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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.

This study aimed to compare the aerosol chemistry and in vitro toxicological profiles of two prototype Heated Tobacco Product (p-HTP) variants to the 1R6F Reference Cigarette. In the neutral red uptake screen the p-HTPs were 37-39-fold less potent than 1R6F, in the micronucleus assay, responses to the p-HTPs were 8-22-fold less, and in the Ames test mutagenicity was weak or removed compared to 1R6F. The cardiovascular scratch wound assay revealed 58-fold greater wound healing impairment following exposure to 1R6F smoke extracts than the p-HTPs. Furthermore, in seven cell stress-related high content screening endpoints (cell count, cytochrome c release, mitochondrial membrane potential, GSH depletion, NFkB translocation, phosphorylation of c-jun and phosphorylation of H2AX), at 4 and 24 h, responses were substantially greater to 1R6F smoke extracts at comparable nicotine levels. The reduced in vitro effects of the p-HTPs were attributed to substantial reductions (90-97%) in selected HPHCs measured compared to in 1R6F smoke. The multiple endpoint in vitro assessment approach provides greater mechanistic insight and the first reported toxicological characterisation of these p-HTPs in the literature. Overall, the findings contribute to the growing weight of evidence that HTPs may offer a reduced harm mode of nicotine delivery to adult smokers.

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...

There is scientific agreement that the detrimental effects of cigarettes are produced by the formation of Harmful and Potentially Harmful Constituents from tobacco combustion and not by nicotine. For this reason numerous public health bodies and governments worldwide have indicated that e-cigarettes have a central role to play in tobacco harm reduction. In this study, high content screening (HCS) was used to compare the effects of neat e-liquids and 3R4F reference cigarette smoke condensate (CSC), which served as a positive control, in Normal Human Bronchial Epithelial (NHBE) cells. The endpoints measured covered cellular health, energy production and oxidative stress. Base liquids, with or without nicotine, and commercial, flavoured, nicotine-containing e-liquids (CFs), had little or no effect on cell viability and most HCS endpoints even at significantly higher concentrations (typically 100 times or higher) than 3R4F CSC. CSC induced a dose-dependent decrease of cell viability and triggered the response in all HCS endpoints. Effects of CFs were typically observed at or above 1%. CF Menthol was the most active flavour, with minimum effective concentrations 43 to 659 times higher than corresponding 3R4F CSC concentrations. Our results show a lower biological activity of e-liquids compared to cigarette smoke condensate in this experimental setting, across wide range of cellular endpoints.

Graphical abstract, Smoking is a cause of serious diseases in smokers including chronic respiratory diseases. This study aimed to evaluate the tobacco harm reduction (THR) potential of an electronic vapor product (EVP, myblu™) compared to a Kentucky Reference Cigarette (3R4F), and assessed endpoints related to chronic respiratory diseases. Endpoints included: cytotoxicity, barrier integrity (TEER), cilia function, immunohistochemistry, and pro-inflammatory markers. In order to more closely represent the user exposure scenario, we have employed the in vitro 3D organotypic model of human airway epithelium (MucilAir™, Epithelix) for respiratory assessment. The model was repeatedly exposed to either whole aerosol of the EVP, or whole 3R4F smoke, at the air liquid interface (ALI), for 4 weeks to either 30, 60 or 90 puffs on 3-exposure-per-week basis. 3R4F smoke generation used the ISO 20778:2018 regime and EVP aerosol used the ISO 20768:2018 vaping regime. Exposure to undiluted whole EVP aerosol did not trigger any significant changes in the level of pro-inflammatory mediators, cilia beating function, barrier integrity and cytotoxicity when compared with air controls. In contrast, exposure to diluted (1:17) whole cigarette smoke caused significant changes to all the endpoints mentioned above. To our knowledge, this is the first study evaluating the effects of repeated whole cigarette smoke and whole EVP aerosol exposure to a 3D lung model at the ALI. Our results add to the growing body of scientific literature supporting the THR potential of EVPs relative to combustible cigarettes and the applicability of the 3D lung models in human-relevant product risk assessments.

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.

