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In vitro (geno)toxicity assessment of electronic vapour products (EVPs), relative to conventional cigarette, currently uses assays, including the micronucleus and Ames tests. Whilst informative on induction of a finite endpoint and relative risk posed by test articles, such assays could benefit from mechanistic supplementation. The ToxTracker and Aneugen Clastogen Evaluation analysis can indicate the activation of reporters associated with (geno)toxicity, including DNA damage, oxidative stress, the p53-related stress response and protein damage. Here, we tested for the different effects of a selection of neat e-liquids, EVP aerosols and Kentucky reference 1R6F cigarette smoke samples in the ToxTracker assay. The assay was initially validated to assess whether a mixture of e-liquid base components, propylene glycol (PG) and vegetable glycerine (VG) had interfering effects within the system. This was achieved by spiking three positive controls into the system with neat PG/VG or phosphate-buffered saline bubbled (bPBS) PG/VG aerosol (nicotine and flavour free). PG/VG did not greatly affect responses induced by the compounds. Next, when compared to cigarette smoke samples, neat e-liquids and bPBS aerosols (tobacco flavour; 1.6% freebase nicotine, 1.6% nicotine salt or 0% nicotine) exhibited reduced and less complex responses. Tested up to a 10% concentration, EVP aerosol bPBS did not induce any ToxTracker reporters. Neat e-liquids, tested up to 1%, induced oxidative stress reporters, thought to be due to their effects on osmolarity in vitro. E-liquid nicotine content did not affect responses induced. Additionally, spiking nicotine alone only induced an oxidative stress response at a supraphysiological level. In conclusion, the ToxTracker assay is a quick, informative screen for genotoxic potential and mechanisms of a variety of (compositionally complex) samples, derived from cigarettes and EVPs. This assay has the potential for future application in the assessment battery for next-generation (smoking alternative) products, including EVPs.

Introduction: Electronic cigarette (e-cigarette) aerosol is understood to provide reduced exposure to harmful toxicants compared with tobacco cigarette smoke, as it delivers nicotine and flavors wi...

Graphical abstract, Highlights • Effective in vitro strategies are required to predict early developmental toxicity. • devTOXqP is a metabolomics biomarker assay using iPSCs. • Sample smoke/aerosol captured in bPBS, was tested up to 10% concentration. • Cigarettes & HTP bPBS extracts were predicted as potentially developmentally toxic. • HYB & EVP aerosols were not predicted as having developmentally toxic potential in devTOXqP., devTOX quickPredict (devTOXqP) is a metabolomics biomarker-based assay that utilises human induced pluripotent stem (iPS) cells to screen for potential early stage embryonic developmental toxicity in vitro. Developmental toxicity potential is assessed based on the assay endpoint of the alteration in the ratio of key unrelated biomarkers, ornithine and cystine (o/c). This work aimed to compare the developmental toxicity potential of tobacco-containing and tobacco-free non-combustible nicotine products to cigarette smoke. Smoke and aerosol from test articles were produced using a Vitrocell VC10 smoke/aerosol exposure system and bubbled into phosphate buffered saline (bPBS). iPS cells were exposed to concentrations of up to 10% bPBS. Assay sensitivity was assessed through a spiking study with a known developmental toxicant, all-trans-retinoic acid (ATRA), in combination with cigarette smoke extract. The bPBS extracts of reference cigarettes (1R6F and 3R4F) and a heated tobacco product (HTP) were predicted to have the potential to induce developmental toxicity, in this screening assay. The bPBS concentration at which these extracts exceeded the developmental toxicity threshold was 0.6% (1R6F), 1.3% (3R4F), and 4.3% (HTP) added to the cell media. Effects from cigarette smoke and HTP aerosol were driven largely by cytotoxicity, with the cell viability and o/c ratio dose–response curves crossing the developmental toxicity thresholds at very similar concentrations of added bPBS. The hybrid product and all the electronic cigarette (e-cigarette) aerosols were not predicted to be potential early developmental toxicants, under the conditions of this screening assay.

