Characterisation of vaping liquids used in vaping devices across four countries: results from an analysis of selected vaping liquids reported by users in the 2016 ITC Four Country Smoking and Vaping Survey

ObjectivesThis study presents an analysis of vaping products (VPs) purchased in the USA, Canada, England and Australia and assesses whether differences in regulations were associated with differences in the chemical composition of the VPs.MethodsApril–September 2017, a total of 234 VP refill liquids and prefilled cartridges were purchased in convenience samples of retail locations in each country. Products were chosen from brands and styles most commonly reported by current VP users in the 2016 ITC Four Country Smoking and Vaping Survey. All products were tested for nicotine, tobacco-specific nitrosamines (TSNAs), minor tobacco alkaloids, organic acids and flavouring chemicals.ResultsConsistent with the laws in Canada and Australia at the time of product purchase, nicotine was not detected in any of the VPs (n=10 in Canada; n=15 in Australia). US liquids (n=54) had a mean nicotine concentration of 16.2 mg/mL, (range=0.0–58.6) and English liquids (n=166) had a mean concentration of 11.9 mg/mL ((range=0.0–31.2) F(3244)=12.32, p<0.001). About 5% of English samples exceeded the UK’s 20 mg/mL nicotine limit. Substantial country differences were observed in levels ofTSNAs, with the USA being higher than elsewhere. Of all products tested, 18.8% contained at least one organic acid. Liquids purchased in England contained far more identifiable flavouring chemicals than those in the other countries.ConclusionsVP composition, particularly with respect to nicotine and flavouring, varies by country, likely reflecting both marketplace preferences and country-specific regulations. There are differences between nicotine levels claimed on the package and actual levels, particularly in England.

Development of an Advanced Multicellular Intestinal Model for Assessing Immunomodulatory Properties of Anti-Inflammatory Compounds

Intestinal inflammation is the collective term for immune system-mediated diseases of unknown, multifactorial etiology, with often complex interactions between genetic and environmental factors. To mechanistically investigate the effect of treatment with compounds possessing immunomodulating properties in the context of intestinal inflammation, we developed an immunocompetent in vitro triculture intestinal model consisting of a differentiated intestinal epithelial layer (Caco-2/HT29-MTX) and immunocompetent cells (differentiated THP-1). The triculture mimicked a healthy intestine with stable barrier integrity. Lipopolysaccharide treatment triggered a controlled and reversible inflammatory state, resulting in significant impairment of barrier integrity and release of pro-inflammatory cytokines and chemokines, which are known hallmarks of intestinal inflammation. Treatment with known anti-inflammatory reference compounds (TPCA-1 and budenoside) prevented the induction of an inflammatory state; the decreasing triculture responses to this treatment measured by cytokine release, transepithelial electric resistance (TEER), and epithelial layer permeability proved the suitability of the intestinal model for anti-inflammatory drug screening. Finally, selected tobacco alkaloids (nicotine and anatabine (R/S and S forms)) were tested in the in vitro triculture for their potential anti-inflammatory properties. Indeed, naturally occurring alkaloids, such as tobacco-derived alkaloids, have shown substantial anti-inflammatory effects in several in vitro and in vivo models of inflammation, gaining increasing interest. Similar to the anti-inflammatory reference compounds, one of the tobacco alkaloids under investigation partially prevented the decrease in the TEER and increase in permeability and reduced the release of pro-inflammatory cytokines and chemokines. Taken together, these data confirm that our in vitro model is suitable for screening potential anti-inflammatory compounds in the context of intestinal inflammation.

A distant pathway-cross regulation designed with transcription factors and JAZ repressors downregulates the biosynthesis of tobacco alkaloids and specific nitrosamines

Decrease of nicotine, other tobacco alkaloids (OTAs), and carcinogenic tobacco specific nitrosamines (TSNAs) is important for health. To reduce these compounds, we selected MRE and G-box elements of promoters of four Nicotiana tabacum Jasmonate ZIM-domain (NtJAZ) repressor genes as targets to screen transcription factors (TFs) and then identified Arabidopsis Production of Anthocyanin Pigment 1 (PAP1) and Transparent Testa 8 (TT8) for a novel regulation design. Electrophoretic mobility shift, cross-linked chromatin immunoprecipitation, and dual luciferase analyses demonstrated that PAP1, TT8, PAP1/TT8, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex bound to and activated NtJAZ promoters. Stacked PAP1 (P) and TT8 (T), which were introduced to commercial tobacco cultivars, Narrow Leaf Madole (NL) and KY171 (KY), upregulated four NtJAZs, downregulated seven/eight nicotine biosynthetic genes, and reduced nicotine and nornicotine in transgenic red P+T-NL and P+L-KY plants. Field trials of red P+T-NL, P+L-KY, and PAP1 tobacco genotypes and metabolic quantification showed significant reduction of nicotine and four OTAs in most or all cured leaves. Three TSNAs and the fourth one were diminished by 63-92% and 30-74% in contents in all leaves of P+T-NL and P+L-KY genotypes, respectively. The total content of each TSNA was significantly reduced by 25-60% in leaves of PAP1 plants. Taken together, this regulation designed crossing from Arabidopsis anthocyanin to tobacco alkaloid pathway is defined as a distant pathway-cross regulation (DPCR). The proof of concept of DPCR unearths new functions of two known TFs, new activators of NtJAZs, and a negative regulation pathway of nicotine biosynthesis in red tobacco.