Evaluation of the Tobacco Heating System 2.2. Part 2: Chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol.

The chemical composition, in vitro genotoxicity, and cytotoxicity of the mainstream aerosol from the Tobacco Heating System 2.2 (THS2.2) were compared with those of the mainstream smoke from the 3R4F reference cigarette. In contrast to the 3R4F, the tobacco plug in the THS2.2 is not burnt. The low operating temperature of THS2.2 caused distinct shifts in the aerosol composition compared with 3R4F. This resulted in a reduction of more than 90% for the majority of the analyzed harmful and potentially harmful constituents (HPHCs), while the mass median aerodynamic diameter of the aerosol remained similar. A reduction of about 90% was also observed when comparing the cytotoxicity determined by the neutral red uptake assay and the mutagenic potency in the mouse lymphoma assay. The THS2.2 aerosol was not mutagenic in the Ames assay. The chemical composition of the THS2.2 aerosol was also evaluated under extreme climatic and puffing conditions. When generating the THS2.2 aerosol under "desert" or "tropical" conditions, the generation of HPHCs was not significantly modified. When using puffing regimens that were more intense than the standard Health Canada Intense (HCI) machine-smoking conditions, the HPHC yields remained lower than when smoking the 3R4F reference cigarette with the HCI regimen.

Evaluation of the Tobacco Heating System 2.2. Part 3: Influence of the tobacco blend on the formation of harmful and potentially harmful constituents of the Tobacco Heating System 2.2 aerosol.

The Tobacco Heating System (THS2.2), which uses "heat-not-burn" technology, generates an aerosol from tobacco heated to a lower temperature than occurs when smoking a combustible cigarette. The concentrations of harmful and potentially harmful constituents (HPHCs) are significantly lower in THS2.2 mainstream aerosol than in smoke produced by combustible cigarettes. Different tobacco types and 43 tobacco blends were investigated to determine how the blend impacted the overall reductions of HPHCs in the THS2.2 mainstream aerosol. The blend composition had minimal effects on the yields of most HPHCs in the aerosol. Blends containing high proportions of nitrogen-rich tobacco, e.g., air-cured, and some Oriental tobaccos, produced higher acetamide, acrylamide, ammonia, and nitrogen oxide yields than did other blends. Most HPHCs were found to be released mainly through the distillation of HPHCs present in the tobacco plug or after being produced in simple thermal reactions. HPHC concentrations in the THS2.2 aerosol may therefore be further minimized by limiting the use of flue- and fire-cured tobaccos which may be contaminated by HPHCs during the curing process and carefully selecting nitrogen rich tobaccos with low concentrations of endogenous HPHCs for use in the tobacco plug blend.

Comparison of the impact of the Tobacco Heating System 2.2 and a cigarette on indoor air quality.

The impact of the Tobacco Heating System 2.2 (THS 2.2) on indoor air quality was evaluated in an environmentally controlled room using ventilation conditions recommended for simulating "Office", "Residential" and "Hospitality" environments and was compared with smoking a lit-end cigarette (Marlboro Gold) under identical experimental conditions. The concentrations of eighteen indoor air constituents (respirable suspended particles (RSP) < 2.5 µm in diameter), ultraviolet particulate matter (UVPM), fluorescent particulate matter (FPM), solanesol, 3-ethenylpyridine, nicotine, 1,3-butadiene, acrylonitrile, benzene, isoprene, toluene, acetaldehyde, acrolein, crotonaldehyde, formaldehyde, carbon monoxide, nitrogen oxide, and combined oxides of nitrogen) were measured. In simulations evaluating THS 2.2, the concentrations of most studied analytes did not exceed the background concentrations determined when non-smoking panelists were present in the environmentally controlled room under equivalent conditions. Only acetaldehyde and nicotine concentrations were increased above background concentrations in the "Office" (3.65 and 1.10 µg/m(3)), "Residential" (5.09 and 1.81 µg/m(3)) and "Hospitality" (1.40 and 0.66 µg/m(3)) simulations, respectively. Smoking Marlboro Gold resulted in greater increases in the concentrations of acetaldehyde (58.8, 83.8 and 33.1 µg/m(3)) and nicotine (34.7, 29.1 and 34.6 µg/m(3)) as well as all other measured indoor air constituents in the "Office", "Residential" and "Hospitality" simulations, respectively.

