Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis

Background\ud DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis. \ud \ud Results\ud A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation. \ud \ud Conclusion\ud The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver. \ud \u

The percutaneous absorption of soman in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant

PURPOSE: The aim of this study was to evaluate a candidate haemostat (WoundStat™), down-selected from previous in vitro studies, for efficacy as a potential skin decontaminant against the chemical warfare agent pinacoyl methylfluorophosphonate (Soman, GD) using an in vivo pig model. MATERIALS AND METHODS: An area of approximately 3 cm2 was dermatomed from the dorsal ear skin to a nominal depth of 100 µm. A discrete droplet of 14C-GD (300 µg kg-1) was applied directly onto the surface of the damaged skin at the centre of the dosing site. Animals assigned to the treatment group were given a 2 g application of WoundStat™ 30 s after GD challenge. The decontamination efficacy of WoundStat™ against GD was measured by the direct quantification of the distribution of 14C-GD, as well as routine determination of whole blood cholinesterase and physiological measurements. RESULTS: WoundStat™ sequestered approximately 70% of the applied 14C-GD. Internal radiolabel recovery from treated animals was approximately 1% of the initially applied dose. Whole blood cholinesterase levels decreased to less than 10% of the original value by 15 min post WoundStat™ treatment and gradually decreased until the onset of apnoea or until euthanasia. All treated animals showed signs of GD intoxication that could be grouped into early (mastication, fasciculations and tremor), intermediate (miosis, salivation and nasal secretions) and late onset (lacrimation, body spasm and apnoea) effects. Two of the six WoundStat™ treated animals survived the study duration. CONCLUSIONS: The current study has shown that the use of WoundStat™ as a decontaminant on damaged pig ear skin was unable to fully protect against GD toxicity. Importantly, the findings indicate that the use of WoundStat™ in GD contaminated wounds would not exacerbate GD toxicity. These data suggest that absorbent haemostatic products may offer some limited functionality as wound decontaminants.Peer reviewedFinal Accepted Versio

Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures

Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation. Trout hepatocyte spheroids are a promising in vitro model for this assessment. In this investigation, the gene expression and function for xenobiotic metabolism and cellular efflux were characterised. Using fluorescence, transport and real time PCR analysis, the expression and functionality of a variety of genes related to xenobiotic metabolism and drug efflux were assessed in a range of trout hepatocyte culture preparations. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques. A transient decline in the expression of genes related to both xenobiotic metabolism and transport was determined during spheroid development, with a subsequent recovery in older spheroids. The most mature spheroids also exhibited an expression profile most comparable to that reported in vivo. Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors. In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux. Together with the enhanced gene expression and longevity of the model, hepatocytes in spheroid culture may prove to be an accurate alternative model to study the mechanisms of these processes in fish liver and provide an assay to determine the bioaccumulation potential of environmental contaminants

Low Doses of the Carcinogen Furan Alter Cell Cycle and Apoptosis Gene Expression in Rat Liver Independent of DNA Methylation

BACKGROUND: Evidence of potent rodent carcinogenicity via an unclear mechanism suggests that furan in various foods [leading to an intake of up to 3.5 mu g/kg body weight (bw)/day] may present a potential risk to human health

Mapping Drug Physico-Chemical Features to Pathway Activity Reveals Molecular Networks Linked to Toxicity Outcome

The identification of predictive biomarkers is at the core of modern toxicology. So far, a number of approaches have been proposed. These rely on statistical inference of toxicity response from either compound features (i.e., QSAR), in vitro cell based assays or molecular profiling of target tissues (i.e., expression profiling). Although these approaches have already shown the potential of predictive toxicology, we still do not have a systematic approach to model the interaction between chemical features, molecular networks and toxicity outcome. Here, we describe a computational strategy designed to address this important need. Its application to a model of renal tubular degeneration has revealed a link between physico-chemical features and signalling components controlling cell communication pathways, which in turn are differentially modulated in response to toxic chemicals. Overall, our findings are consistent with the existence of a general toxicity mechanism operating in synergy with more specific single-target based mode of actions (MOAs) and provide a general framework for the development of an integrative approach to predictive toxicology

