The genotoxicity of mainstream and sidestream marijuana and tobacco smoke condensates.

While the prevalence of tobacco use has decreased in Canada over the past decade, that of marijuana use has increased, particularly among youth. However, the risks of adverse health effects from marijuana smoke exposure, specifically as compared to tobacco, are currently not well understood. The objectives of this study were to evaluate the relative ability of matched marijuana and tobacco condensates to induce (geno)toxic responses in three in vitro test systems. This study provides comparative data for matched sidestream and mainstream condensates, as well as condensates prepared under both a standard and an extreme smoking regime designed to mimic marijuana smoking habits. The results indicate that tobacco and marijuana smoke differ substantially in terms of their cytotoxicity, Salmonella mutagenicity, and ability to induce chromosomal damage (i.e., micronucleus formation). Specifically, the marijuana condensates were all found to be more cytotoxic and more mutagenic in the presence of S9 than the matched tobacco condensates. In contrast, the tobacco condensates appeared to induce cytogenetic damage in a concentration-dependent manner, whereas the matched marijuana condensates did not. In addition, when corrected for total particulate matter yield, little difference was observed in the mutagenic activity of samples smoked under the extreme vs the standard regime for both tobacco and marijuana condensates.

Effect of smoking conditions and methods of collection on the mutagenicity and cytotoxicity of cigarette mainstream smoke.

There is a history for the use of in vitro bioassays to assess the toxicological properties of mainstream cigarette smoke (MSS). The results described in the literature were, for the most part, obtained with MSS collected under Federal Trade Commission (FTC) or International Organization for Standardization (ISO) conditions. However, numerous studies have shown that smokers smoke their cigarettes more intensely (e.g., they take larger puffs and/or more frequent puffs and/or partially occlude filter ventilation) than they are smoked on smoking machines operated under FTC (or ISO) conditions. It has also been reported that MSS composition changes with changes in smoking conditions. Furthermore, some governmental agencies have adopted regulations that specify more intensive protocols (i.e., Health Canada Intensive, HCI) for the collection of MSS for in vitro toxicological assays. Consequently, the performance of the Ames assay (TA98+S9, TA100+S9) and neutral red uptake assay under ISO and HCI protocols was studied with two blended (KR1R4F/KR2R4F, KR1R5F) and one flue-cured (CIM-7) reference cigarettes. The main outcome was when results were reported on a per milligram TPM (that portion of the mainstream smoke which is trapped in the smoke trap, expressed as milligrams per cigarette) basis generated under ISO conditions was more mutagenic and more cytotoxic than was TPM generated under HCI conditions. However, the decrease in biological activity could not be explained only by the increased in the water content of the TPM on going from ISO to HCI smoking conditions, and the results may be influenced by differences in smoke chemistry as a result of differing smoke collection systems.

An improved high performance liquid chromatography-fluorescence detection method for the analysis of major phenolic compounds in cigarette smoke and smokeless tobacco products.

An improved HPLC method has been developed for the determination of major phenolic compounds in cigarette smoke. A novel reversed phase column with a pentafluorophenylpropyl (PFP) ligand in the stationary phase was chosen to separate the positional isomers (p-, m-, and o-cresols). Methanol instead of acetonitrile was used as the organic mobile phase component to improve the separation of the isomers and cope with the crisis of global acetonitrile shortage in 2009. A shorter analytical column with smaller particle size was used to further increase separation efficiency and reduces solvent consumption. These improvements have led to a new HPLC method that is simpler and faster than the GC-MS method and more sensitive, selective and efficient than the widely used traditional HPLC method. The limit of detection (LOD) and limit of quantification (LOQ) of this method are at the ng/mL level for most of the phenols with good linearity (R(2) ≥ 0.999) and precision (RSD<15%). The method has been validated using reference tobacco products. The mainstream and sidestream cigarette smoke yields of phenolic compounds obtained by this method are comparable to those obtained by traditional HPLC method with the advantage that p-, m-, and o-cresols can be determined and reported separately by the new method. The method can also be applied for analysis of phenols in smokeless tobacco product.

