Gregory M. Polzin

Determination of tar, nicotine, and carbon monoxide yields in the mainstream smoke of selected international cigarettes

Objective: Survey of nicotine, tar, and carbon monoxide (CO) smoke deliveries from 77 cigarette brands purchased in 35 countries was conducted using a standardised machine smoking method. The goal of this study was to determine regional variations and differences in the tar, nicotine, and CO smoke yields of a cigarette brand manufactured by a leading transnational corporation and of non-US locally popular cigarette brands. Design: The majority of the cigarettes were purchased in each of the participating countries by delegate members of the World Health Organization and forwarded to the Centers for Disease Control and Prevention for analysis. Smoke deliveries were determined using a standardised smoking machine method and subsequent gravimetric and gas chromatography analysis. Results: The smoke deliveries varied widely. Mainstream smoke deliveries varied from 6.8 to 21.6 mg tar/cigarette, 0.5 to 1.6 mg nicotine/cigarette, and 5.9 to 17.4 mg CO/cigarette. In addition to the smoke deliveries, the cigarettes were examined to determine physical parameters such as filter composition, length, and ventilation levels. Conclusion: Analysis of the smoke deliveries suggested that cigarettes from the Eastern Mediterranean, Southeast Asia, and Western Pacific WHO regions tended to have higher tar, nicotine, and CO smoke deliveries than did brands from the European, American, or African WHO regions surveyed.

Concentrations of nine alkenylbenzenes, coumarin, piperonal and pulegone in Indian bidi cigarette tobacco.

Indian-made bidi cigarettes sold in the United States are available in a variety of exotic (e.g. clove, mango) and candy-like (e.g. chocolate, raspberry) flavors. Because certain tobacco flavorings contain alkenylbenzenes and other toxic or carcinogenic chemicals, we measured the concentration of flavor-related compounds in bidi tobacco using a previously developed method. Twenty-three brands of bidis were sampled using automated headspace solid-phase microextraction and subsequently analyzed for 12 compounds by gas chromatography-mass spectrometry. Two alkenylbenzene compounds, trans-anethole and eugenol, were found in greater than 90% of the brands analyzed. Methyleugenol, pulegone and estragole were each detected in 30% or more of the brands, whereas safrole and elemicin were not detected in any of the brands. The flavor-related compounds with the highest tobacco concentrations were eugenol (12,000 microg/g tobacco) and trans-anethole (2200 microg/g tobacco). The highest eugenol and trans-anethole concentrations found in bidi tobacco were about 70,000 and 7500 times greater, respectively, than the highest levels previously found in US cigarette brands. Measurement of these compounds is crucial to evaluation of potential risks associated with inhaling highly concentrated flavor-related compounds from bidis or other tobacco products.

Effects of 10 Cigarette Smoke Condensates on Primary Human Airway Epithelial Cells by Comparative Gene and Cytokine Expression Studies

Cigarettes vary in tobacco blend, filter ventilation, additives, and other physical and chemical properties, but little is known regarding potential differences in toxicity to a smokers airway epithelia. We compared changes in gene expression and cytokine production in primary normal human bronchial epithelial cells following treatment for 18 h with cigarette smoke condensates (CSCs) prepared from five commercial and four research cigarettes, at doses of approximately 4 microg/ml nicotine. Nine of the CSCs were produced under a standard International Organization for Standardization smoking machine regimen and one was produced by a more intense smoking machine regimen. Isolated messenger RNA (mRNA) was analyzed by microarray hybridization, and media was analyzed for secreted cytokines and chemokines. Twenty-one genes were differentially expressed by at least 9 of the 10 CSCs by more than twofold, including genes encoding detoxifying and antioxidant proteins. Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and NAD(P)H dehydrogenase, quinone 1 (NQO-1) were selected for validation with quantitative real-time PCR (qRT-PCR) and Western blot analyses. NQO-1 expression determined with microarrays, qRT-PCR, and Western blotting differed among the CSC types, with good correlation among the different assays. CYP1A1 mRNA levels varied substantially, but there was little correlation with the protein levels. For each CSC, the three most induced and three most repressed genes were identified. These genes may be useful as markers of exposure to that particular cigarette type. Furthermore, differences in interleukin-8 secretion were observed. These studies lay the foundation for future investigations to analyze differences in the responses of in vivo systems to tobacco products marketed with claims of reduced exposure or reduced harm.

Effect of Differing Levels of Tobacco-Specific Nitrosamines in Cigarette Smoke on the Levels of Biomarkers in Smokers

Background: Smokers are exposed to significant doses of carcinogens, including tobacco-specific nitrosamines (TSNA). Previous studies have shown significant global differences in the levels of TSNAs in cigarette smoke because of the variation in tobacco blending and curing practices around the world. Methods: Mouth-level exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) measured in cigarette butts and urinary concentrations of its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) were examined among 126 daily smokers in four countries over a 24-hour study period. Results: As mouth-level exposure of NNK increased, the urinary NNAL increased even after adjustment for other covariates (β = 0.46, P = 0.004). The relationship between mouth-level exposure to nicotine and its salivary metabolite, cotinine, was not statistically significant (β = 0.29, P = 0.057), likely because of the very limited range of differences in mouth-level nicotine exposure in this population. Conclusions: We have shown a direct association between the 24-hour mouth-level exposure of NNK resulting from cigarette smoking and the concentration of its primary metabolite, NNAL, in the urine of smokers. Internal dose concentrations of urinary NNAL are significantly lower in smokers in countries that have lower TSNA levels in cigarettes such as Canada and Australia in contrast to countries that have high levels of these carcinogens in cigarettes, such as the United States. Impact: Lowering the levels of NNK in the mainstream smoke of cigarettes through the use of specific tobacco types and known curing practices can significantly affect the exposure of smokers to this known carcinogen. Cancer Epidemiol Biomarkers Prev; 19(6); 1389–98. ©2010 AACR.

Mutagenicity of 11 cigarette smoke condensates in two versions of the mouse lymphoma assay

Cigarette smoke condensate (CSC) is genotoxic in nearly all assays in which it has been tested. In this study, we investigated the mutagenicity of 11 CSCs using the microwell and soft-agar versions of the mouse lymphoma assay (MLA). These CSCs were prepared from commercial or experimental cigarettes, 10 of them were produced using International Organisation for Standardisation (ISO) conditions and one CSC was generated using intense Massachusetts Department of Public Health (MDPH) conditions. In the presence of rat liver S9, the L5178Y/Tk(+/-) mouse lymphoma cells were treated with 11 CSCs at different concentrations (25-200 μg/ml) for 4 h. All CSCs resulted in dose-dependent increases of both cytotoxicity and mutagenicity in both versions of the MLA. The mutagenic potencies of the CSCs were calculated as mutant frequency per microgram CSC from the slope of the linear regression of the dose-response curves and showed no correlations with the tar yield of the cigarette or nicotine concentrations of the CSCs. Comparing two CSCs produced from the same commercial cigarettes using two different smoking conditions, the one generated under ISO conditions was more mutagenic than the other generated under intense conditions on a per microgram CSC basis. We also examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for the mutants induced by 11 CSCs. The most common type of mutation observed was LOH with chromosome damage spanning less than ∼34 Mbp. These results indicate that the MLA identifies different genotoxic potencies among a variety of CSCs and that the results from both versions of the assay are comparable.

Cytotoxicity of eight cigarette smoke condensates in three test systems: Comparisons between assays and condensates

Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose-response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells.

Estimating smokers' mouth-level exposure to select mainstream smoke constituents from discarded cigarette filter butts.

INTRODUCTION Standardized machine smoking measurements are poor predictors of exposure. We have refined a method using the solanesol deposited in discarded cigarette butts as a marker for estimating deliveries of mainstream smoke constituents. Developing a fast and accurate method for measuring solanesol in cigarette filters to assess tobacco smoke intake could provide a way to assess how people smoke under natural conditions. We have developed and validated a new, lower-cost, high-throughput method to measure the solanesol content in discarded cigarette filter butts and correlated these measurements with mainstream smoke deliveries of nicotine and tobacco-specific nitrosamines (TSNAs). METHODS Cigarettes were machine smoked under a variety of conditions to cover a wide range of nicotine deliveries and solanesol levels in the spent cigarette filter. Following machine smoking, a 1-cm portion of filter material, measured from the mouth end, was removed from the cigarette butts for analysis. Although an isotopically labeled solanesol analog is currently not commercially available, we achieved excellent quantitative results using a structurally similar compound, geranylgeraniol, as an internal standard (IS). After spiking with IS and solvent extracted, solanesol extracts were then analyzed using liquid chromatography coupled with a single-quadrupole mass analyzer. Analysis was carried out using manual preparation as well as a high-throughput 48-well format using automated liquid handlers. RESULTS Recoveries of solanesol from cigarette butts exceeded 95% with excellent precision and exhibited excellent linearity for both preparation methods. In addition, we show that the mouth-level exposure for both nicotine and TSNAs may be estimated by their relation to the solanesol retained in the cigarette filter. DISCUSSION We believe that this method provides excellent versatility and throughput for the estimation of mouth-level exposure to a wide range of toxins in cigarette smoke under naturalistic conditions. In addition, this method allows a far more accurate measure of exposure both from a single cigarette as well as from daily smoking.