Given the complexity of inhaled substances, the aerosol exposure environment has seen diversification and development of setups in conjunction with the evolving in vitro toxicology space. Each laboratory uses its in vitro exposure system differently (different protocols, adaptations, and biological analysis). Unfortunately, as systems diversify, so does the complexity of comparing multiple systems in a “standardized” manner. As yet, no one has compared simply whether these diverse systems can all generate a consistent aerosol stream, which is paramount prior to transit and exposure. This study has compared, at source, aerosol generation (using nicotine as an exposure marker) in nine in vitro whole-aerosol exposure setups (seven different systems) across five distinct geographically independent locations, including the UK, the USA, Switzerland, Germany, and Japan. The results demonstrate that, despite system-wide differences (adaptations, nuances, and application), these systems—when appropriately maintained and used under a prescribed set of established conditions can all generate a consistent and statistically comparable aerosol stream. These data will be invaluable for new researchers and established laboratories, so they may benchmark against this study. Finally, this interlaboratory comparison combined with the wealth of transit and exposure interface data, may help the environment move towards a truly validated and consistent approach to aerosol exposure. Such an approach could be replicated for other aerosolized products, such as e-cigarettes and heated tobacco products.

Total particulate matter (TPM) and the gas-vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.

Meiosis is central to the formation of haploid gametes or spores in that it segregates homologous chromosomes and halves the chromosome number. A prerequisite of this genome bisection is the pairing of homologous chromosomes during the first meiotic prophase. When budding yeast cells are induced to undergo meiosis, this has profound consequences for nuclear structure: after premeiotic DNA replication centromeres disperse, while telomeres move about the nuclear periphery and temporarily cluster during the leptotene/zygotene transition (bouquet stage) of the prophase to first meiotic division. In vegetative cells, Hdf1p (yKu) and the myosin-like proteins Mlp1p and Mlp2p have been suggested to contribute to the organization of silent chromatin, tethering of telomeres to the nuclear periphery, DNA repair, and telomere maintenance. Here, we investigated by molecular cytology whether yKu and Mlp proteins contribute to telomere and chromosome dynamics in meiosis. It was found that mlp1 Delta mlp2 Delta double-mutant cells undergo centromere dispersion, telomere clustering, homologue pairing, and sporulation like wild type. On the other hand, cells deficient for yKu underwent meiosis-specific chromosomal events with a delay, while they eventually sporulated like wild type. These results suggest that the absence of yKu not only affects vegetative nuclear architecture (Laroche et al., 1998) but also interferes with the ordered occurrence of chromosome dynamics during first meiotic prophase.

We investigated the sequence of chromosomal events during meiotic prophase in haploid, diploid and autotetraploid SK1 strains of Saccharomyces cerevisiae. Using molecular cytology, we found that meiosis-specific nuclear topology (i.e. dissolution of centromere clustering, bouquet formation and meiotic divisions) are significantly delayed in polyploid SK1 meiosis. Thus, and in contrast to the situation in plants, an increase in ploidy extends prophase I in budding yeast. Moreover, we found that bouquet formation also occurs in haploid and diploid SK1 meiosis deficient in the telomeric heterochromatin protein Sir3p. Diploid sir3Delta SK1 meiosis showed pleiotropic defects such as delayed centromere cluster resolution in a proportion of cells and impeded downstream events (i.e. bouquet formation, homologue pairing and meiotic divisions). Meiotic telomere clustering occurred in diploid and haploid sir3Delta strains. Using the haploid system, we further show that a bouquet forms at the kar3Delta SPB. Comparison of the expression of meiosis-specific Ndj1p-HA and Zip1p in haploid control and kar3Delta time courses revealed that fewer cells enter the meiotic cycle in absence of Kar3p. Elevated frequencies of bouquets in kar3Delta haploid meiosis suggest a role for Kar3p in regulation of telomere dynamics.

We have studied the repair of a DNA-DSB created by the VMA1-derived endonuclease in mutants that have different levels of Spo11-DSBs: WT (sae2), few (hop1), and none (spo11-Y135F). In spo11-Y135F and hop1 cells, intrachromosomal repair is more frequent than in WT and sae2 cells. In spo11-Y135F cells there was no chromosome pairing or synapsis and a faster turnover of resected DNA. Compared to WT and sae2 cells, spo11-Y135F and hop1 cells have a greater proportion of long resection tracts. The data suggest that high levels of Spo11-DSBs are required for normal regulation of resection, even at a DSB created by another protein. WT control over resection could be important for directing repair to be interchromosomal, increasing the chance of creating interhomolog connections essential to meiotic segregation.

