to evaluate the safety of e-cigarettes. While some researchers have modified smoking machines for e-cigarette aerosol generation, these machines are either not readily available, not automated for e-cigarette testing or have not been adequately described. The objective of this study was to build an e-cigarette vaping machine that can be used to test, under standard conditions, e-liquid aerosolization and nicotine and toxicant delivery.MethodsThe vaping machine was assembled from commercially available parts, including a puff controller, vacuum pump, power supply, switch to control current flow to the atomizer, three-way value to direct air flow to the atomizer, and three gas dispersion tubes for aerosol trapping. To validate and illustrate its use, the variation in aerosol generation was assessed within and between KangerTech Mini ProTank 3 clearomizers, and the effect of voltage on aerosolization and toxic aldehyde generation were assessed.ResultsWhen using one ProTank 3 clearomizer and different e-liquid flavors, the coefficient of variation (CV) of aerosol generated ranged between 11.5% and 19.3%. The variation in aerosol generated between ProTank 3 clearomizers with different e-liquid flavors and voltage settings ranged between 8.3% and 16.3% CV. Aerosol generation increased linearly at 3-6V across e-liquids and clearomizer brands. Acetaldehyde, acrolein, and formaldehyde generation increased markedly at voltages at or above 5V.ConclusionThe vaping machine that we describe reproducibly aerosolizes e-liquids from e-cigarette atomizers under controlled conditions and is useful for testing of nicotine and toxicant delivery.ImplicationsThis study describes an electronic cigarette vaping machine that was assembled from commercially available parts. The vaping machine can be replicated by researchers and used under standard conditions to generate e-cigarette aerosols and characterize nicotine and toxicant delivery.

The regulation of mevalonic acid synthesis requires both nonsterol isopentenoid and sterol regulatory signal molecules. A primary target of this multivalent control process is the enzyme which catalyzes mevalonate synthesis: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). In this report Staphylococcus aureus alpha-toxin perforated Chinese hamster ovary cells were used to facilitate the identification of isopentenoidogenic reactions and metabolites required for mevalonate-mediated loss of HMG-CoA reductase activity. alpha-Toxin-perforated cells retained the capacity to decrease, upon demand, HMG-CoA reductase activity and protein in response to mevalonate or isopentenoid pyrophosphate esters. Also, it was deduced with highly specific metabolic inhibitors, that conversion of farnesyl 1-diphosphate to squalene was required for mevalonate-mediated suppression of reductase activity. Since squalene (2 microM) did not downregulate reductase activity, pre-squalene pyrophosphate or a derivative, or polyprenyl-1-pyrophosphate-generated inorganic pyrophosphate, or a combination of these metabolites are proposed as candidate regulatory nonsterol isopentenoid signal molecules.

Background: Smoking tobacco preparations in a water pipe (hookah) is widespread in many places of the world, including the United States, where it is especially popular among young people. Many perceive water pipe smoking to be less hazardous than cigarette smoking. We studied systemic absorption of nicotine, carbon monoxide, and carcinogens from one water pipe smoking session. Methods: Sixteen subjects smoked a water pipe on a clinical research ward. Expired carbon monoxide and carboxyhemoglobin were measured, plasma samples were analyzed for nicotine concentrations, and urine samples were analyzed for the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanol (NNAL) and polycyclic aromatic hydrocarbon (PAH) metabolite biomarker concentrations. Results: We found substantial increases in plasma nicotine concentrations, comparable to cigarette smoking, and increases in carbon monoxide levels that are much higher than those typically observed from cigarette smoking, as previously published. Urinary excretion of NNAL and PAH biomarkers increased significantly following water pipe smoking. Conclusions: Absorption of nicotine in amounts comparable to cigarette smoking indicates a potential for addiction, and absorption of significant amounts of carcinogens raise concerns of cancer risk in people who smoke tobacco products in water pipes. Impact: Our data contribute to an understanding of the health impact of water pipe use. Cancer Epidemiol Biomarkers Prev; 20(11); 2345–53. ©2011 AACR.

Cotinine is the most widely used biomarker to distinguish active versus passive smoking. However, there is an overlap in cotinine levels when comparing light or occasional smokers versus heavily exposed passive smokers. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a tobacco-specific nitrosamine measurable in urine with a much longer half-life than cotinine. The aim of the study was to determine optimal cutoff points to discriminate active versus passive smokers and to compare sensitivity and specificity for the use of cotinine, NNAL, and the ratio of the NNAL/cotinine in urine.Cotinine and NNAL were measured in urine of 373 active smokers and 228 passive smokers.Geometric mean cotinine levels were 2.03 ng/ml (interquartile interval: 0.43-8.60) and 1,043 ng/ml (658-2,251) and NNAL levels were 5.80 pg/ml (2.28-15.4) and 165 pg/ml (90.8-360) pg/ml in passive and active smokers, respectively. NNAL/cotinine ratio in urine was significantly higher for passive smokers when compared with active smokers (2.85 vs. 0.16, p.01). The receiver operating characteristics analysis determined optimal cutoff points to discriminate passive versus active smokers: 31.5 ng/ml for cotinine (sensitivity: 97.1% and specificity: 93.9%), 47.3 pg/ml for NNAL (87.4% and 96.5%), and 0.74 x 10⁻³ for NNAL/cotinine ratio (97.3% and 87.3%).Both urine cotinine and NNAL are sensitive and specific biomarkers for discriminating the source of tobacco smoke exposure. Cotinine is the best overall discriminator when biomarkers are measured while a person has ongoing exposure to tobacco smoke. NNAL because of its long half-life would be particularly useful when there is a delay between exposure and biomarker measurement. The NNAL/cotinine ratio provides similar sensitivity but poorer specificity at discriminating passive versus active smokers when compared with NNAL alone.

Using insect (Drosophila) and rodent mammalian cell cultures we demonstrate that acute mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A was initiated in the absence of protein synthesis. In addition, insect and mammalian cells depleted (1 h) of putative regulatory post-isopentenyl- I -pyrophosphate metabolites by incubation with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A synthase (L-659,699) accumulated 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme units. This accumulation resulted primarily from a decrease in the loss of 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme units rather than from an increase in enzyme synthesis. These observations suggest that the initial phase of mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was governed primarily by post-translational processes. An unknown rapidly turning over post-isopentenyl-1-pyrophosphate metabolite(s) is proposed as the agonist for these post-translational processes.

Objectives: Menthol cigarettes are smoked by 27% of U.S. smokers, and there are concerns that menthol might enhance toxicity of cigarette smoking by increasing systemic absorption of smoke toxins. We measured urine menthol concentrations in relation to biomarkers of exposure to nicotine and tobacco carcinogens. Methods: Concentrations of menthol glucuronide (using a novel analytical method), nicotine plus metabolites (nicotine equivalents, NE), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and polycyclic aromatic hydrocarbon (PAH) metabolites were measured in the urine of 60 menthol and 67 regular cigarette smokers. Results: Urine menthol was measurable in 82% of menthol and 54% in regular cigarette smokers. Among menthol smokers, urine menthol was highly correlated with NE, NNAL, and PAHs. In a multiple regression model NE but not menthol was significantly associated with NNAL and PAHs. Conclusions: Urine menthol concentration is a novel biomarker of exposure in menthol cigarette smokers, and is highly correlated with exposure to nicotine and carcinogens. Menthol is not independently associated with carcinogen exposure when nicotine intake is considered. Cancer Epidemiol Biomarkers Prev; 19(12); 3013–9. ©2010 AACR.

Active smoking and secondhand smoke (SHS) exposure can cause or worsen many acute and chronic cardiovascular and pulmonary diseases (1, 2). SHS is a combination of mainstream smoke (exhaled by smokers) and sidestream smoke given off by the burning end of the tobacco product. Even brief exposure to SHS has significant immediate effects on endothelial cell function, inflammation, and lung function (3). Despite public health efforts to discourage smoking, U.S. smoking prevalence has recently plateaued at 20%, and the number of smokers internationally is increasing (4, 5). In addition, even though smoking has been increasingly restricted in public places and workplaces, an estimated 40% of U.S. nonsmokers have biological evidence of significant SHS exposure (6). Thus, cigarette smoke exposure remains an important cause of preventable chronic and acute disease in both smokers and those exposed to SHS. The association between recent tobacco smoke exposure and critical illness has been poorly studied. The lack of accurate methods to quantify exposure to tobacco smoke, and SHS in particular, in the critically ill has been a major hindrance. Historically, studies have commonly used questionnaires to measure exposure to tobacco smoke. This method is particularly difficult to use in the intensive care unit, because most critically ill patients have altered levels of consciousness or are endotracheally intubated or both. When patients are unable to provide a history, surrogate and chart reporting are often substituted; however, these sources can be subject to social desirability bias and are frequently inaccurate, out of date, or missing (7–9). Thus, to study the effects of cigarette smoke exposure on the incidence or outcomes of critical illness, a more quantitative approach to measuring cigarette smoke exposure in critically ill patients is needed. Biomarkers of cigarette smoke exposure quantify the biologically active dose of nicotine and toxins to which patients have been exposed, overcoming the subjectivity and inaccuracy of self-reporting or surrogate reporting. Such markers have been used to distinguish active and passive smokers from nonsmokers in epidemiologic studies and to establish causal relationships between both active smoking and SHS exposure and cardiovascular and lung disease (1, 10, 11). Nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a potent lung carcinogen), and their metabolites are markers specific for tobacco exposure in smokers, smokeless tobacco users, and nonsmokers exposed to SHS. Determining the optimal biomarker or combination of biomarkers for measuring cigarette smoke exposure in critically ill patients will be key for future studies of this population. The primary objective of our study was to compare cigarette smoke exposure, as measured by biomarkers, to exposure by self-report in a cohort of critically ill patients. A secondary objective was to determine how well biomarkers of exposure taken from different biological specimens (serum, urine, hair, and nails) correlate with each other in this population.

Objectives: Cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are widely used biomarkers for tobacco-derived nicotine and the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), respectively. The discrepancy between cotinine levels in relation to disease risk comparing active versus passive smoking suggests a nonlinear tobacco smoke dose-response and/or that cotinine is not providing an accurate measure of exposure to the toxic constituents of secondhand tobacco smoke. Methods: Cotinine and NNAL were measured in the urine of 373 active smokers and 228 passive smokers. Results: Average cotinine levels were 1,155 (interquartile range, 703-2,715) for active smokers and 1.82 (0.45-7.33) ng/mg creatinine for passive smokers. Average NNAL levels were 183 (103-393) and 5.19 (2.04-11.6) pg/mg creatinine, respectively. NNAL/cotinine ratio in urine was significantly higher for passive smokers when compared with active smokers (2.85 × 103 versus 0.16 × 103, P < 0.0001). Conclusions: Passive smoking is associated with a much higher ratio of NNAL/cotinine in the urine compared with active smoking. Impact: Cotinine measurement leads to an underestimation of exposure to the carcinogen NNK from secondhand smoke when compared with active smoking. Cancer Epidemiol Biomarkers Prev; 19(11); 2795–800. ©2010 AACR.

The relationship between absolute isopentenoidogenesis (total and specific) and 3-hydroxy-3-methylglutaryl-coenzyme A suppression in response to increased mevalonate availability is unknown. We determined absolute isopentenoidogenesis values for the nonsterologenic Drosophila Kc cell incubated (2 h) with increasing [3H]mevalonate concentrations. At least 80% of the maximum suppression of 3-hydroxy-3-methyl glutaryl-co-enzyme A activity was achieved when total isopentenoidogenesis was increased only 2-fold. However, a 12-fold increase in total isopentenoidogenesis was achieved at higher exogenous [3H]mevalonate concentrations. Thus, modulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was coupled to physiological changes in mevalonate/nonsterol isopentenoid availability. In contrast, isopentenoid accumulation, oxidation, and secretion were enhanced with pharmacological increases in mevalonate availability. Furthermore, an apparent constancy of total isopentenoidogenesis values plus increased metabolism of exogenous mevalonate and a significant (35-45%) suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, in response to exogenous substrate concentrations (less than 150 microM), supported a partial or complete compensatory dimunition in endogenous substrate synthesis. Since these responses occurred within the 2-h study, earlier time periods must be assessed to (i) define the initial nonsterol-mediated regulatory response and (ii) to trap the nonsterol isopentenoid regulatory signal molecule(s).

It has been established that vertebrates and yeasts modified a unique subset of polypeptides with farnesyl and geranylgeranyl residues. This observation has been extended to Drosophila K c cells. [ 3 H]Mevalonate was incorporated into 54 K c cell peptides (18–92 kDa). As reported for mammalian cells, most of the labeled peptides had molecular weights between 21 and 27 kDa. C 18 radio-HPLC tryptic digest profiles for delipidized, [ 3 H]mevalonate-labeled (a) insect ( Drosophila and Spodoptera frugiperda ) and mammalian (Chinese hamster ovary met 18-2b) cells, (b) K c cell nuclear lamin, and (c) a 23.5-kDa purified K c cell GTP-binding protein were compared and analyzed. [ 35 S]Cysteine-labeled K c cells yielded a tryptic digest radio-HPLC profile which was congruent with that for [ 3 H]mevalonate-labeled cells. A significant fraction (30–33%) of the doubly labeled tryptic peptides were eluted with ⩾93% acetonitrile. K c cell nuclear lamin tryptic digests yielded a single 3 H-labeled product which migrated as S -farnesylcysteine. The K c cell 23.5-kDa GTP-binding protein's 3 H-labeled oligopeptide(s)/amino acid(s) was geranylgeranylated and its tryptic digest profile was representative of prenylated proteins whose oligopeptides eluted with ⩾93% acetonitrile. Moreover, the 3 H-labeled oligopeptide/amino acid profiles plus prenyl group patterns for [ 3 H]mevalonate-labeled K c and mammalian cell total extracts were similar. Collectively, these observations supported a prenylated protein spectrum and prenyl group usage as highly conserved eukaryotic cellular characteristics.

Embryonic Drosophila cells (K c cells) and [5- 3 H] mevalonate (⩽10 μ m ) were used to determine the absolute basal in vivo rate of total mevalonic acid synthesis/ utilization. An absolute in vivo mevalonic acid synthesis rate of 0.69 nmol/h/mg total cell protein was measured. Absolute mevalonate utilization was obtained by correcting for the extent of endogenous dilution of exogenous [ 3 H]mevalonate at isotopic equilibrium. Cellular [ 3 H]farnesol specific radioactivity was used as representative of a rapidly turning over isopentenoid pool. Although our previous K c cell study ( Havel, C. M., Rector, E. R. II, Watson, J. A., 1986, J. Biol. Chem. 261, 10,150–10,156 ) demonstrated that ⩾40% of the metabolized [ 3 H]mevalonate appeared as 3 H-labeled media water, this report established that t,t -3,7,11-[ 3 H]trimethyl-2,6,10-dodecatriene 1,12 dioic acid was also secreted. Media accumulation of the C 15 -α,ω-prenyl dioic acid and 3 H 2 O was related directly to [ 3 H]mevalonic acid availability. This is the first mevalonate carbon balance study reported for a eukaryotic organism. It was concluded that (i) K c cells synthesized more mevalonate than needed for normal growth and essential isopentenoids and (ii) excess mevalonate carbon accumulated intra- and extracellularly as isopentenoid compounds distal to C 5 products. Finally, this study emphasized the need to measure total mevalonate utilization and not mevalonate conversion to a single isopentenoid end product in carbon balance investigations.

Exposure to secondhand tobacco smoke (SHS) has been linked to increased risk for a number of diseases, including lung cancer. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is of particular interest due to its potency and its specificity in producing lung tumors in animals. The NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine is frequently used as a biomarker for exposure. Due to its long half-life (40-45 days), NNAL may provide a long-term, time-averaged measure of exposure. We developed a highly sensitive liquid chromatography – tandem mass spectrometry (LC-MS/MS) method for determination of NNAL in human urine. The method involves liquid-liquid extraction followed by conversion to the hexanoate ester derivative. This derivative facilitates separation from interfering urinary constituents by extraction and chromatography, and enhances detection with electrospray ionization mass spectrometry. The lower limit of quantitation is 0.25 pg/mL for 5 mL urine specimens. Applications to studies of people with a range of different SHS exposure levels is described.

During replication, hepatitis delta virus (HDV) switches from production of small to large delta antigen. Both antigen isoforms have an HDV genome binding domain and are packaged into hepatitis B virus (HBV)-derived envelopes but differ at their carboxy termini. The large antigen was shown to contain a terminal CXXX box and undergo prenylation. The large, but not the small, antigen formed secreted particles when expressed singly with HBV surface antigen. Mutation of Cys211 in the CXXX box of the large antigen abolished both prenylation and particle formation, suggesting that this site is important for virion morphogenesis.

The mad2-1 mutation inactivates the cell-cycle feedback control that prevents budding yeast cells from leaving mitosis until spindle assembly is complete. The gene product of MAD2 shows significant sequence similarity to the alpha-subunit of prenyltransferases. Here we isolate a new temperature-sensitive mad2 mutant, mad2-2ts, and find that Mad2p is required for the membrane association of Ypt1p and Sec4p, two prenylated small GTP-binding proteins involved in protein trafficking. Extracts from mad2-2ts mutant cells fail to geranylgeranylate a number of substrates at the non-permissive temperature. mad2-2ts is synthetically lethal with bet2-1, a mutation in the gene that encodes for the beta-subunit of the Ypt1p and Sec4p geranylgeranyl transferase. Therefore MAD2 and BET2 gene products may physically interact to form a geranylgeranyl transferase complex. In addition, the difference between the phenotypes of mad2-1 and mad2-2ts suggests that MAD2 has distinct roles in protein transport and the mitotic feedback control.