Reducing attractiveness of e-liquids: proposal for a restrictive list of tobacco-related flavourings.

OBJECTIVE: Electronic cigarettes are addictive and harmful, and flavour is a key factor determining their abuse liability. Both adult smokers and young non-smokers like sweet and fruity flavours in particular. In order to discourage e-cigarette use among youth, the Dutch government announced in 2020 to only allow tobacco flavours in e-liquids. We propose a restrictive list of flavourings that will only enable the production of e-liquids with a tobacco flavour. METHODS: We used e-liquid ingredient data notified via the European Common Entry Gate system before the government's announcement. First, we classified all e-liquids into flavour categories, and continued with the set of flavourings present in tobacco e-liquids. Five selection criteria related to prevalence of use, chemical composition, flavour description and health effects were defined to compile a restrictive list of tobacco flavourings. RESULTS: E-liquids marketed as having tobacco flavour contained 503 different flavourings, some with tobacco flavour, but also other (such as sweet) flavours. We excluded (1) 330 flavourings used in <0.5% of e-liquids, (2) 77 used less frequently in tobacco than in all e-liquids, (3) 13 plant extracts, (4) 60 that are sweet or not associated with a tobacco flavour and (5) 7 flavourings with hazardous properties. This resulted in a final list of 16 flavourings. CONCLUSIONS: Implementing this restrictive list will likely discourage e-cigarette use among youth, but could also make e-cigarettes less attractive as smoking cessation aid.

Flavours and flavourings in waterpipe products: a comparison between tobacco, herbal molasses and steam stones.

ObjectivesFlavoured products are especially appealing to youth and contribute to the onset of waterpipe smoking and continued use of waterpipe tobacco. The goal of database and chemical analysis was to provide a clear overview of commonly used flavours and flavourings in tobacco and related waterpipe products, that is, herbal molasses and steam stones. METHODS: In 2019, 249 waterpipe tobacco products were registered in the European Common Entry Gate by manufacturers to be marketed in The Netherlands. Flavour categories were assigned to the registered products based on their brand names and product descriptions. Nicotine and eleven 1111 flavourings were identified and quantified in waterpipe tobacco (n=8), herbal molasses (n=7) and steam stones (n=4) by extraction and gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: Flavour categories could be assigned to 237 of 249 registered waterpipe tobacco products. Eight flavour main categories and 48 unique subcategories were identified and presented in a flavour wheel. All registered waterpipe tobacco products were flavoured, and the majority (78%) was fruit flavoured. Herbal molasses contained similar median flavouring levels, and steam stones contained lower median levels compared with waterpipe tobacco. Flavourings in waterpipe products were almost exclusively fruity and sweet, often in combination with menthol/mint flavourings. CONCLUSIONS: This study is the first to present a waterpipe tobacco flavour wheel, providing a quick overview of waterpipe tobacco flavours and thereby aiding communication among experts around the globe. GC-MS analysis revealed that the most prevalent flavourings are present in similar levels in herbal and tobacco waterpipe products. Banning flavourings in all waterpipe products would be a good strategy to reduce waterpipe smoking among youth.

Aluminum Hydroxide And Aluminum Phosphate Adjuvants Elicit A Different Innate Immune Response.

Aluminum hydroxide (Al(OH)3) and aluminum phosphate (AlPO4) are widely used adjuvants in human vaccines. However, a rationale to choose one or the other is lacking since the differences between molecular mechanisms of action of these adjuvants are unknown. In the current study, we compared the innate immune response induced by both adjuvants in vitro and in vivo. Proteome analysis of human primary monocytes was used to determine the immunological pathways activated by these adjuvants. Subsequently, analysis of immune cells present at the site of injection and proteome analysis of the muscle tissue revealed the differentially regulated processes related to the innate immune response in vivo. Incubation with Al(OH)3 specifically enhanced the activation of antigen processing and presentation pathways in vitro. In vivo experiments showed that only intramuscular (I.M.) immunization with Al(OH)3 attracted neutrophils, while I.M. immunization with AlPO4 attracted monocytes/macrophages to the site of injection. In addition, only I.M. immunization with Al(OH)3 enhanced the process of hemostasis after 96 hours, possibly related to neutrophilic extracellular trap formation. Both adjuvants differentially regulated various immune system-related processes. The results show that Al(OH)3 and AlPO4 act differently on the innate immune system. We speculate that these different regulations affect the interaction with cells, due to the different physicochemical properties of both adjuvants.

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis.

Valproic acid-induced gene expression responses in rat whole embryo culture and comparison across in vitro developmental and non-developmental models.

Transcriptomic evaluations may improve toxicity prediction of in vitro-based developmental models. In this study, transcriptomics was used to identify VPA-induced gene expression changes in rat whole embryo culture (WEC). Furthermore, VPA-induced responses were compared across in vitro-based developmental models, such as the cardiac and neural embryonic stem cells (ESTc and ESTn, respectively) and the zebrafish embryotoxicity model. VPA-induced gene regulation in WEC corresponded with observed morphological effects and previously suggested mechanisms of toxicity. Gene Ontology term-directed analysis showed conservation of VPA-induced gene expression changes across in vitro-based developmental models, with ESTc and ESTn exhibiting complementary responses. Furthermore, comparison of in vitro-based developmental and non-developmental models revealed that more generalized VPA-induced effects can be detected using non-developmental models whereas developmental models provide added value when assessing developmental-specific effects. These analyses can be used to optimize test batteries for the detection of developmental toxicants in vitro.