The entry into meiosis is characterized by a lengthy premeiotic S phase and a reorganization of the nuclear architecture. Analysis of centromere and telomere dynamics in wild-type Saccharomyces cerevisiae meiosis suggests that resolution of vegetative centromere and telomere clusters are independent events differently connected to premeiotic S phase. Absence of the B-type cyclin Clb5 or the Set1 histone methyltransferase leads to a delay of premeiotic S phase by separate mechanisms. In clb5Δ cells, centromere cluster resolution appears normal, whereas dissolution of the vegetative telomere clusters is impaired and meiosis-specific clustering of telomeres, i.e. bouquet formation, is grossly delayed. In set1Δ cells, centromere and telomere redistribution are both impaired and bouquet nuclei are absent, despite proper location of the meiosis-specific telomere protein Ndj1. Thus, centromere and telomere redistribution at the onset of prophase I is differentially regulated, with centromere dispersion occurring independently of premeiotic S phase. The normal kinetics of dissolution of the vegetative telomere clusters in a set1Δ mec1-1 mutant suggests the presence of a checkpoint that limits the dispersion of telomeres in absence of Set1.

International audience; Recent experiments have shown that gene repression can be correlated with relocation of genes to heterochromatin-rich silent domains. Here, we investigate whether nuclear architecture and spatial positioning can contribute directly to the transcriptional activity of a genetic locus in Saccharomyces cerevisiae. By disassembling telomeric silent domains without altering the chromatin-mediated silencing machinery, we show that the transcriptional activity of silencer--reporter constructs depends on intranuclear position. This demonstrates that telomeric silent domains are actively involved in transcriptional silencing. Employing fluorescent in situ hybridization (FISH) in combination with genetic assays, we demonstrate that telomeres control the establishment of transcriptional states by reversible partitioning with the perinuclear silencing domains. Anchoring telomeres interferes with their ability to assume an active state, whereas disassembly of silencing domains prevents telomeres from assuming a repressed state. Our data support a model in which domains of enriched transcriptional regulators allow genes to determine transcriptional states by spatial positioning.

Fluorescence in situ hybridization provides an effective means to delineate chromosomes and chromosomal regions of interest, during all stages of the cell cycle, irrespective of chromatin composition and compaction. This property makes FISH particularly useful for studying chromosome behavior in species with minute genomes and/or ill-defined metaphase chromosomes. Genetic model organisms like fission and budding yeast display such cytological characteristics. Consequently, chromosome painting and locus-specific yeast FISH (Scherthan et al. 1992, Scherthan et al. 1994, Uzawa et al. 1992, Guacci et al. 1994, Weiner and Kleckner 1994, Gotta et al. 1996) has become an indispensable tool in the analysis of chromosome dynamics and organization in wild-type and mutant yeast strains.

We have investigated the requirements for NDJ1 in meiotic telomere redistribution and clustering in synchronized cultures of Saccharomyces cerevisiae. On induction of wild-type meiosis, telomeres disperse from premeiotic aggregates over the nuclear periphery, and then cluster near the spindle pole body (bouquet arrangement) before dispersing again. In ndj1Δ meiocytes, telomeres are scattered throughout the nucleus and fail to form perinuclear meiosis-specific distribution patterns, suggesting that Ndj1p may function to tether meiotic telomeres to the nuclear periphery. Since ndj1Δ meiocytes fail to cluster their telomeres at any prophase stage, Ndj1p is the first protein shown to be required for bouquet formation in a synaptic organism. Analysis of homologue pairing by two-color fluorescence in situ hybridization with cosmid probes to regions on III, IX, and XI revealed that disruption of bouquet formation is associated with a significant delay (>2 h) of homologue pairing. An increased and persistent fraction of ndj1Δ meiocytes with Zip1p polycomplexes suggests that chromosome polarization is important for synapsis progression. Thus, our observations support the hypothesis that meiotic telomere clustering contributes to efficient homologue alignment and synaptic pairing. Under naturally occurring conditions, bouquet formation may allow for rapid sporulation and confer a selective advantage.

Chromosome arrangement in spread nuclei of the budding yeast, Saccharomyces cerevisiae was studied by fluorescence in situ hybridization with probes to centromeres and telomeric chromosome regions. We found that during interphase centromeres are tightly clustered in a peripheral region of the nucleus, whereas telomeres tend to occupy the area outside the centromeric domain. In vigorously growing cultures, centromere clustering occurred in ∼90% of cells and it appeared to be maintained throughout interphase. It was reduced when cells were kept under stationary conditions for an extended period. In meiosis, centromere clusters disintegrated before the emergence of the earliest precursors of the synaptonemal complex. Evidence for the contribution of centromere clustering to other aspects of suprachromosomal nuclear order, in particular the vegetative association of homologous chromosomes, is provided, and a possible supporting role in meiotic homology searching is discussed.