An improved headspace solid-phase microextraction method for the analysis of free-base nicotine in particulate phase of mainstream cigarette smoke.

The content of free-base nicotine in cigarette smoke is a controversial subject, partly due to methodological issues. In this investigation, an improved method to measure free-base nicotine in cigarette smoke using headspace solid-phase microextraction (HS-SPME) combined with GC/MS analysis, was developed and validated for this purpose. Cigarette smoke particulate phase (PP) was collected onto a 44mm glass fiber filter pad. The pad was cut in halves with one half used to determine the concentrations of total nicotine and water. The remaining half was analyzed by HS-SPME for free-base nicotine. The following factors were found to have a significant impact on the responses of free-base nicotine: SPME fiber type, pre-equilibrium time before HS-SPME, extraction time and temperature, PP water content, and the solvent used for the preparation of standards. It was also found that the impact of PP water content on the determination of free-base nicotine from smoke sample could be corrected by a water correction factor calculated based on an experimentally determined reciprocal model. The precision of the method was evaluated with smoke samples of reference cigarettes: Canadian flue-cured monitor and Kentucky reference 2R4F. The RSD values obtained were in the 12.8-16.8% range.

Quantitative method for the analysis of tobacco-specific nitrosamines in cigarette tobacco and mainstream cigarette smoke by use of isotope dilution liquid chromatography tandem mass spectrometry.

An improved liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of tobacco specific nitrosamines (TSNA). It utilizes four stable isotope-labeled internal standards instead of two as reported by others. A separate internal standard for each analyte is required to minimize sample matrix effects on each analyte, which can lead to poor analyte recoveries and decreases in method accuracy and precision if only one or two of the internal standards are used, especially for complex sample matrixes and when no sample cleanup steps are performed as in this study. In addition, two ion-transition pairs (instead of one) are used for each analyte for the confirmation and quantification, further enhancing the method's accuracy and robustness. These improvements have led to a new LC-MS/MS method that is faster, more sensitive, and selective than the traditional methods and more accurate and robust than the published LC-MS/MS methods. The linear range of the method was from 0.2 to 250 ng/mL with a limit of detection of each TSNA varied from 0.027 to 0.049 ng/mL. Good correlations between the results obtained by the new method and the traditional method were observed for the samples studied.

A comparison of mainstream and sidestream marijuana and tobacco cigarette smoke produced under two machine smoking conditions.

The chemical composition of tobacco smoke has been extensively examined, and the presence of known and suspected carcinogens in such smoke has contributed to the link between tobacco smoking and adverse health effects. The consumption of marijuana through smoking remains a reality and, among youth, seems to be increasing. There have been only limited examinations of marijuana smoke, including for cannabinoid content and for tar generation. There have not been extensive studies of the chemistry of marijuana smoke, especially in direct comparison to tobacco smoke. In this study, a systematic comparison of the smoke composition of both mainstream and sidestream smoke from marijuana and tobacco cigarettes prepared in the same way and consumed under two sets of smoking conditions, was undertaken. This study examined the suite of chemicals routinely analyzed in tobacco smoke. As expected, the results showed qualitative similarities with some quantitative differences. In this study, ammonia was found in mainstream marijuana smoke at levels up to 20-fold greater than that found in tobacco. Hydrogen cyanide, NO, NO x , and some aromatic amines were found in marijuana smoke at concentrations 3-5 times those found in tobacco smoke. Mainstream marijuana smoke contained selected polycyclic aromatic hydrocarbons (PAHs) at concentrations lower than those found in mainstream tobacco smoke, while the reverse was the case for sidestream smoke, with PAHs present at higher concentrations in marijuana smoke. The confirmation of the presence, in both mainstream and sidestream smoke of marijuana cigarettes, of known carcinogens and other chemicals implicated in respiratory diseases is important information for public health and communication of the risk related to exposure to such materials.