Smoking-Associated Exposure of Human Primary Bronchial Epithelial Cells to Aldehydes: Impact on Molecular Mechanisms Controlling Mitochondrial Content and Function.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease primarily caused by exposure to cigarette smoke (CS). During the pyrolysis and combustion of tobacco, reactive aldehydes such as acetaldehyde, acrolein, and formaldehyde are formed, which are known to be involved in respiratory toxicity. Although CS-induced mitochondrial dysfunction has been implicated in the pathophysiology of COPD, the role of aldehydes therein is incompletely understood. To investigate this, we used a physiologically relevant in vitro exposure model of differentiated human primary bronchial epithelial cells (PBEC) exposed to CS (one cigarette) or a mixture of acetaldehyde, acrolein, and formaldehyde (at relevant concentrations of one cigarette) or air, in a continuous flow system using a puff-like exposure protocol. Exposure of PBEC to CS resulted in elevated IL-8 cytokine and mRNA levels, increased abundance of constituents associated with autophagy, decreased protein levels of molecules associated with the mitophagy machinery, and alterations in the abundance of regulators of mitochondrial biogenesis. Furthermore, decreased transcript levels of basal epithelial cell marker KRT5 were reported after CS exposure. Only parts of these changes were replicated in PBEC upon exposure to a combination of acetaldehyde, acrolein, and formaldehyde. More specifically, aldehydes decreased MAP1LC3A mRNA (autophagy) and BNIP3 protein (mitophagy) and increased ESRRA protein (mitochondrial biogenesis). These data suggest that other compounds in addition to aldehydes in CS contribute to CS-induced dysregulation of constituents controlling mitochondrial content and function in airway epithelial cells.

GC-MS analysis of e-cigarette refill solutions: A comparison of flavoring composition between flavor categories.

OBJECTIVES: Electronic cigarette refill solutions (e-liquids) are available in various flavor descriptions that can be categorized as fruit, tobacco, and more. Flavors increase sensory appeal, thereby stimulating e-cigarette use, and flavoring ingredients can contribute to e-cigarette toxicity. We aim to inform toxicologists, sensory scientists, and regulators by determining flavoring compounds in e-liquids with various flavors, and compare results between flavor categories. METHODS: Gas chromatography - mass spectrometry (GC-MS) was used to identify 79 flavorings in 320 e-liquids, classified in 15 flavor categories. Ten flavorings highly prevalent in e-liquids according to information from manufacturers were quantified. Flavoring prevalence was defined as the number of e-liquids with the flavoring as percentage of the total number of e-liquids. The method was validated in terms of specificity, linearity, repeatability, recovery, and sensitivity. RESULTS: The mean number of flavorings per e-liquid was 6 ± 4. Flavoring prevalence was highest for vanillin (creamy/vanilla flavor), ethyl butyrate (ethereal/fruity), and cis-3-hexenol (fresh/green). Based on similarities in flavoring prevalence, four clusters of categories were distinguished: (1) fruit, candy, alcohol, beverages; (2) dessert, coffee/tea, nuts, sweets; (3) menthol/mint; and (4) spices, tobacco, and unflavored. Categories from cluster 4 generally had less flavorings per e-liquid than fruit, candy, alcohol, beverages (cluster 1) and dessert (cluster 2) (p < 0.05). Flavoring concentrations varied between e-liquids within the categories. CONCLUSIONS: We evaluated flavoring compositions of 320 e-liquids using a simple GC-MS method. Flavoring prevalence was similar within four clusters of typically fresh/sweet, warm/sweet, fresh/cooling, and non-sweet flavor categories. To compare flavoring concentrations between individual flavor categories, additional research is needed.

Aldehyde and Volatile Organic Compound Yields in Commercial Cigarette Mainstream Smoke Are Mutually Related and Depend on the Sugar and Humectant Content in Tobacco.

INTRODUCTION: The World Health Organization (WHO) Framework Convention on Tobacco control recognizes the need for tobacco product regulation. In line with that, the WHO Study Group on Tobacco Product Regulation (TobReg) proposed to regulate nine toxicants in mainstream cigarette smoke, including aldehydes, volatile organic compounds (VOCs), and carbon monoxide (CO). We analyzed their relations in 50 commercially available cigarette brands, using two different smoking regimes, and their dependence on sugar and humectant concentrations in tobacco filler. METHODS: We measured sugar and humectant in tobacco filler and aldehydes, VOCs, and tar, nicotine, and CO (TNCO) in mainstream smoke. The general statistics, correlations between emission yields, and correlations between contents and emissions yields were determined for these data. RESULTS: For aldehydes, several significant correlations were found with precursor ingredients in unburnt tobacco when smoked with the Intense regime, most prominently for formaldehyde with sucrose, glucose, total sugars, and glycerol. For VOCs, 2,5-dimethylfuran significantly correlates with several sugars under both International Standards Organization (ISO) and Intense smoking conditions. A correlation network visualization shows connectivity between a sugar cluster, an ISO cluster, and an Intense cluster, with Intense formaldehyde as a central highest connected hub. CONCLUSIONS: Our multivariate analysis showed several strong connections between the compounds determined. The toxicants proposed by WHO, in particular, formaldehyde, can be used to monitor yields of other toxicants under Intense conditions. Emissions of formaldehyde, acetaldehyde, acrolein, and 2,5-dimethylfuran may decrease when sugar and humectants contents are lowered in tobacco filler. IMPLICATIONS: Our findings suggest that the aldehydes and VOCs proposed by TobReg are a representative selection for smoke component market monitoring purposes. In particular, formaldehyde yields may be useful to monitor emissions of other toxicants under Intense conditions. Since the most and strongest correlations were observed with the Intense regime, policymakers are advised to prescribe this regime for regulatory purposes. Policymakers should also consider sugars and humectants contents as targets for future tobacco product regulations, with the additional advantage that consumer acceptance of cigarette smoke is proportional to their concentrations in the tobacco blend.