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 6: 6-Day randomized clinical trial of a menthol cigarette in Japan.

A randomized, controlled, open-label, parallel-group, single-center study to determine biomarkers of exposure to 12 selected harmful and potentially harmful constituents (HPHC) in cigarette smoke, excretion of mutagenic material in urine, and serum Clara cell 16-kDa protein (CC16) in 102 male and female Japanese subjects who smoked Marlboro Ultra Lights Menthol cigarettes (M4J(M); 4 mg tar and 0.3mg nicotine) at baseline. Subjects were randomized to continue smoking M4J(M), or switch to smoking either the Electrically Heated Cigarette Smoking System menthol cigarette (EHCSS-K6(M); 5mg tar and 0.3mg nicotine) or the Lark One menthol cigarette (Lark1(M); 1mg tar and 0.1mg nicotine), or to no-smoking. The mean decreases from baseline to Day 5/6 were statistically significant (p ≤ 0.05) for exposure to 10 of 12 cigarette smoke HPHC including the primary endpoint (carbon monoxide) and urinary excretion of mutagenic material in the EHCSS-K6(M) group (-12.3% to -83.4%). Smaller, but statistically significant reductions (p ≤ 0.05) occurred in the Lark1(M) group (-3.3% to -35.2%), with the exception of urinary mutagens. The largest mean reductions (all p ≤ 0.05) in exposure to cigarette smoke HPHC and excretion of mutagenic material occurred in the no-smoking group (-1.4% to -93.6%). Serum CC16, an indicator of lung epithelial injury, was not significantly different between groups.

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 4: Eight-day randomized clinical trial in Korea.

A randomized, controlled, open-label parallel-group, single-center study to determine biomarkers of exposure to 12 selected harmful and potentially harmful constituents (HPHC) in cigarette smoke and urinary excretion of mutagenic material in 72 male and female Korean subjects smoking Lark One cigarettes (1.0mg tar, 0.1mg nicotine, and 1.5mg CO) at baseline. Subjects were randomized to continue smoking Lark One cigarettes, or switch to an Electrically Heated Cigarette Smoking System (EHCSS) and EHCSS-K3 cigarette (3mg tar, 0.2mg nicotine, and 0.6 mg CO), or to no-smoking. The mean decreases from baseline to Day 8 were statistically significant (all p<0.05) for 10 of 12 HPHC in mainstream cigarette smoke including CO (the primary objective) in the EHCSS-K3 group (range: -1.5% to -74.2%). Exposure to the other determined HPHC was not significantly different. In the Lark One group, the mean exposure to 6 of 12 HPHC in cigarette smoke was significantly (all p<0.05) decreased; however, exposure to CO was significantly increased. The largest mean reductions in biomarkers of exposure to HPHC occurred in smokers who switched to no-smoking (-3.4% to -98.9%). The mean excretion of mutagenic material was significantly decreased (p<0.05) in the EHCSS-K3 and no-smoking groups (-31.8% and -45.3%, respectively), and increased in the Lark One group (+31.5%).

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 8: Nicotine bridging--estimating smoke constituent exposure by their relationships to both nicotine levels in mainstream cigarette smoke and in smokers.

A modeling approach termed 'nicotine bridging' is presented to estimate exposure to mainstream smoke constituents. The method is based on: (1) determination of harmful and potentially harmful constituents (HPHC) and in vitro toxicity parameter-to-nicotine regressions obtained using multiple machine-smoking protocols, (2) nicotine uptake distributions determined from 24-h excretion of nicotine metabolites in a clinical study, and (3) modeled HPHC uptake distributions using steps 1 and 2. An example of 'nicotine bridging' is provided, using a subset of the data reported in Part 2 of this supplement (Zenzen et al., 2012) for two conventional lit-end cigarettes (CC) and the Electrically Heated Cigarette Smoking System (EHCSS) series-K6 cigarette. The bridging method provides justified extrapolations of HPHC exposure distributions that cannot be obtained for smoke constituents due to the lack of specific biomarkers of exposure to cigarette smoke constituents in clinical evaluations. Using this modeling approach, exposure reduction is evident when the HPHC exposure distribution curves between the MRTP and the CC users are substantially separated with little or no overlap between the distribution curves.

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 1: Non-clinical and clinical insights.

The following series of papers presents an extensive assessment of the Electrically Heated Cigarette Smoking System EHCSS series-K cigarette vs. conventional lit-end cigarettes (CC) as an example for an extended testing strategy for evaluation of reduced exposure. The EHCSS produces smoke through electrical heating of tobacco. The EHCSS series-K heater was designed for exclusive use with EHCSS cigarettes, and cannot be used to smoke (CC). Compared to the University of Kentucky Reference Research cigarette 2R4F and a series of commercial CC, mainstream cigarette smoke of both the non-menthol and menthol-flavored EHCSS cigarettes showed a reduced delivery of a series of selected harmful and potentially harmful constituents (HPHC), mutagenic activity determined using the Salmonella typhimurium Reverse Mutation (Ames) assay, and cytotoxicity in the Neutral Red Uptake Assay. Clinical evaluations confirmed reduced exposure to HPHC and excretion of mutagenic material under controlled clinical conditions. Reductions in HPHC exposure were confirmed in a real-world ambulatory clinical study. Potential biomarkers of cardiovascular risk were also reduced under real-world ambulatory conditions. A modeling approach, 'nicotine bridging', was developed based on the determination of nicotine exposure in clinical evaluations which indicated that exposure to HPHC for which biomarkers of exposure do not exist would also be reduced.

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 3: Eight-day randomized clinical trial in the UK.

A randomized, controlled, open-label, parallel-group, single-center study to determine biomarkers of exposure to nine selected harmful and potentially harmful constituents (HPHC) in cigarette smoke and urinary excretion of mutagenic material in 160 male and female subjects smoking Marlboro cigarettes (6 mg tar, 0.5mg nicotine, and 7.0mg CO) at baseline. Subjects were randomized to continue smoking Marlboro cigarettes, or switch to using an Electrically Heated Cigarette Smoking System (EHCSS) smoking one of two EHCSS series-K cigarettes, the EHCSS-K6 cigarette (5mg tar, 0.3mg nicotine, and 0.6 mg CO) or the EHCSS-K3 cigarette (3mg tar, 0.2mg nicotine, and 0.6 mg CO), or switch to smoking Philip Morris One cigarettes (1mg tar, 0.1mg nicotine, and 2.0mg CO), or to no-smoking. The mean decreases from baseline to Day 8 were statistically significant (p ≤ 0.05) for all determined HPHC including benzene and CO (the primary objectives), and urinary excretion of mutagenic material in the EHCSS-K6 (range -35.5 ± 29.2% to -79.4 ± 14.6% [mean ± standard deviation]), EHCSS-K3 (range -41.2 ± 26.6% to -83.1 ± 9.2%), and PM1 (range -14.6 ± 24.1% to -39.4 ± 17.5%) groups. The largest reductions in exposure occurred in the no-smoking group (range -55.4 ± 45.0% to -100.0 ± 0.0%).

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 5: 8-Day randomized clinical trial in Japan.

