Health Considerations Regarding Horizontal Transfer of Microbial Transgenes Present in Genetically Modified Crops

The potential effects of horizontal gene transfer on human health are an important item in the safety assessment of genetically modified organisms. Horizontal gene transfer from genetically modified crops to gut microflora most likely occurs with transgenes of microbial origin. The characteristics of microbial transgenes other than antibiotic-resistance genes in market-approved genetically modified crops are reviewed. These characteristics include the microbial source, natural function, function in genetically modified crops, natural prevalence, geographical distribution, similarity to other microbial genes, known horizontal transfer activity, selective conditions and environments for horizontally transferred genes, and potential contribution to pathogenicity and virulence in humans and animals. The assessment of this set of data for each of the microbial genes reviewed does not give rise to health concerns. We recommend including the above-mentioned items into the premarket safety assessment of genetically modified crops carrying transgenes other than those reviewed in the present study

Синкретизм квантитативних конструювань (на матеріалі числівників англійської мови)

У статті осмислюється явище синкретизму на матеріалі англійських квантитативних слів, нумеральних і денумеральних словосполучень. Мають місце порівняння з димензіональною лексико. Синкрети розглядаються на матеріалі синкретсемії. Актуальність теми мотивується зростаючим інтересом учених до нової наукової парадигми – синергетики.Объектом исследования является феномен синкретизма на материале квантитативных единиц в модусах языка, речи и речевого поведения (предмет исследования). Цель исследования состоит в осмыслении упомянутой парадигмы, ее идей, процессов самоорганизации и саморазвития. В работе верифицируются гипотезы: - номинативные единицы являются билатеральными и синкретичными; - их системная организация и эволюция интегрируется триадой материала, энергии, информации; - основным методологическим механизмом является онтогносиологический подход к исследуемым референтам. Ценным вкладом в синергетику представляется дальнейшая разработка основных положений и понятий новой научной парадигмы – лингвосинергетики.The article in question deals with the English quantitative units, i.e. numerals, numeric words, on the one hand, and dimension units – words of weight and measure, on the other hand. This object is dealt with in the endozones of language, speech and speech behaviour. The aim of investigation consists in identification of the markers of systematic arrangement of the paradigm – its selforganization and evolution: - verification of hypotheses that: - bilateral and syncretic; - aspects of words go together; - incorporated subject mater, energy and information of syncretas make the triad go; - ontognosiological method is at work. Topicality of the theme is objectivized by the growing interest to the problem by scientists of this country and abroad. This is a new scientific paradigm (synergetics), let alone many a lacunar items within its domain. Valour scenes remain undiscovered yet and await their solution in terms of mayor ideas and categories. Lacunarity is indebted to the process of cognition, cognized and non-cognized phenomenas. The choise of complex ontognosiological approach is determined by the nature of referents. Missing items come into being due to the problem of nothingness. The syncretas come archaic due to losses in the vocabulary or fall out of concepts. Some words are prohibited by taboo. Some syncretas work in the speech behaviour (silence, hesitation, pauses)

Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line

Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3¿h to 0.5¿µM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF-¿B (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit¿¿¿ribotoxic stress¿¿¿ER stress¿¿¿calcium release from the ER into cytoplasm¿¿¿T-cell activation and oxidative stress¿¿¿apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell activation response by increased intracellular calcium levels

The effects of deoxynivalenol on gene expression in the murine thymus

AbstractDeoxynivalenol (DON) is a mycotoxin produced by several Fusarium species and is often detected in grains. Because of its high abundance, there has been a large interest in the effects of DON in animals and humans. DON is known to be immunosuppressive at high concentrations and immunostimulatory at low concentrations. The present study aimed to acquire insight into the modes of action of DON. For this, C57Bl6 mice were orally exposed to 5, 10, or 25mg/kg bw DON for 3, 6, or 24h and thymuses were subjected to genome-wide expression microarray analysis. Gene set enrichment analysis (GSEA) demonstrated that DON downregulated genes involved in proliferation, mitochondria, protein synthesis, and ribosomal proteins. Furthermore, GSEA showed a selective downregulation of genes highly expressed at the early precursor thymocytes stage. This indicates that early precursor thymocytes, particularly at the double-positive CD4+CD8+ stage, are more vulnerable to DON than very early or late precursor thymocytes. There was a large overlap of genes upregulated by DON with genes previously reported to be either upregulated during T cell activation or upregulated during negative selection of thymocytes that recognize “self-antigens”. This indicates that DON induces cellular events that also occur after activation of the T cell receptor, for example, release of calcium from the endoplasmatic reticulum. This T cell activation in the thymus then evokes negative selection and depletion of thymocytes, which provides a plausible explanation for the high sensitivity of the thymus for DON exposure. The expression patterns of four genes indicative for some of the processes that were affected after DON treatment were confirmed using real-time PCR. Immunocytological experiments with primary mouse thymocytes demonstrated the translocation of NFAT from the cytoplasm into the nucleus upon exposure top DON, thus providing further evidence for the involvement of T cell activation

The effects of tributyltin oxide and deoxynivalenol on the transcriptome of the mouse thymoma cell line EL-4

The main goal of this study was to assess the potential of the mouse thymoma EL-4 cell line in screening for chemical induced immunotoxicity. Therefore, EL-4 cells were exposed to two well-known immunotoxicants, organotin compound tributyltin oxide (TBTO, 0.5 and 1 mu M for 3 or 6 h) and the mycotoxin deoxynivalenol (DON, 0.25, 0.5 and 1 mu M for 3, 6 or 11 h). Previous studies in human Jurkat T cells and mouse thymus in vivo showed that the primary mode of action of TBTO is induction of endoplasmic reticulum (ER) stress, T cell activation and apoptosis. DON induces ribotoxic stress and, similarly to TBTO, induces ER stress, T cell activation and apoptosis. In the present study, the effects of TBTO and DON on EL-4 mRNA expression were assessed by whole genome microarray analysis. The microarray data were then compared to those obtained with mouse thymuses in vivo, mouse thymocytes in vitro, and CTLL-2 cells and human Jurkat cells in vitro exposed to TBTO or DON. Analysis at the level of gene sets revealed that part of the previously detected modes of action of TBTO and DON were not observed in the EL-4 cell line. In EL-4 cells, TBTO induced genes involved in calcium signalling and ER stress but did not induce genes involved in T cell activation and apoptosis. DON induced RNA related processes and ribosome biogenesis. Furthermore, DON downregulated ER stress, T cell activation and apoptosis which is opposite to the mechanism of DON observed in the mouse thymus in vivo and in Jurkat T cells in vitro. Apparently, EL-4 cells lack factors that are necessary to link ribotoxic stress to ER stress. In addition, of the lack of T cell activation response of EL-4 cells to TBTO is likely due to the fact that these cells are in a constitutively activated state already. Based on the results obtained for TBTO and DON, it can be concluded that the EL-4 cell line has limited value for immunotoxicogenomics based screening

Transcriptome-based functional classifiers for direct immunotoxicity

Current screening methods for direct immunotoxic chemicals are mainly based on general toxicity studies with rodents. The present study aimed to identify transcriptome-based functional classifiers that can eventually be exploited for the development of in vitro screening assays for direct immunotoxicity. To this end, a toxicogenomics approach was applied in which gene expression changes in human Jurkat lymphoblastic T cells were investigated in response to a wide range of compounds, including direct immunotoxicants, immunosuppressive drugs, and non-immunotoxic control chemicals. On the basis of DNA microarray data previously obtained by the exposure of Jurkat cells to 31 test compounds (Shao et al. in Toxicol Sci 135(2):328-346, 2013), we identified a set of 93 genes, of which 80 were significantly regulated (|numerical ratio| a parts per thousand yen1.62) by at least three compounds and the other 13 genes were significantly regulated by either one single compound or compound class. A total of 28 most differentially regulated genes were selected for qRT-PCR verification using a training set of 44 compounds consisting of the above-mentioned 31 compounds (23 immunotoxic and 8 non-immunotoxic) and 13 additional immunotoxicants. Good correlation between the results of microarray and qRT-PCR (Pearson's correlation, R a parts per thousand yen 0.69) was found for 27 out of the 28 genes. Redundancy analysis of these 27 potential classifiers led to a final set of 25 genes. To assess the performance of these genes, Jurkat cells were exposed to 20 additional compounds (external verification set) followed by qRT-PCR. The classifier set of 25 genes gave a good performance in the external verification: accuracy 85 %, true positive rate (sensitivity) 88 %, and true negative rate (specificity) 67 %. Furthermore, on the basis of the gene ontology annotation of the 25 classifier genes, the immunotoxicants examined in this study could be categorized into distinct functional subclasses. In conclusion, we have identified and validated classifier genes that can be used for the development of an in vitro assay for the identification and initial characterization of hazards for direct immunotoxicity of chemicals and drugs. This assay promises to complement animal-free toxicity testing approaches within the field of direct immunotoxicity

Towards harmonization of test methods for in vitro hepatic clearance studies.

Non-animal methods for toxicokinetics, such as in vitro hepatic metabolic clearance studies, play an important role in chemical risk evaluations. To gain regulatory acceptance of such clearance data, the development of a test guideline for performing in vitro hepatic clearance studies is crucial. The aim of the present study was to obtain insight in the experimental conditions of clearance studies that influence obtained intrinsic clearance (CLint) values. To that end, in vitro hepatic CLint data obtained with rat or human hepatocytes and methodological aspects of the experiments, were collected from 42 different suitable studies published between 1995 and 2018. The CLint values for the majority of chemicals differed by more than one order of magnitude. We estimated the systematic effect of different experimental setups on the CLint values using a random forest regression analysis, revealing that 'hepatocyte concentration', 'species' (rat or human hepatocytes) and 'culture medium' have the largest impact. Calculating unbound CLint (CLint,u) values slightly reduced the variation for most chemicals. Given that in vivo clearance is in general underpredicted based on in vitro clearance data, a harmonized protocol is preferably based on a protocol that provides relatively high in vitro CLint values.</p