Sensory analysis of characterising flavours: evaluating tobacco product odours using an expert panel.

OBJECTIVES: Tobacco flavours are an important regulatory concept in several jurisdictions, for example in the USA, Canada and Europe. The European Tobacco Products Directive 2014/40/EU prohibits cigarettes and roll-your-own tobacco having a characterising flavour. This directive defines characterising flavour as 'a clearly noticeable smell or taste other than one of tobacco […]'. To distinguish between products with and without a characterising flavour, we trained an expert panel to identify characterising flavours by smelling. METHODS: An expert panel (n=18) evaluated the smell of 20 tobacco products using self-defined odour attributes, following Quantitative Descriptive Analysis. The panel was trained during 14 attribute training, consensus training and performance monitoring sessions. Products were assessed during six test sessions. Principal component analysis, hierarchical clustering (four and six clusters) and Hotelling's T-tests (95% and 99% CIs) were used to determine differences and similarities between tobacco products based on odour attributes. RESULTS: The final attribute list contained 13 odour descriptors. Panel performance was sufficient after 14 training sessions. Products marketed as unflavoured that formed a cluster were considered reference products. A four-cluster method distinguished cherry-flavoured, vanilla-flavoured and menthol-flavoured products from reference products. Six clusters subdivided reference products into tobacco leaves, roll-your-own and commercial products. CONCLUSIONS: An expert panel was successfully trained to assess characterising odours in cigarettes and roll-your-own tobacco. This method could be applied to other product types such as e-cigarettes. Regulatory decisions on the choice of reference products and significance level are needed which directly influences the products being assessed as having a characterising odour.

The Sensory Difference Threshold of Menthol Odor in Flavored Tobacco Determined by Combining Sensory and Chemical Analysis.

Cigarettes are an often-used consumer product, and flavor is an important determinant of their product appeal. Cigarettes with strong nontobacco flavors are popular among young people, and may facilitate smoking initiation. Discriminating flavors in tobacco is important for regulation purposes, for instance to set upper limits to the levels of important flavor additives. We provide a simple and fast method to determine the human odor difference threshold for flavor additives in a tobacco matrix, using a combination of chemical and sensory analysis. For an example, the human difference threshold for menthol odor, one of the most frequently used tobacco flavors, was determined. A consumer panel consisting of 20 women compared different concentrations of menthol-flavored tobacco to unflavored cigarette tobacco using the 2-alternative forced choice method. Components contributing to menthol odor were quantified using headspace GC-MS. The sensory difference threshold of menthol odor corresponded to a mixture of 43 (37-50)% menthol-flavored tobacco, containing 1.8 (1.6-2.1) mg menthol, 2.7 (2.3-3.1) µg menthone, and 1.0 (0.9-1.2) µg neomenthyl acetate per gram of tobacco. Such a method is important in the context of the European Tobacco Product Directive, and the US Food and Drug Administration Tobacco Control Act, that both prohibit cigarettes and roll-your-own tobacco with a characterizing flavor other than tobacco. Our method can also be adapted for matrices other than tobacco, such as food.

Long-term (in)stability of folate and vitamin B12 in human serum.

BACKGROUND: In epidemiological research it is very important to test the stability of biomarkers as a function of both storage time and temperature. In this study the stability of both folate and vitamin B12 in human serum samples have been tested after storage at three different temperatures up to 1 year. METHODS: Serum samples of 16 individuals were used in this study. The concentration of folate and vitamin B12 has been determined at T=0 and at several time points up to 1 year after storage at -20°C, -70°C and -196°C. The statistical difference from the initial value at T=0 were determined with a t-test. RESULTS: Folate in serum samples remained stable at -70°C but was not stable during storage at -20°C. A fast decrease was observed after Day 4 which resulted in a stable level of about 60% of the original value measured at T=0 (p<0.001). The rank order of folate concentration in the samples, however, was not affected. The stability of vitamin B12 was good at all temperatures tested. CONCLUSIONS: Measurements of folate concentrations in serum stored at -20°C are not reliable. The rank order, however, was not changed. Vitamin B12 was stable at all temperatures tested. For both folate and vitamin B12 storage at -70°C is sufficient to maintain the original concentration for 1 year. Storage at -196°C in liquid nitrogen is not necessary for these nutrients.