A randomized, controlled, open-label, parallel-group, single-center study to determine biomarkers of exposure to twelve selected harmful and potentially harmful constituents (HPHCs) in cigarette smoke and urinary excretion of mutagenic material in 128 male and female Japanese subjects smoking Marlboro cigarettes (6 mg tar, 0.5mg nicotine, and 7.0mg CO) at baseline. Subjects were randomized to continue smoking Marlboro cigarettes, or switch to the Electrically Heated Cigarette Smoking System (EHCSS) and smoke either the EHCSS-K6 (5mg tar, 0.3mg nicotine, and 0.6 mg CO) or the EHCSS-K3 (3mg tar, 0.2mg nicotine, and 0.6 mg CO) cigarette, or switch to smoking Lark One cigarettes (1mg tar, 0.1mg nicotine, and 2.0mg CO), or to no-smoking. The mean decreases from baseline to Day 8 were statistically significant (p ≤ 0.05) for all cigarette smoke HPHC including CO (the primary objective) and excretion of mutagenic material in the EHCSS-K6 (range: -14.6% to -75.6%) and EHCSS-K3 (range: -9.8% to -73.0%) groups. Statistically significant reductions (all p ≤ 0.05) in exposure to ten cigarette smoke HPHC (range: -5.9% to -34.6%), but not urinary mutagenicity, were observed in the Lark One group. The largest mean reductions in exposure to HPHC (all p ≤ 0.01 level) occurred in the no-smoking group (range: -13.7% to -97.6%).

Comparison of exposure to selected cigarette smoke constituents in adult smokers and nonsmokers in a European, multicenter, observational study.

BACKGROUND: This multicenter, observational study was conducted in three European countries (Germany, Switzerland, and the United Kingdom) to determine the exposure of adult cigarette smokers and nonsmokers to selected cigarette smoke constituents: 1,3-butadiene, 2-naphthylamine, 4-aminobiphenyl, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acrolein, benzene, carbon monoxide, nicotine, pyrene, and o-toluidine. METHODS: Smokers were grouped by tar category (TC) according to the tar yield of their regular cigarette brand: TC1: ≤4 mg tar, TC2: 5-7 mg tar, and TC3: ≥8 mg tar [to the legal tar yield ceiling in the respective countries (10 or 12 mg tar)]. Levels of biomarkers of exposure to the aforementioned cigarette smoke constituents were compared between smokers and nonsmokers, and within smokers across tar categories. RESULTS: The full population consisted of 1,631 subjects (1,223 smokers and 408 nonsmokers). Biomarkers of exposure were analyzed for 1,558 subjects (valid case population) as follows: 1,159 smokers (TC1: n = 402, TC2: n = 379, TC3: n = 378), and 399 nonsmokers. Exposure levels were higher in smokers than nonsmokers and increased with increasing tar yield and cigarette consumption. An association of tar category and exposure level was observed for all smoke constituents, except pyrene, 4-aminobiphenyl, and o-toluidine, whereas only NNK exposure was different in all three tar categories. CONCLUSIONS: Smoking status and, among smokers, daily cigarette consumption and tar yield were observed to affect biomarker of exposure levels. IMPACT: This research provides a comprehensive evaluation of smoke constituent exposure of adult cigarette smokers and nonsmokers in three European countries.

Biotransformation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in lung tissue from mouse, rat, hamster, and man.

