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4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), two tobacco specific nitrosamine carcinogens, can form adducts with DNA and proteins via pyridyloxobutylation upon phase I enzyme-mediated bioactivation. Such DNA modifications have been proposed as the root cause to initiate carcinogenesis. Upon hydrolysis, both DNA and protein modifications would release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). The released HPB, being tobacco carcinogen specific, has the potential to serve as a surrogate biomarker for both tobacco exposure and carcinogen bioactivation. Because of its easy access, blood is a great source of such investigations with the potential in epidemiological application. HPB quantification from haemoglobin (Hb), however, has been demonstrated with limited success. To further explore this potentially paradigm-shift opportunity, we reported, for the first time, the detection and quantification of HPB from albumin (Alb) adducts formed by the tobacco-specific nitrosamines in mice and in human smokers. The time-course quantitative analysis of HPB from mouse Alb upon NNK exposure suggests that such an Alb adduct is stable. The amounts of HPB from Alb adducts in smoker plasma averaged 1.82 ± 0.19 pg/mg Alb (0.42 to 3.11 pg/mg Alb), which was 36 times the value in nonsmokers (0.05 ± 0.01 pg/mg Alb). Importantly, HPB level from Alb correlated positively with the level of human tobacco exposure estimated by urinary total nicotine equivalent (TNE) (R2 = 0.6170). For comparison, HPB level from Alb was 16.5 times that of Hb (0.12 ± 0.02 pg/mg Hb) in the plasma and red blood cell (RBC) samples of the same smokers. In addition, there was no significant correlation between HPB levels from Hb and TNE (R2 = 0.0719). These data overall suggest that HPB from Alb adducts can serve as a surrogate biomarker to monitor the level of tobacco exposure and carcinogenic nitrosamine bioactivation.

The Aboriginal population of Central Australia use endemic Nicotiana species to make a smokeless tobacco product known usually as pituri or mingkulpa. Nicotiana leaves are masticated with wood ash into a ‘quid’ that is chewed/sucked for absorption of nicotine. In addition to nicotine, smokeless tobacco products contain a spectrum of biologically active compounds that may contribute to effects on health. The objective of this study was to quantify nicotine, and related alkaloids and tobacco specific nitrosamines (TSNAs), in Nicotiana leaves used in pituri, and compare in vitro toxicity of pure nicotine with Nicotiana leaf extract at the same concentration of nicotine. An aqueous extract of dry leaves of Nicotiana gossei and a reference smokeless tobacco (CORESTA CRP2) were quantified for major pyridine alkaloids and TSNAs using HPLC-UV and LC–MS/MS. A range of extract concentrations and corresponding concentrations of nicotine standard were tested using an MTS assay to measure human lung epithelium cell (A549) survival. Cells treated for 24 h with the maximum concentration of 1.5 mg/ml of nicotine resulted in 77% viability. In contrast, extracts from N. gossei leaves and CRP2 containing a similar concentration of nicotine (1.3 mg/ml) resulted in remarkably lower viability of 1.5 and 6%, respectively. Comparison of cytotoxicity of pure nicotine with that of the extracts revealed that nicotine was not the source of their cytotoxicity. Other biologically active compounds such as the known carcinogens NNK and NNN, derived from nicotine and nornicotine and found to be present in the smokeless tobacco extracts, may be responsible.

In recent years the number of waterpipe smokers has increased substantially worldwide. Here we report on the concentrations of tobacco-specific nitrosamines (TSNAs) and polycyclic aromatic hydrocarbons (PAHs) in waterpipe smoke and the analysis of selected biomarkers indicative for the body burden in waterpipe users. We further identify high amounts of unburned humectants (glycerol and propylene glycol) in the waterpipe smoke as main part of the so-called "tar" fraction. These results give cause for serious concern. For standardization we applied a machine smoking protocol. Smoke was collected on glass fiber filters and analyzed for nicotine, water, humectants, TSNAs, and PAHs. In addition, we determined carbon monoxide and found high amounts in the smoke being causative for high levels of carboxyhemoglobin (COHb) in the blood of smokers. In comparison to the reference cigarette 3R4F, the nicotine contents were 10-times higher, but TSNA levels were found lower in waterpipe smoke. This finding explained the low levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol detected in the urine of waterpipe smokers. Finally, the levels of benzo[a]pyrene were three times higher in waterpipe smoke compared to the reference cigarette. Altogether, the data presented in this study point to the health hazards associated with the consumption of waterpipes.