Comparative Testing Report on the Detection and Quantification of GM events in compound feedstuff - Comparative testing round: ILC-EURL-GMFF-CT-02/12

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF (Regulation (EC) No 1829/2003(1)) that is also mandated as EU-RL by Regulation (EC) 882/2006(2) organised a comparative testing round for National Reference Laboratories (NRLs) nominated under Regulation (EC) No 882/2004(2). Participation was open and free of charge for NRLs nominated under Regulation (EC) No 1981/2006(3), for all members of the European Network of GMO Laboratories (ENGL), and for official control laboratories from the EU and third countries. The EU-RL is accredited under ISO 17043 (‘General requirements for proficiency testing’(4)) and this comparative testing round met this ISO Standard(4). The test items were produced in-house from dried leaves of MON 88017 (MON-88Ø17-3) and seeds of soybean event 40-3-2 (MON-Ø4Ø32-6) provided by Monsanto, by spiking a compound feedstuff provided by a Belgian NRL. Participants were required to screen two test items (feedstuff Levels 1 and 2) for the presence of maize events Maize MON 88017, MON 89034 and soybean events 356043, 40-3-2 and MON 89788. Any event detected then had to be quantified. Participants could report the results in mass/mass % or copy/copy % and the EU-RL calculated the robust means (R) of Level 1 and 2 test items accordingly. In addition, "target" values () were assigned by the EU-RL on the basis of its homogeneity study(8) for m/m % data. These values were included in the uncertainty budget. The target standard deviation for CT was fixed by the Advisory Board for Comparative Testing at 0.15 (log10 value) for soybean event 40-3-2 and at 0.20 for maize event MON 88017 based on experience from previous CT rounds. This target standard deviation was used to derive z-scores for the participants’ results. Ninety laboratories from 43 countries registered for this CT round of which 82 from 35 countries returned at least qualitative test results. The results of the qualitative evaluation of the GM content indicated that most of the laboratories correctly detected soybean event 40-3-2 and maize event MON 88017 thus resulting in a very good performance overall. The results of the quantitative evaluation of GM content were found to be satisfactory overall for both events, with 94% of the laboratories submitting results in mass/mass % with a z-score, estimated on the basis of the robust mean, lying within the range of -2 to +2. This percentage decreased to 79% for results expressed in copy/copy %. When asked to repeat experimental work, most of the underperforming laboratories obtained satisfactory results. Only ~57% of participants provided information on measurement uncertainty in a complete and consistent manner, it is apparent therefore that despite the overall satisfactory outcome of this CT round, there is still improvement needed in this crucial area.JRC.I.3-Molecular Biology and Genomic

Comparative Testing Report on the Detection and Quantification of GM Events in Rice Noodles

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF), accredited under ISO/IEC 17043, organised a comparative testing (CT) round for National Reference Laboratories (NRLs) nominated under Regulation (EC) No 882/2004, with voluntary participation of other official control laboratories. The test items consisted of rice noodles and commercial soybeans spiked with ground powder of soybean event DP-356043-5 in two different concentrations (Level 1 and 2). Participants were required to perform species identification and test for the presence of any GM event in the two test items. Any event detected then had to be quantified. Participants could report the results in mass/mass % or copy/copy % and the EU-RL GMFF calculated the robust means (R) for Level 1 and 2 test items accordingly. The target standard deviation for CT was fixed by the Advisory Board for Comparative Testing at 0.2 for the event, based on the experience of previous CT rounds. The robust means and target standard deviation were used to derive z-scores for the participants’ results. Eighty-eight laboratories from 42 countries registered for this CT round, of which 85 laboratories from 41 countries returned at least qualitative test results. When performing species identification, almost all laboratories correctly identified soybean and rice in both test items, and a few laboratories also detected maize and/or oilseed rape. In total 71 laboratories reported the presence of GM material in the test items, but 14 laboratories failed in this task. All of the 71 laboratories, except eight, correctly identified soybean event DP-356043-5 in the test items. Results of the quantitative evaluation of the GM content were satisfactory for both measurement units, with only two NRLs appointed under Regulation (EC) No 1981/2006 (one measuring in m/m % and one in cp/cp %) obtaining unsatisfactory z-scores (|z| ≥ 2.0) for both test items. Despite the overall satisfactory outcome of this CT round, only 58 % of participants provided information on measurement uncertainty in a complete and consistent manner, and further improvement in this crucial area is needed.JRC.I.3-Molecular Biology and Genomic