Exposure to the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be an important etiological risk factor for lung cancer in tobacco users. The metabolism of NNK via carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), alpha-hydroxylation to form both DNA methylating and pyridyloxobutylating intermediates, and detoxification by pyridyl N-oxidation and glucuronide formation are well-characterized in laboratory animals but less so in man. The in vitro kinetics of 0.03-250 microM [5-(3)H]NNK metabolism were determined under identical experimental conditions using female A/J mouse, male Fischer 344 rat, female Syrian golden hamster, and human lung tissue explants in tissue culture. The concentration-dependent percentage contribution of the three major pathways of NNK metabolism (carbonyl reduction, alpha-hydroxylation, and N-oxidation) showed large interspecies variation. Quantitatively, in mouse, carbonyl reduction to NNAL increased steadily with an increasing substrate concentration (10-74% total NNK metabolism), while concurrent decreases occurred in end products of alpha-hydroxylation (60 to 18%) and N-oxidation (42 to 5%). In rat lung, there were no apparent concentration-dependent trends (NNAL, 42 +/- 4%; alpha-hydroxylation, 35 +/- 2%; and N-oxidation, 24 +/- 3%). In hamster lung, a clear concentration-dependent increase in the contribution of NNAL to total NNK metabolism (from 47 to 87%) was paralleled by a steady decline in end products of alpha-hydroxylation (31 to 11%) and N-oxidation (22 to 2%). Human lung metabolism showed no concentration-dependent tendencies (NNAL, 89 +/- 1%; alpha-hydroxylation, 8.8 +/- 1.1%; and N-oxidation, 2.1 +/- 0.3%). The major alpha-hydroxylation product in human lung was 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), thus supporting the potential pyridyloxobutylation of lung DNA. Metabolism to 4-(3-pyridyl)-4-oxobutanoic acid (keto acid), which could result in lung DNA methylation, was only sporadically seen in human lung but present to a far greater extent in rodent lung. No evidence for glucuronidation was found in any species. Generally, the rate of formation of all NNK metabolites showed two different enzyme kinetics, resulting in large differences between apparent K(m) and V(max) values in the low (up to 2.8 microM) and high substrate concentration ranges. The metabolism of NNK by alpha-hydroxylation is considerably lower in human lung as compared to that observed in rodent species, suggesting that extrapolation of in vitro rodent data to man may result in invalid conclusions about the capacity of the human lung to activate NNK under realistic conditions of NNK exposure expected to occur in man.

Comparison of environmental tobacco smoke (ETS) concentrations generated by an electrically heated cigarette smoking system and a conventional cigarette.

Smoking conventional lit-end cigarettes results in exposure of nonsmokers to potentially harmful cigarette smoke constituents present in environmental tobacco smoke (ETS) generated by sidestream smoke emissions and exhaled mainstream smoke. ETS constituent concentrations generated by a conventional lit-end cigarette and a newly developed electrically heated cigarette smoking system (EHCSS) that produces only mainstream smoke and no sidestream smoke emissions were investigated in simulated "office" and "hospitality" environments with different levels of baseline indoor air quality. Smoking the EHCSS (International Organisation for Standardization yields: 5 mg tar, 0.3 mg nicotine, and 0.6 mg carbon monoxide) in simulated indoor environments resulted in significant reductions in ETS constituent concentrations compared to when smoking a representative lit-end cigarette (Marlboro: 6 mg tar, 0.5 mg nicotine, and 7 mg carbon monoxide). In direct comparisons, 24 of 29 measured smoke constituents (83%) showed mean reductions of greater than 90%, and 5 smoke constituents (17%) showed mean reductions between 80% and 90%. Gas-vapor phase ETS markers (nicotine and 3-ethenylpyridine) were reduced by an average of 97% (range 94-99%). Total respirable suspended particles, determined by online particle measurements and as gravimetric respirable suspended particles, were reduced by 90% (range 82-100%). The mean and standard deviation of the reduction of all constituents was 94 +/- 4%, indicating that smoking the new EHCSS in simulated "office" and "hospitality" indoor environments resulted in substantial reductions of ETS constituents in indoor air.

4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in lung, lower esophagus and cardia of sudden death victims.

4-Hydroxy-l-(3-pyridyl)-l-butanone (HPB)-releasing adducts are formed by metabolic activation of N'-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone and have been proposed as specific biomarkers for exposure to tobacco smoke. However, in several studies hemoglobin adducts releasing HPB were on average less than threefold higher in smokers compared to nonsmokers. Using an improved analytical method we have recently found a sevenfold difference in DNA adduct levels in the lung from smoking and nonsmoking lung cancer patients. In the present study we extended the determination of HPB-releasing DNA adducts by gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) to samples of peripheral lung, lower esophagus and cardia from tumor-free sudden death victims (primarily road traffic accidents, suicide and sudden cardiac arrest). The donors were classified as either current smokers or nonsmokers based on cotinine in either blood or urine (cut-off values for active smoking: >15 ng cotinine/ml blood or >100 ng cotinine/ml urine). Contrary to our expectation, DNA adduct levels (fmol HPB/mg DNA) in lung tissue from tumor-free smokers (N=32, 92+/-148) were not significantly different from values in nonsmokers (N=56, 61+/-66). The values in tumor-free smokers were on average more than fourfold lower compared to smoking lung cancer patients in our previous study. Adduct levels in the mucosa of esophagus (N=82; 133+/-160) and cardia (N=30; 108+/-102) of sudden death victims did not show any difference according to the current smoking status. HPB-releasing DNA adduct levels in cardia and esophagus were significantly correlated (N=29; Spearman r=0.609; p<0.001). In contrast, adduct levels in lung did not correlate with either esophagus (77 cases) or cardia (28 cases). Further studies are necessary to elucidate the discrepancies in lung DNA adduct levels in smokers with or without lung cancer and to identify obvious additional sources other than tobacco for these adducts.

Mass spectrometric analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human lung.

An improved analytical method was developed for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung samples of patients undergoing surgery for lung cancer. HPB-releasing adducts can be formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, and have been reported to play an important role in tobacco carcinogenesis. [2,2,3,3-D(4)]HPB (D(4)-HPB) was used as an internal standard, and HPB released by acid hydrolysis of DNA was determined by gas chromatography/mass spectrometry in the negative ion chemical ionisation mode. The method is sensitive with a limit of detection of 5.9 fmol HPB and a limit of quantification of 15.2 fmol HBP/mg DNA. The recovery of HPB was 82+/-17% and the background response was 10.1+/-1.8 fmol HPB/sample. The concentration of HPB-releasing lung DNA adducts was significantly higher (p<0.0001) in 21 self-reported smokers compared to in 11 self-reported nonsmokers (404+/-258 fmol versus 59+/-56 fmol HPB/mg DNA, respectively). HPB-releasing hemoglobin adduct concentrations were only marginally higher in a subset of 12 smokers compared to in 7 nonsmokers (63+/-53 fmol versus 42+/-34 fmol HPB/g hemoglobin; p=0.36). No correlation was found between HPB-releasing adducts in DNA and hemoglobin (p=0.074).

Determination of three carcinogenic aromatic amines in urine of smokers and nonsmokers.

Aromatic amines (arylamines) such as o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl occur in the environment and are constituents of tobacco smoke. Human exposure to these aromatic amines has long been associated with an elevated risk of bladder cancer. A validated, specific, and sensitive method for measuring o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in cigarette smokers and nonsmokers was developed. The method uses acid hydrolysis of the arylamine conjugates in urine, extraction with n-hexane, derivatization with pentafluoropropionic anhydride, and subsequent analysis with gas chromatography combined with mass spectrometry using negative ion chemical ionization. The limits of detection were 4 ng/L for o-toluidine and 1 ng/L for 2-aminonaphthalene and 4-aminobiphenyl. Smokers (N = 10) excreted significantly higher amounts of o-toluidine (204 versus 104 ng/24 h), 2-aminonaphthalene (20.8 versus 10.7 ng/24 h), and 4-aminobiphenyl (15.3 versus 9.6 ng/24 h) than nonsmokers (N = 10). Urinary arylamine excretion in smokers was associated with the extent of smoking as assessed by daily cigarette consumption, urinary excretion of nicotine equivalents (nicotine plus its five major metabolites), cotinine in saliva, and carbon monoxide in exhaled breath. All nonsmokers investigated had quantifiable amounts of o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in their urine, confirming that other environmental sources of exposure to these compounds also occur. In conclusion, the analytical method is suitable for measuring short-term exposure to arylamines in urine of non-occupationally exposed smokers and nonsmokers.

Urinary mercapturic acids and a hemoglobin adduct for the dosimetry of acrylamide exposure in smokers and nonsmokers.

Acrylamide, used in the manufacture of polyacrylamide and grouting agents, is also present in the diet and tobacco smoke. It is a neurotoxin and a probable human carcinogen. Analytical methods were established to determine the mercapturic acids of acrylamide (N-acetyl-S-(2-carbamoylethyl)-L-cysteine, AAMA) and its metabolite glycidamide (N-(R/S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine, GAMA) by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), as well as the N-terminal valine adduct of acrylamide (N-2-carbamoylethylvaline, AAVal) released by N-alkyl Edman degradation of hemoglobin by gas chromatography-mass spectrometry (GC-MS). Twenty-four-hour urine samples from 60 smokers and 60 nonsmokers were analyzed for AAMA and GAMA, and blood samples were analyzed for AAVal. Smokers excreted 2.5-fold higher amounts of AAMA and 1.7-fold higher amounts of GAMA in their urine and had 3-fold higher levels of AAVal in their blood. All three biomarkers of acrylamide exposure were strongly correlated with the smoking dose as determined by the daily cigarette consumption, nicotine equivalents (the molar sum of nicotine, cotinine, trans-3'-hydroxycotinine, and their respective glucuronides) in urine, salivary cotinine, and carbon monoxide in expired breath. In nonsmokers, a weak but significant correlation between AAMA and the estimated dietary intake of acrylamide was found. It is concluded that all three biomarkers of acrylamide are suitable for the determination of exposure in both smokers and nonsmokers.

Nicotine metabolism, human drug metabolism polymorphisms, and smoking behaviour.

Large interindividual differences occur in human nicotine disposition, and it has been proposed that genetic polymorphisms in nicotine metabolism may be a major determinant of an individual's smoking behaviour. Hepatic cytochrome P4502A6 (CYP2A6) catalyses the major route of nicotine metabolism: C-oxidation to cotinine, followed by hydroxylation to trans-3'-hydroxycotinine. Nicotine and cotinine both undergo N-oxidation and pyridine N-glucuronidation. Nicotine N-1-oxide formation is catalysed by hepatic flavin-containing monooxygenase form 3 (FMO3), but the enzyme(s) required for cotinine N-1'-oxide formation has not been identified. trans-3'-Hydroxycotinine is conjugated by O-glucuronidation. The uridine diphosphate-glucuronosyltransferase (UGT) enzyme(s) required for N- and O-glucuronidation have not been identified. CYP2A6 is highly polymorphic resulting in functional differences in nicotine C-oxidation both in vitro and in vivo; however, population studies fail to consistently and conclusively demonstrate any associations between variant CYP2A6 alleles encoding for either reduced or enhanced enzyme activity with self-reported smoking behaviour. The functional consequences of FMO3 and UGT polymorphisms on nicotine disposition have not been investigated, but are unlikely to significantly affect smoking behaviour. Therefore, current evidence does not support the hypothesis that genetic polymorphisms associated with nicotine metabolism are a major determinant of an individual's smoking behaviour and exposure to tobacco smoke.

Effect of nicotine, cotinine and phenethyl isothiocyanate on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism in the Syrian golden hamster.

The effect of nicotine, cotinine and phenethyl isothiocyanate (PEITC) on metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was studied in the Syrian golden hamster. Urinary metabolite profiles were determined in 24 h urine after a single subcutaneous (s.c.) administration of [5-(3)H]NNK (80 nmol/kg, s.c.). Co-administration of either a 500-fold higher dose of nicotine (40 micromol/kg, s.c.) or a 5000-fold higher dose of cotinine (400 micromol/kg, s.c.) significantly (P<0.001) reduced metabolic activation of NNK by alpha-hydroxylation to 85 and 71% of control, respectively. Co-administration of a 300-fold higher dose of PEITC (1 micromol/g diet) slightly reduced alpha-hydroxylation of NNK (94% of control). Metabolism of NNK by reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was increased by nicotine (155%), and significantly increased by cotinine (670%, P<0.001) and PEITC (219%, P<0.01). Detoxification of NNAL by glucuronidation was also increased by all three test agents. Detoxification of NNK and NNAL by N-oxidation was marginally increased by nicotine, reduced by PEITC, and significantly reduced by cotinine. The urinary metabolite profiles suggest that nicotine, which occurs in concentrations up to 30000-fold higher than NNK in mainstream cigarette smoke, and cotinine, its proximal metabolite, may have a significant protective effect against in vivo metabolic activation of NNK.