Determination of tar, nicotine, and carbon monoxide yields in the smoke of bidi cigarettes

A survey of the nicotine, tar, and carbon monoxide (CO) levels in mainstream smoke from 21 brands of bidi cigarettes and five brands of traditional cigarettes was conducted using a variation of the Federal Trade Commission (FTC) standardized cigarette smoking machine method. The primary difference between this method and the FTC method was a reduction of the 60-s puff interval to 15 s. The shorter puff interval was required to prevent the bidi cigarettes from self-extinguishing and may represent a closer approximation to human usage. The goal of this study was to evaluate the smoke-delivery potential for tar, nicotine, and CO in mainstream smoke from bidi cigarettes compared with traditional domestic cigarettes smoked under identical conditions. Approximately half of the bidi brands examined were marketed as filtered varieties. Unlike traditional cigarettes, the filtered and unfiltered bidi brands yielded comparable smoke deliveries. Thus, a filtered bidi cigarette brand does not provide any harm-reduction benefit that might result from a reduction in levels of tar, nicotine, and CO compared with an unfiltered variety. Our findings indicate that bidi cigarettes can deliver high levels of tar (77.9+/-9.5 mg/bidi), nicotine (2.7+/-.4 mg/bidi), and CO (39.2+/-5.7 mg/bidi). In comparison, traditional cigarettes smoked using the bidi cigarette protocol have lower tar and CO yields, but have nicotine deliveries comparable with bidi cigarettes.

Effect of charcoal-containing cigarette filters on gas phase volatile organic compounds in mainstream cigarette smoke

Of the chemicals identified to date in mainstream cigarette smoke with known toxicological properties, the volatile organic compounds (VOCs) are considered the most hazardous group owing to their high abundance and toxicity. In this research we evaluate a recently introduced line of cigarettes that contain charcoal in their filters. The amount of charcoal in these filters ranged from 45 mg to 180 mg and were either dispersed among the filter material or contained in a small cavity in the filter segment. Charcoal has long been used for removing VOCs from both water and air. Our findings indicate that these cigarettes reduce machine generated mainstream smoke deliveries of a wide range of VOCs compared to a similar, non-charcoal filtered, cigarette. However, this reduction is dependent not only on the amount of charcoal present but also on the volume of smoke being drawn through the filter. While a brand with 45 mg charcoal reduces VOC delivery under ISO smoking conditions, charcoal saturation and breakthrough occur under more intense smoking conditions. Breakthrough is minimised for brands with the most charcoal. Overall, the brands with the most charcoal are effective at reducing VOC deliveries under even intense smoking conditions.

Automated determination of seven phenolic compounds in mainstream tobacco smoke

Exposure to hydroxyl-substituted arenes, commonly referred to as phenols or phenolic compounds, can have serious health consequences. Select phenols present in tobacco smoke are cardiovascular toxins, act as tumor co-promoters and show genotoxic activity. To examine the mainstream smoke levels of these compounds, we developed and applied a method for quantitative analysis of seven phenols (phenol, o-cresol, m-cresol, p-cresol, catechol, resorcinol, and hydroquinone) in mainstream smoke. Total mainstream smoke particulate matter was collected on a Cambridge filter pad and spiked with an isotopically labeled internal standard solution. This pad underwent an automated phenol derivatization procedure to increase analyte volatility and enhance detection. Following the derivatization step, phenols from the particulate matter were sampled using solid-phase microextraction with subsequent gas chromatography/mass spectrometric detection. Sensitivity, selectivity, accuracy, and reproducibility were more than adequate for routine detection of phenols in mainstream smoke. Detection limits ranged from 0.04-0.57 microg, with a quantification range of 0.1-710 microg. Higher sensitivity and sample throughput were achieved compared with previously described methods. Mainstream smoke from 28 brands of domestic commercial cigarettes was evaluated to assess typical levels, and reference cigarettes containing single tobacco blends were examined to ascertain the phenolic profile from different types of tobaccos. As expected under machine smoking conditions using the Federal Trade Commission parameters, full-flavored cigarettes deliver more phenols than the light varieties, followed by the ultra light varieties. Differences were seen in relative levels of phenolic compounds in the mainstream smoke from unfiltered cigarettes made with a single type of tobacco.