Genomic and Phenotypic Alterations of the Neuronal-Like Cells Derived from Human Embryonal Carcinoma Stem Cells (NT2) Caused by Exposure to Organophosphorus Compounds Paraoxon and Mipafox

Organophosphorus compounds (OPs) are pesticides of worldwide use due to the acute insecticidal effects mediated by the inhibition of esterases of the central nervous system (mainly acetylcholinesterase and neuropathy target esterase (NTE)). OPs need to inhibit acetylcholinesterase to be effective insecticides, but not NTE since its inhibition might cause progressive, irreversible delayed neuropathy in humans and other species. Additionally, other neurological and neurodevelopmental toxic effects with unknown targets have been reported in humans or animals chronically exposed to OPs. We used a mixed neuronal/glia culture derived from well-characterised human embryonal carcinoma stem cells (hNT2) to determine if neuropathic OP mipafox and non-neuropathic OP paraoxon are able to alter the neuronal differentiation process evaluated by gene expression studies, neuronal electrical activity measurements and neural cell morphology quantification. Exposure to paraoxon at non-cytotoxic concentrations altered the expression of different genes involved mainly in signalling pathways related to chromatin assembly and nucleosome integrity, generating cultures with a larger number of differentiated neurons-like cells and branching points than in the control. Moreover, these paraoxon-exposed neuronal-like cells displayed reduced electrical activity when compared with the control neurons as measured by Micro Electrode Array Chips. Similarly, exposure to mipafox, a known inhibitor of NTE activity, also reduced the electrical activity of hNT2 cultures differentiated into neurons-like cells, but no significant changes in cell morphology or gene expression were detected. Therefore, we conclude that paraoxon is able to strongly disturb in vitro neurodifferentiation, while the absence of morphological and transcriptional alterations did not allow us to conclude if the electrophysiological alterations detected in mipafox-exposed neurons are due to neurodevelopmental toxicity or to effects on mature neurons.JRC.I.2-Public Health Policy Suppor

Changes in Caco-2 cells transcriptome profiles upon exposure to gold nanoparticles

Abstract Higher efficacy and safety of nano gold therapeutics require examination of cellular responses to gold nanoparticles (AuNPs). In this work we compared cellular uptake, cytotoxicity and RNA expression patterns induced in Caco-2 cells exposed to AuNP (5 and 30 nm). Cellular internalization was dose and time-dependent for both AuNPs. The toxicity was observed by colony forming efficiency (CFE) and not by Trypan blue assay, and exclusively for 5 nm AuNPs, starting at the concentration of 200 μM (24 and 72 h of exposure). The most pronounced changes in gene expression (Agilent microarrays) were detected at 72 h (300 μM) of exposure to AuNPs (5 nm). The biological processes affected by smaller AuNPs were: RNA/zinc ion/transition metal ion binding (decreased), cadmium/copper ion binding and glutathione metabolism (increased). Some Nrf2 responsive genes (several metallothioneins, HMOX, G6PD, OSGIN1 and GPX2) were highly up regulated. Members of the selenoproteins were also differentially expressed. Our findings indicate that exposure to high concentration of AuNPs (5 nm) induces metal exposure, oxidative stress signaling pathways, and might influence selenium homeostasis. Some of detected cellular responses might be explored as potential enhancers of anti-cancer properties of AuNPs based nanomedicines

Sex as a major determinant of gene expression in tissues of mice exposed to arsenate.

