Acrylonitrile Induction of Rodent Neoplasia: Potential Mechanism of Action and Relevance to Humans

Acrylonitrile, an industrial chemical, is a multisite carcinogen in rats and mice, producing tumors in four tissues with barrier function, that is, brain, forestomach, Zymbal’s gland, and Harderian gland. To assess mechanism(s) of action (MoA) for induction of neoplasia and to evaluate whether the findings in rodents are indicative of human hazard, data on the potential key effects produced by acrylonitrile in the four rodent target tissues of carcinogenicity were evaluated. A notable finding was depletion of glutathione in various organs, including two target tissues, the brain, and forestomach, suggesting that this effect could be a critical initiating event. An additional combination of oxidative DNA damage and cytotoxic effects of acrylonitrile and its metabolites, cyanide, and 2-cyanoethylene oxide, could initiate pro-inflammatory signaling and sustained cell and tissue injury, leading to compensatory cell proliferation and neoplastic development. The in vivo DNA-binding and genotoxicity of acrylonitrile has been studied in several target tissues with no compelling positive results. Thus, while some mutagenic effects were reported in acrylonitrile-exposed rodents, data to determine whether this mutagenicity stems from direct DNA reactivity of acrylonitrile are insufficient. Accordingly, the induction of tumors in rodents is consistent primarily with a non-genotoxic MoA, although a contribution from weak mutagenicity cannot be ruled out. Mechanistic data to support conclusions regarding human hazard from acrylonitrile exposure is weak. Comparison of metabolism of acrylonitrile between rodents and humans provide little support for human hazard. Three of the tissues affected in bioassays (forestomach, Zymbal’s gland, and Harderian gland) are present only in rodents, while the brain is anatomically different between rodents and humans, diminishing relevance of tumor induction in these tissues to human hazard. Extensive epidemiological data has not revealed causation of human cancer by acrylonitrile

PET/CT Imaging of c-Myc Transgenic Mice Identifies the Genotoxic N-Nitroso-Diethylamine as Carcinogen in a Short-Term Cancer Bioassay

Background: More than 100,000 chemicals are in use but have not been tested for their safety. To overcome limitations in the cancer bioassay several alternative testing strategies are explored. The inability to monitor non-invasively onset and progression of disease limits, however, the value of current testing strategies. Here, we report the application of in vivo imaging to a c-Myc transgenic mouse model of liver cancer for the development of a short-term cancer bioassay. Methodology/Principal Findings: mCT and 18 F-FDG mPET were used to detect and quantify tumor lesions after treatment with the genotoxic carcinogen NDEA, the tumor promoting agent BHT or the hepatotoxin paracetamol. Tumor growth was investigated between the ages of 4 to 8.5 months and contrast-enhanced mCT imaging detected liver lesions as well as metastatic spread with high sensitivity and accuracy as confirmed by histopathology. Significant differences in the onset of tumor growth, tumor load and glucose metabolism were observed when the NDEA treatment group was compared with any of the other treatment groups. NDEA treatment of c-Myc transgenic mice significantly accelerated tumor growth and caused metastatic spread of HCC in to lung but this treatment also induced primary lung cancer growth. In contrast, BHT and paracetamol did not promote hepatocarcinogenesis. Conclusions/Significance: The present study evidences the accuracy of in vivo imaging in defining tumor growth, tumor load, lesion number and metastatic spread. Consequently, the application of in vivo imaging techniques to transgeni

Optimisation of Over-Expression in E. coli and Biophysical Characterisation of Human Membrane Protein Synaptogyrin 1

Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs

C4 nephritic factor in patients with immune-complex-mediated membranoproliferative glomerulonephritis and C3-glomerulopathy

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Mechanisms of DNA-Reactive and Epigenetic Chemical Carcinogens: Applications to Carcinogenicity Testing and Risk Assessment

Chemicals with carcinogenic activity in either animals or humans produce increases in neoplasia through diverse mechanisms. One mechanism is reaction with nuclear DNA. Other mechanisms consist of epigenetic effects involving either modifications of regulatory macromolecules or perturbation of cellular regulatory processes. The basis for distinguishing between carcinogens that have either DNA reactivity or an epigenetic activity as their primary mechanism of action is detailed in this review. In addition, important applications of information on these mechanisms of action to carcinogenicity testing and human risk assessment are discussed

Assessment of DNA Binding and Oxidative DNA Damage by Acrylonitrile in Two Rat Target Tissues of Carcinogenicity: Implications for the Mechanism of Action

Exposure to acrylonitrile induces formation of tumors at multiple sites in rats, with females being more sensitive. The present study assessed possible mechanisms of acrylonitrile tumorigenicity, covalent DNA binding, DNA breakage, and oxidative DNA damage, in two target tissues, the brain and Zymbal\u27s glands, of sensitive female Fischer (F344) and Sprague-Dawley (SD) rats. One group received acrylonitrile in drinking water at 100 ppm for 28 days. Two other groups were administered either acrylonitrile in drinking water at 100 ppm or drinking water alone for 27 days, followed by a single oral gavage dose of 11 mg/kg b

Testing of Flavor and Fragrance Materials in Turkey Egg Genotoxicity Assay (TEGA) and Comparison of the Results in Ovo, in Vitro and in Vivo

The aim of the study was to investigate the use of the Turkey Egg Genotoxicity Assay (TEGA) as a non-animal alternative to in vivo follow-up studies. The genotoxic potential of 19 diverse flavor and fragrance (F&F) agents was assessed in the TEGA using 32P-nucleotide postlabeling (NPL) and comet assays to detect hepatic DNA adducts and strand breaks, respectively. The compounds were selected for testing based on their chemical structures and results in the GADD45a-Gluc ‘BlueScreen HC’ (BSHC) genotoxicity and the Ames mutagenicity assays. Two F&F materials (BDHCA and MEU) produced DNA adducts, and four materials (BDHCA, BMHCA, HEX and MAL) produced DNA strand breaks in the NPL and comet assays, respectively. Fourteen other tested compounds were negative in both NPL and comet assays. Of these14 materials, only 3 are negative in vitro, yet the majority of these materials are not shown to be genotoxic. Based on reports of oxidative DNA damage for two of the materials (MAL & HDMF), these compounds were tested in the enhanced comet assay using repair enzymes to identify oxidative DNA damage. In the enhanced comet assay positive comet findings for MAL were not confirmed, and only equivocal evidence of oxidative damage was found. Meanwhile, HDMF, produced positive results in the enhanced comet assay with formamidopyrimidine DNA glycosylase (FPG) enzyme digestion. In the test set, the TEGA had better specificity and sensitivity for in vivo data compared to in vitro test results. The observed non-concordance with in vitro data could be due to differences in the endpoints measured by the TEGA compared to those in vitro or due to the acknowledged higher rate of false positive results in in vitro systems. The TEGA showed high accuracy compared to standard in vivo genotoxicity assays for the prediction of genotoxic carcinogens as positive and the non-(genotoxic) carcinogens as negative. This findings are of high importance, since the end result of standard genotoxicity assays is typically the prediction of cancer risk