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

Trichostatin A Enhances Gap Junctional Intercellular Communication in Primary Cultures of Adult Rat Hepatocytes

The effects of histone deacetylase inhibitor Trichostatin A (TSA) on connexin (Cx) expression and gap junctional intercellular communication (GJIC) were investigated in primary cultures of adult rat hepatocytes. GJIC was monitored by using the scrape-loading/dye transfer method. Immunoblotting and immunocytochemistry were used to investigate Cx protein levels and localization. Cx gene expression was studied by means of quantitative reverse transcriptase-polymerase chain reaction. TSA increased Cx32 protein levels and affected negatively the Cx26 protein levels. The latter was preferentially located in the cytosol of cultured cells. TSA also promoted the appearance of Cx43 in the nuclear compartment of primary cultured hepatocytes. Overall, this resulted in enhanced GJIC activity. It is important to note that the time of onset of TSA treatment was crucial for the extent of its outcome and that the effects of TSA on Cx protein levels occurred independently of transcriptional changes. TSA differentially affects Cx proteins in primary rat hepatocyte cultures, suggesting distinct regulation and/or distinct roles of the different Cx species in the control of hepatic homeostasis. TSA enhances GJIC between primary cultured rat hepatocytes, an interesting finding supporting its use to further optimize liver-based in vitro models for pharmacotoxicological purpose

Providing access to risk prediction tools via the HL7 XML-formatted risk web service

Background: Cancer risk prediction tools provide valuable information to clinicians but remain computationally challenging. Many clinics find that Ca Gene or Hughes Risk Apps fit their needs for easy- and ready-to-use software to obtain cancer risks; however, these resources may not fit all clinics’ needs. The Hughes Risk Apps Group and Bayes Mendel Lab therefore developed a web service, called “Risk Service", which may be integrated into any client software to quickly obtain standardized and up-to-date risk predictions for Bayes Mendel tools (BRCAPRO, MMRpro, PancPRO, and MelaPRO), the Tyrer-Cuzick IBIS Breast Cancer Risk Evaluation Tool, and the Colorectal Cancer Risk Assessment Tool. Findings: Software clients that can convert their local structured data into the HL7 XML-formatted family and clinical patient history (Pedigree model) may integrate with the Risk Service. The Risk Service uses Apache Tomcat and Apache Axis2 technologies to provide an all Java web service. The software client sends HL7 XML information containing anonymized family and clinical history to a Dana-Farber Cancer Institute (DFCI) server where it is parsed, interpreted, and processed by multiple risk tools. The Risk Service then formats the results into an HL7 style message and returns the risk predictions to the originating software client. Upon consent, users may allow DFCI to maintain the data for future research. The Risk Service implementation is exemplified through Hughes Risk Apps. Conclusions: The Risk Service broadens the availability of valuable, up-to-date cancer risk tools and allows clinics and researchers to integrate risk prediction tools into their own software interface designed for their needs. Each software package can collect risk data using its own interface, and display the results using its own interface, while using a central, up-to-date risk calculator. This allows users to choose from multiple interfaces while always getting the latest risk calculations. Consenting users contribute their data for future research, thus building a rich multi-center resource

Use of 5-azacytidine in a proof-of-concept study to evaluate the impact of pre-natal and post-natal exposures, as well as within generation persistent DNA methylation changes in Daphnia

Short-term exposures at critical stages of development can lead to delayed adverse effects long after the initial stressor has been removed, a concept referred to as developmental origin of adult disease. This indicates that organisms’ phenotypes may epigenetically reflect their past exposure history as well as reflecting chemicals currently present in their environment. This concept has significant implications for environmental monitoring. However, there is as yet little or no implementation of epigenetics in environmental risk assessment. In a proof-of-principle study we exposed Daphnia magna to 5-azacytidine, a known DNA de-methylating agent. Exposures covered combinations of prenatal and postnatal exposures as well as different exposure durations and recovery stages. Growth, the transcription of genes and levels of metabolites involved in regulating DNA methylation, and methylation levels of several genes were measured. Our data shows that prenatal exposures caused significant changes in the methylome of target genes, indicating that prenatal stages of Daphnia are also susceptible to same level of change as post-natal stages of Daphnia. While the combination of pre- and postnatal exposures caused the most extreme reduction in DNA methylation compared to the control group. Furthermore, some of the changes in the methylation patterns were persistent even after the initial stressor was removed. Our results suggest that epigenetic biomarkers have the potential to be used as indicators of past chemical exposure history of organisms and provide strong support for implementing changes to the current regimes for chemical risk assessment to mimic realistic environmental scenarios

Environmentally relevant iron oxide nanoparticles produce limited acute pulmonary effects in rats at realistic exposure levels

Iron is typically the dominant metal in the ultrafine fraction of airborne particulate matter. Various studies have investigated the toxicity of inhaled nano-sized iron oxide particles (FeOxNPs) but their results have been contradictory, with some indicating no or minor effects and others finding effects including oxidative stress and inflammation. Most studies, however, did not use materials reflecting the characteristics of FeOxNPs present in the environment. We, therefore, analysed the potential toxicity of FeOxNPs of different forms (Fe3O4 , α-Fe2O3 and γ-Fe2O3 ) reflecting the characteristics of high iron content nano-sized particles sampled from the environment, both individually and in a mixture (FeOx-mix). A preliminary in vitro study indicated Fe3O4 and FeOx-mix were more cytotoxic than either form of Fe2O3 in human bronchial epithelial cells (BEAS-2B). Follow-up in vitro (0.003, 0.03, 0.3 µg/mL, 24 h) and in vivo (Sprague–Dawley rats, nose-only exposure, 50 µg/m3 and 500 µg/m3 , 3 h/d × 3 d) studies therefore focused on these materials. Experiments in vitro explored responses at the molecular level via multi-omics analyses at concentrations below those at which significant cytotoxicity was evident to avoid detection of responses secondary to toxicity. Inhalation experiments used aerosol concentrations chosen to produce similar levels of particle deposition on the airway surface as were delivered in vitro. These were markedly higher than environmental concentrations. No clinical signs of toxicity were seen nor effects on BALF cell counts or LDH levels. There were also no significant changes in transcriptomic or metabolomic responses in lung or BEAS-2B cells to suggest adverse effects