Exploring the zebrafish embryo as an alternative model for the evaluation of liver toxicity by histopathology and expression profiling.

The whole zebrafish embryo model (ZFE) has proven its applicability in developmental toxicity testing. Since functional hepatocytes are already present from 36 h post fertilization onwards, whole ZFE have been proposed as an attractive alternative to mammalian in vivo models in hepatotoxicity testing. The goal of the present study is to further underpin the applicability of whole ZFE for hepatotoxicity testing by combining histopathology and next-generation sequencing-based gene expression profiling. To this aim, whole ZFE and adult zebrafish were exposed to a set of hepatotoxic reference compounds. Histopathology revealed compound and life-stage-specific effects indicative of toxic injury in livers of whole ZFE and adult zebrafish. Next-generation sequencing (NGS) was used to compare transcript profiles in pooled individual RNA samples of whole ZFE and livers of adult zebrafish. This revealed that hepatotoxicity-associated expression can be detected beyond the overall transcription noise in the whole embryo. In situ hybridization verified liver specificity of selected highly expressed markers in whole ZFE. Finally, cyclosporine A (CsA) was used as an illustrative case to support applicability of ZFE in hepatotoxicity testing by comparing CsA-induced gene expression between ZFE, in vivo mouse liver and HepaRG cells on the levels of single genes, pathways and transcription factors. While there was no clear overlap on single gene level between the whole ZFE and in vivo mouse liver, strong similarities were observed between whole ZFE and in vivo mouse liver in regulated pathways related to hepatotoxicity, as well as in relevant overrepresented transcription factors. In conclusion, both the use of NGS of pooled RNA extracts analysis combined with histopathology and traditional microarray in single case showed the potential to detect liver-related genes and processes within the transcriptome of a whole zebrafish embryo. This supports the applicability of the whole ZFE model for compound-induced hepatotoxicity screening.

Lasting effects on body weight and mammary gland gene expression in female mice upon early life exposure to n-3 but not n-6 high-fat diets.

Exposure to an imbalance of nutrients prior to conception and during critical developmental periods can have lasting consequences on physiological processes resulting in chronic diseases later in life. Developmental programming has been shown to involve structural and functional changes in important tissues. The aim of the present study was to investigate whether early life diet has a programming effect on the mammary gland. Wild-type mice were exposed from 2 weeks prior to conception to 6 weeks of age to a regular low-fat diet, or to high-fat diets based on either corn oil or flaxseed oil. At 6 weeks of age, all mice were shifted to the regular low-fat diet until termination at 10 weeks of age. Early life exposure to a high-fat diet, either high in n-6 (corn oil) or in n-3 (flaxseed oil) polyunsaturated fatty acids, did not affect birth weight, but resulted in an increased body weight at 10 weeks of age. Transcriptome analyses of the fourth abdominal mammary gland revealed differentially expressed genes between the different treatment groups. Exposure to high-fat diet based on flaxseed oil, but not on corn oil, resulted in regulation of pathways involved in energy metabolism, immune response and inflammation. Our findings suggest that diet during early life indeed has a lasting effect on the mammary gland and significantly influences postnatal body weight gain, metabolic status, and signaling networks in the mammary gland of female offspring.

A comparison of gene expression responses in rat whole embryo culture and in vivo: time-dependent retinoic acid-induced teratogenic response.

The whole embryo culture (WEC) model serves as a potential alternative for classical in vivo developmental toxicity testing. In the WEC, cultured rat embryos are exposed during neurulation and early organogenesis and evaluated for morphological effects. Toxicogenomic-based approaches may improve the predictive ability of WEC by providing molecular-based markers associated with chemical exposure, which can be compared across multiple parameters (e.g., exposure duration, developmental time, experimental model). Additionally, comparisons between in vitro and in vivo models may identify objective relevant molecular responses linked with developmental toxicity endpoints in vivo. In this study, using a transcriptomic approach, we compared all-trans retinoic acid (RA)-exposed and nonexposed Wistar rat embryos derived using WEC (RA, 0.5 µg/ml) or in vivo (RA, 50 mg/kg, oral gavage) to identify overlapping and nonoverlapping effects of RA on RNA expression in parallel with morphological changes. Across six time points (gestational day 10 + 2-48 h), we observed strong similarities in RA response at the gene (directionality, significance) and functional (e.g., embryonic development, cell differentiation) level which associated with RA-induced adverse morphological effects, including growth reduction as well as alterations in neural tube, limb, branchial, and mandible development. We observed differences between models in the timing of RA-induced effects on genes related to embryonic development and RA metabolism. These observations on the gene expression level were associated with specific differential morphological outcomes. This study supports the use of WEC to examine compound-induced molecular responses relative to in vivo and, furthermore, assists in defining the applicability domain of the WEC in determining complementary windows of sensitivity for developmental toxicological investigations.