Hepatic gene expression in flounder chronically exposed to multiply polluted estuarine sediment: Absence of classical exposure ‘biomarker’ signals and induction of inflammatory, innate immune and apoptotic pathways

The effects of chronic long term exposure to multiply-polluted environments on fish are not well understood, but environmental surveys suggest that such exposure may cause a variety of pathologies, including cancers. Transcriptomic profiling has recently been used to assess gene expression in European flounder (Platichthys flesus) living in several polluted and clean estuaries. However, the gene expression changes detected were not unequivocally elicited by pollution, most likely due to the confounding effects of natural estuarine ecosystem variables. In this study flounder from an uncontaminated estuary were held on clean or polluted sediments in mesocosms, allowing control of variables such as salinity, temperature, and diet. After 7 months flounder were removed from each mesocosm and hepatocytes prepared from fish exposed to clean or polluted sediments. The hepatocytes were treated with benzo(a)pyrene (BAP), estradiol (E2), copper, a mixture of these three, or with the vehicle DMSO. A flounder cDNA microarray was then used to measure hepatocyte transcript abundance after each treatment. The results show that long term chronic exposure to a multiply-polluted sediment causes increases in the expression of mRNAs coding for proteins of the endogenous apoptotic program, of innate immunity and inflammation. Contrary to expectation, the expression of mRNAs which are commonly used as biomarkers of environmental exposure to particular contaminants were not changed, or were changed contrary to expectation. However, acute treatment of hepatocytes from flounder from both clean and polluted sediments with BAP or E2 caused the expected changes in the expression of these biomarkers. Thus transcriptomic analysis of flounder exposed long-term to chronic pollution causes a different pattern of gene expression than in fish acutely treated with single chemicals, and reveals novel potential biomarkers of environmental contaminant exposure. These novel biomarkers include Diablo, a gene involved in apoptotic pathways and highly differentially regulated by both chronic and acute exposure to multiple pollutants

Scientific opinion on flavouring group evaluation 415 (FGE.415): (E)-3-benzo[1,3]dioxol-5-yl-N,N-diphenyl-2-propenamide

The EFSA Panel on Food Additives and Flavourings (FAF) was requested to evaluate the safety of the substance (E)-3-benzo[1,3]dioxol-5-yl-N,N-diphenyl-2-propenamide [FL-no: 16.135] as a new flavouring substance, in accordance with Regulation (EC) No 1331/2008. The substance has not been reported to occur naturally and it is chemically synthesised. It is intended to be used as a flavouring substance in specific categories of food, but not intended to be used in beverages. The chronic dietary exposure to [FL-no: 16.135] estimated using the added portions exposure technique (APET), is calculated to be 780 mu g/person per day for a 60-kg adult and 480 mu g/person per day for a 15-kg 3-year-old child. [FL-no: 16.135] did not show genotoxic effects in bacterial mutagenicity and mammalian cell micronucleus assays in vitro. Developmental toxicity was not observed in a study in rats at the dose levels up to 1,000 mg/kg body weight (bw) per day. The Panel derived a BMDL of 101 mg/kg bw per day from a 90-day toxicity study. Based on this BMDL, adequate margins of exposure of 7,800 and 3,200 could be calculated for adults and children, respectively. The Panel concluded that there is no safety concern for [FL-no: 16.135], when used as a flavouring substance at the estimated level of dietary exposure calculated using the APET approach, based on the intended uses and use levels as specified in Appendix B. The Panel further concluded that the combined exposure to [FL-no: 16.135] from its use as a food flavouring substance and from its presence in toothpaste is also not of safety concern. (C) 2022 Wiley-VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority