Tobacco Nitrate and Free Radical Levels in the Mainstream Smoke of US Cigarette Brands.

Tobacco nitrate levels have been known to impact the levels of toxicants such as polyaromatic hydrocarbons and tobacco-specific nitrosamines (TSNAs) produced during smoking. Recent work in our group showed that the intrinsic nitrate levels in individual tobacco varieties also have a large influence on the formation of gas-phase (GP) free radicals in the mainstream smoke of cigarettes produced with a single tobacco variety. As tobacco nitrate content is a potential target for future regulatory policies, we investigated whether the levels of GP free radicals in the smoke from commercially available cigarettes is also dependent on the nitrate content in the corresponding tobacco blends. GP and particulate-phase (PP) free radical yields in mainstream smoke produced from 25 popular US cigarette brands were measured by electron paramagnetic resonance (EPR) spectroscopy. For each brand, we also measured levels of the TSNAs NNN (N'-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) via HPLC-MS and the nicotine content via GC-FID. Our results show that the intrinsic nitrate levels varied >15-fold and GP radicals varied 4-fold among the 25 brands tested. The GP radicals were correlated with intrinsic nitrate levels (r = 0.87, p < 0.001). NNK and NNN levels varied >8-fold and 12-fold, respectively. We found that NNK was moderately correlated to nitrate content (r = 0.42, p = 0.03) while the NNN was strongly correlated to the nitrate content (r = 0.65, p < 0.001). Nicotine levels varied the least (<3-fold) but showed a moderate negative correlation to nitrate content (r = -0.47, p = 0.02). No statistically significant correlation was observed between nicotine and TSNA levels in mainstream smoke. Overall, this demonstrates that the nitrate content of tobacco blends used in US cigarette brands impacts toxicant output in the mainstream smoke, although other proprietary variables (total ventilation, additives, filter type, etc.) may also modulate these results.

Influence of Tobacco Variety and Curing on Free Radical Production in Cigarette Smoke.

INTRODUCTION: Cigarette smoke contains highly reactive free radicals thought to play an important role in tobacco smoke-induced harm. Previously, large variations in free radical and toxicant output have been observed in commercial cigarettes. These variations are likely because of cigarette design features (paper, filter, and additives), tobacco variety (burley, bright, oriental, etc.), and tobacco curing methods (air, sun, flue, and fire). Previous reports show that tobacco varieties and curing methods influence the production of tobacco smoke constituents like the tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK). AIMS AND METHODS: We evaluated free radical, nicotine, and NNK production in cigarette smoke from cigarettes produced with 15 different types of tobacco. Gas-phase free radicals were captured by spin trapping with N-tert-butyl-α-phenylnitrone and particulate-phase radicals were captured on a Cambridge Filter pad (CFP). Both types of radicals were analyzed using electron paramagnetic resonance spectroscopy. Nicotine and NNK were extracted from the CFP and analyzed by gas chromatography flame ionization detection and liquid chromatography-mass spectrometry, respectively. RESULTS: Gas-phase radicals varied nearly 8-fold among tobacco types with Saint James Perique tobacco producing the highest levels (42 ±â€…7 nmol/g) and Canadian Virginia tobacco-producing the lowest levels (5 ±â€…2 nmol/g). Nicotine and NNK levels in smoke varied 14-fold and 192-fold, respectively, by type. Gas-phase free radicals were highly correlated with NNK levels (r = 0.92, p < .0001) and appeared to be most impacted by tobacco curing method. CONCLUSIONS: Altogether, these data suggest that tobacco types used in cigarette production may serve as a target for regulation to reduce harm from cigarette smoking. IMPLICATIONS: Variations in cigarette free radical and NNK levels vary based on the tobacco variety and curing method. Reducing the ratio of high-producing free radical and NNK tobacco types offer a potential tool for regulators and producers looking to reduce toxicant output from cigarettes.

Building comprehensive glucosinolate profiles for brassica varieties.

