Project description:Parabens have been used for decades as preservatives in food, drugs and cosmetics. The majority however, were banned in 2009 and 2014 leaving only methyl-, ethyl-, propyl-, and butyl-derivates available for subsequent use. Methyl- and propylparaben have been extensively tested in vivo, with no resulting evidence for developmental and reproductive toxicity (DART). In contrast, ethylparaben has not yet been tested for DART in animal experiments, and it is currently debated if additional animal studies are warranted. In order to perform a comparison of the four currently-approved parabens, we used a previously established in vitro test based on human induced pluripotent stem cells (iPSC) that are exposed to test substances during their differentiation to neuroectodermal cells. EC50 values for cytotoxicity were 906 µM, 698 µM, 216 µM and 63 µM for methyl-, ethyl-, propyl- and butylparaben, respectively, demonstrating that cytotoxicity increases with increasing alkyl chain length. Genome-wide analysis demonstrated that FDR-adjusted significant gene expression changes occurred only at cytotoxic or close to cytotoxic concentrations, for example 1,720 differentially expressed genes (DEG) at 1,000 µM ethylparaben, 1 DEG at 316 µM, and no DEG at 100 µM or lower concentrations. The highest concentration of ethylparaben that did not induce any cytotoxicity nor DEG was 1670-fold above the highest published concentrations reported in biomonitoring studies (60 nM ethylparaben in cord blood). In conclusion, cytotoxicity and gene expression alterations of ethylparaben occurred at concentrations of approximately three orders of magnitude above human blood concentrations; moreover, the substance fitted well into a scenario where toxicity increases with the alkyl chain length, and gene expression changes only occur at cytotoxic or close to cytotoxic concentrations. Therefore, no evidence was obtained suggesting that in vivo DART with ethylparaben would lead to different results as the methyl- or propyl derivates.

Project description:In vitro toxicology approaches have evolved, from a focus on the molecular changes within a cell to understanding of toxicity-related mechanisms that simulate the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offer a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the relevancy and application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. The biological impact was assessed following exposure to aerosol generated from a candidate modified risk tobacco product (MRTP), the Tobacco Heating System (THS) 2.2, as compared with smoke generated from reference cigarette 3R4F using an in vitro human 3-D nasal epithelial cultures. A series of experimental repetitions where multiple doses of the aerosol and smoke were applied, were conducted to obtain reproducible measurements and reliable observations to understand the cellular/molecular changes that occur following exposure. Aligned with the 3Rs Strategy and the Vision-and-Strategy of the Toxicity Testing in the 21st Century, this study implemented a systems toxicology approach and found that for all tested concentrations, the impact of 3R4F smoke was considerably greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in the tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles. In addition, to evaluate further the possible adverse effects of THS2.2 aerosol, a dose range assessment was conducted. A broader range of THS2.2 concentrations were exposed to the nasal cultures. Various dilutions of THS2.2 were applied to the cultures using the Vitrocell® 24/48 exposure system, corresponding to the concentrations of nicotine between 0.15 mg/L and 1.79 mg/L.

Project description:In vitro toxicology approaches have evolved, from a focus on the molecular changes within a cell to understanding of toxicity-related mechanisms that simulate the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offer a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the relevancy and application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. The biological impact was assessed following exposure to aerosol generated from a candidate modified risk tobacco product (MRTP), the Tobacco Heating System (THS) 2.2, as compared with smoke generated from reference cigarette 3R4F using an in vitro human 3-D nasal epithelial cultures. A series of experimental repetitions where multiple doses of the aerosol and smoke were applied, were conducted to obtain reproducible measurements and reliable observations to understand the cellular/molecular changes that occur following exposure. Aligned with the 3Rs Strategy and the Vision-and-Strategy of the Toxicity Testing in the 21st Century, this study implemented a systems toxicology approach and found that for all tested concentrations, the impact of 3R4F smoke was considerably greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in the tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles. In addition, to evaluate further the possible adverse effects of THS2.2 aerosol, a dose range assessment was conducted. A broader range of THS2.2 concentrations were exposed to the nasal cultures. Various dilutions of THS2.2 were applied to the cultures using the Vitrocellï¾® 24/48 exposure system, corresponding to the concentrations of nicotine between 0.15 mg/L and 1.79 mg/L

Project description:In vitro toxicology approaches have evolved, from a focus on the molecular changes within a cell to understanding of toxicity-related mechanisms that simulate the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offer a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the relevancy and application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. The biological impact was assessed following exposure to aerosol generated from a candidate modified risk tobacco product (MRTP), the Tobacco Heating System (THS) 2.2, as compared with smoke generated from reference cigarette 3R4F using an in vitro human 3-D nasal epithelial cultures. A series of experimental repetitions where multiple doses of the aerosol and smoke were applied, were conducted to obtain reproducible measurements and reliable observations to understand the cellular/molecular changes that occur following exposure. Aligned with the 3Rs Strategy and the Vision-and-Strategy of the Toxicity Testing in the 21st Century, this study implemented a systems toxicology approach and found that for all tested concentrations, the impact of 3R4F smoke was considerably greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in the tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles. In addition, to evaluate further the possible adverse effects of THS2.2 aerosol, a dose range assessment was conducted. A broader range of THS2.2 concentrations were exposed to the nasal cultures. Various dilutions of THS2.2 were applied to the cultures using the VitrocellÂ® 24/48 exposure system, corresponding to the concentrations of nicotine between 0.15 mg/L and 1.79 mg/L

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Risk assessment of parabens in a transcriptomics-based in vitro system

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