The use of electronic vapour products (EVPs) continues to increase worldwide and with advances in cell culture systems, molecular biology and the computational sciences there is also accumulating evidence of their potential reduced toxicity and reduced potential harm when compared to cigarette smoke. To further understand the potential risks and health effects associated with exposure to EVP aerosols we have assessed the cellular and transcriptomic response from a commercially available lung tissue culture system (MucilAirTM) following a single sub-cytotoxic exposure to cigarette smoke and the equivalent nicotine delivered dose of EVP aerosol. The transcriptomic, cellular (cilia beat frequency (CBF) and percent active area (%AA), trans epithelial electrical resistance (TEER), histology) and cytokine release were assessed at 4- and 48- hours following recovery from air, EVP aerosol (8.4% V/V: mybluTM blueberry flavour, 2.4% nicotine) and 3R4F smoke (3.5% V/V: exposure). No pathological changes were observed at either recovery time point from any exposure. Air and EVP aerosol exposure had no effect on CBF, %AA nor TEER at 48 hours. Exposure to cigarette smoke resulted in a decrease in TEER, an increase in CBF and the release of proinflammatory cytokines at both recovery time points. Although the number of significantly expressed genes was minimal following exposure to EVP aerosol, exposure to 3R4F smoke resulted in a significant upregulation of several disease relevant pathways. These data provide evidence that following an acute exposure to EVP aerosol there is significantly less damage to lung cells in culture than the equivalent, nicotine based, dose of cigarette smoke.

The use of electronic vaping products (EVPs) continues to increase worldwide among adult smokers in parallel with accumulating information on their potential toxicity and relative safety compared to tobacco smoke. At this time, in vitro assessments of many widely available EVPs are limited. In this study, an in vitro battery of established assays was used to examine the cytotoxic (Neutral red uptake), genotoxic (In vitro micronucleus) and mutagenic (Bacterial reverse mutation) responses of two commercial EVPs (blu GO™ disposable and blu PLUS+™ rechargeable) when compared to smoke from a reference cigarette (3R4F). In total, 12 commercial products were tested as e-liquids and as aerosols. In addition, two experimental base liquids containing 1.2% and 2.4% nicotine were also assessed to determine the effect of flavour and nicotine on all three assays. In the bacterial reverse mutation (Ames) and in vitro micronucleus (IVM) assays, exposures to e-liquids and EVP aerosols, with and without nicotine and in a range of flavourings, showed no mutagenic or genotoxic effects compared to tobacco smoke. The neutral red uptake (NRU) assay showed significantly reduced cytotoxicity (P .05) for whole undiluted EVP aerosols compared to tobacco smoke, which by contrast was markedly cytotoxic even when diluted. The reduced in vitro toxicological responses of the EVPs add to the increasing body of scientific weight-of-evidence supporting the role of high-quality EVPs as a harm reduction tool for adult smokers.

Some mathematical calculations were done that provided information about the structure and biochemistry of polyhydroxyalkanoic acid (PHA) granules and about the amounts of the different constituents that contribute to the PHA granules. The data obtained from these calculations are compared with data from the literature, which show that PHA granules consist not only of the polyester but also of phospholipids and proteins. The latter are referred to as granule-associated proteins, and they are always located at the surface of the PHA granules. A concept is proposed that distinguishes four classes of structurally and functionally different granule-associated proteins: (i) class I comprises the PHA synthases, which catalyze the formation of ester linkages between the constituents; (ii) class II comprises the PHA depolymerases, which are responsible for the intracellular degradation of PHA, (iii) class III comprises a new type of protein, which is referred to as phasins and which has most probably a function analogous to that of oleosins in oilseed plants, and (iv) class IV comprises all other proteins, which have been found to be associated with the granules but do not belong to classes I–III. Particular emphasis is placed on the phasins, which constitute a significant fraction of the total cellular protein. Phasins are assumed to form a close protein layer at the surface of the granules, providing the interface between the hydrophilic cytoplasm and the much more hydrophobic core of the PHA inclusion.Key words: PHA granules, polyhydroxyalkanoic acids, PHA synthase, Alcaligenes eutrophus, Pseudomonas oleovorans.

Polyhydroxyalkanoic acids (PHA) represent a storage compound for carbon and/or energy and occur as insoluble inclusions in the cells of a wide range of different bacteria (Steinbuchel 1991) mostly during cultivation of the cells in the presence of an excess of carbon source and if one other essential nutrient limits growth (Anderson, Dawes 1990). In addition to poly(3-hydroxybutyric acid), poly(3HB), which is the most well-known representative of PHA, 90 different hydroxyalkanoic acids have been detected as constituents of PHA from bacterial origin (Steinbuchel, Valentin 1995). These polyesters have been attracted much attention during the last years since they are biodegradable thermoplastics and/or elastomers which exhibit many other interesting properties and which therefore lend themselves for a wide range of different technical applications (Hocking, Marchessault 1994).