Brassica plants play an important role in common agricultural practices, such as livestock feed or biofumigation, due to the bioactivity of the natural degradation products of glucosinolate metabolites. Therefore, the ability to survey comprehensive glucosinolate profiles for individual brassicas is essential for informing proper species selection for the intended application. Current methods for glucosinolate identification and quantification involve complex or unconventional procedures, and proper reference materials are not readily available. Therefore, researchers with limited resources that require glucosinolate profiles are at an extreme disadvantage. In this work, a simple and accurate HPLC-MS method was developed and validated to build preliminary glucosinolate profiles for three agriculturally relevant forage brassica varieties [turnip (B. rapa L.), canola (B. napus L.), and rapeseed (B. napus L.)]. The average glucosinolate content across three herbage collection dates for canola, rapeseed and turnip were 2.9 ± 0.9 mg g-1, 6.4 ± 1.3 mg g-1, and 14 ± 3.4 mg g-1, respectively. GLS concentrations are reported in milligrams of glucosinolate, calculated as sinigrin equivalents, per gram of dry plant material. This semi-quantitative approach for reporting total GLS content in brassicas is accurate within 15%. Several minor individual glucosinolates were identified that have not been previously reported in canola, rapeseed and turnip species, including glucotropaeolin and 4-hydroxyglucobrassicin (canola), glucoraphanin and glucoberteroin (rapeseed), and glucosinalbin and glucobarbarin (turnip). This non-targeted screen of several forage brassica varieties demonstrates the inherent variation in both the individual glucosinolate content and the total glucosinolate profile among brassicas, and highlights the importance of such glucosinolate characterization in agricultural practices. Additionally, the method developed in this study can be used as a tool for researchers with limited resources to build accurate glucosinolate profiles of brassica plants.

Effects of tobacco nitrate content on free radical levels in mainstream smoke.

Tobacco smoke free radicals play an important role in the development of smoking related adverse health effects. We previously reported that gas phase (GP) radicals vary greatly by cigarette brand and tobacco variety and are highly correlated with levels of NNK in smoke. Since NNK production in tobacco is dependent on nitrate, we proposed that GP radical production may also be associated with tobacco nitrate content. To test this, we examined the relationship between intrinsic nitrate levels in 15 individual tobacco types and the levels of free radicals delivered in mainstream smoke from cigarettes produced from these tobaccos. Intrinsic nitrate levels varied >250-fold among the tobacco types, ranging from <0.1 mg/g tobacco in the Bright Leaf types to 24.1 ±â€¯0.4 mg/g in Light Fire Cured Virginia tobacco. Among the tobacco types tested, GP radicals were highly correlated with nitrate levels (r = 0.96, p < 0.0001). To investigate nitrate-specific changes to free radical production during smoking, different concentrations of exogenous sodium nitrate were added to unsmoked shredded leaves of 4 different tobacco types (Bright Leaf Sweet Virginia, American Virginia, Semi-Oriental 456, and reconstituted). Nitrate addition resulted in dose-dependent increases in GP radicals in the corresponding smoke, supporting our hypothesis that intrinsic nitrate levels are responsible for GP radical production in cigarette smoke. We also observed increases in NNK levels as a function of added nitrate that varied significantly among the 4 tobacco types tested, implying that other tobacco-type related factors may be impacting nicotine nitrosation during pyrolysis. Altogether, these findings have identified tobacco nitrate as a key factor in the production of GP radicals, but to a lesser extent with PP radicals, as well as NNK during combustion and highlight its potential implication as a target for regulation.

Lifetimes of the Aglycone Substrates of Specifier Proteins, the Autonomous Iron Enzymes That Dictate the Products of the Glucosinolate-Myrosinase Defense System in Brassica Plants.

Specifier proteins (SPs) are components of the glucosinolate-myrosinase defense system found in plants of the order Brassicales (brassicas). Glucosinolates (GLSs) comprise at least 150 known S-(ß-d-glucopyranosyl)thiohydroximate-O-sulfonate compounds, each with a distinguishing side chain linked to the central carbon. Following tissue injury, the enzyme myrosinase (MYR) promiscuously hydrolyzes the common thioglycosidic linkage of GLSs to produce unstable aglycone intermediates, which can readily undergo a Lossen-like rearrangement to the corresponding organoisothiocyanates. The known SPs share a common protein architecture but redirect the breakdown of aglycones to different stable products: epithionitrile (ESP), nitrile (NSP), or thiocyanate (TFP). The different effects of these products on brassica consumers motivate efforts to understand the defense response in chemical detail. Experimental analysis of SP mechanisms is challenged by the instability of the aglycones and would be facilitated by knowledge of their lifetimes. We developed a spectrophotometric method that we used to monitor the rearrangement reactions of the MYR-generated aglycones from nine GLSs, discovering that their half-lives (t1/2) vary by a factor of more than 50, from <3 to 150 s (22 °C). The t1/2 of the sinigrin-derived allyl aglycone (34 s), which can form the epithionitrile product (1-cyano-2,3-epithiopropane) in the presence of ESP, proved to be sufficient to enable spatial and temporal separation of the MYR and ESP reactions. The results confirm recent proposals that ESP is an autonomous iron-dependent enzyme that intercepts the unstable aglycone rather than a direct effector of MYR. Knowledge of aglycone lifetimes will enable elucidation of how the various SPs reroute aglycones to different products.