Inorganic arsenic, frequently found as contaminant of ground water used for drinking purposes in many areas of the world, is a well-known potent human toxicant and carcinogen. Chronic exposure to inorganic arsenic has been associated with cancer of skin, lung, bladder and kidney and, probably, liver. The mechanism of arsenic action in vivo is poorly understood, in particular in relation to dose, type of tissue and gender. To elucidate tissue- and gender dependent biological responses in the genome of mice, we have used cDNA macroarrays for investigation on the expression of 1185 cancer-related genes in mice after exposure to arsenate in drinking water. Continuous exposures of mice to arsenate in drinking water modulate the gene expression in tissues. Interestingly, there were remarkable sex differences: male and female mice show completely different changes in the expression of cancer-related genes. The main gene functional families modulated, were covering a wide range of biochemical and physiological regulations, like cell cycle modulation, cell adhesion, apoptosis, xenobiotic metabolism, DNA repair, protein turnover and proto-oncogens. This result demonstrates important gene-environmental interactions: the molecular mechanisms triggered by arsenic levels frequently experienced following exposure via drinking water, are totally different in males and females. The results obtained using cancer-related genes will be compared with the profiles of over 30.000 genes using the Applied Biosystems expression Array System, to clarify the sex-specific gene pathways

The arsenic in mice as experimental model for risk modifiers.

Studies on the relevance of host factors in modulating the physiological responses following chronic exposure to xenobiotics were carried out according to a \u201cToxicogenomic Model on Arsenic in Mice\u201d developed at thte JRC. This model is focused on chronic exposure to arsenate given alone or in combination with other xenobiotics, to assess potential \u201ccocktail effects\u201d and related cumulative risks. DNA-macroarrays technology is applied to evaluate physiological responses at transcriptional level and assessing possible biochemical responses. A cluster of 1200 cancer genes was used for screening purposes, while quantitative PCR on selected genes applied for validation. The exposure varied from in-utero and post-lactation up to adult age (4 months), the chemical forms (arsenate and dimethylarsenate) and doses from 0.1 up to 10 mg As/L in drinking water. Comparison between acute single doses and chronic exposure was also performed. Chronic exposure to arsenate and atrazine in drinking water was selected as an example of multiple chronic exposure. The liver, kidney, lung, bone marrow, adrenals, uterus, and testis were the tissues considered. In the tissues of mice chronically exposed to arsenate, the modulation of gene expression was not only depending on the levels, types and length of exposure, while differently regulated also by the sex, age and diet. The main gene functional families modulated were covering a wide range of biochemical and physiological regulations, like cell cycle modulation, cell adhesion, apoptosis, xenobiotic metabolism, DNA repair, protein turnover, and proto-oncogenes. The patterns of gene expression were strongly influenced by co-exposure to other xenobiotics like atrazine and naphthalene, particularly for genes involved in the metabolism and in neuroendocrine regulation. These effects varied according to the tissue considered, supporting the needs for coherent and specifically designed studies to assess relevant biomarkers of long-term exposure to low levels of xenobiotics and their mixtures

Dietary proteins modulates the gene expression in mice chronically exposed to arsenate.

In the frame of a project on the assessment of risk modifying factors modulating the health effects of environmental chemicals we are developing a toxicogenomic approach using an \u201carsenic in mice\u201d experimental model, considering multistressors exposure, genetics, age, levels and length of exposure, etc. In the present study, we used cDNA Macroarrays to investigate the effects of low protein intake on the expression of 1185 cancer-related genes in the liver of male and female mice transplacentary exposed to different levels of arsenate in drinking water during gestation and developmental age. The results of this study support the relevance of dietary factors in modulating the physiological responses in gene expression following chronic exposure to xenobiotics. In mice chronically exposed to arsenate in drinking water, the modulation of gene expression in different tissues was not only depending on the levels of the xenobiotic under investigation, but mainly regulated by the content of proteins in diet

Clonogenicity and gene expression modulation in the bone marrow of mice chronically exposed to arsenic and atrazine.

The clonogenicity of myeloid progenitors (CFU-GM) and the modulation of gene expression of 1185 cancer-related genes by DNA-macroarrays in bone marrow were used to investigate in male and female mice the combined effects of continuous exposure to arsenate and atrazine in drinking water. In male mice, the exposure to arsenate or to atrazine alone and the combined exposure did not change the clonogenicity of the progenitors. In females the percentage of CFU-GM decreased significantly after atrazine exposure, did not change with arsenic treatment, but dramatically increased after the combined exposure to the two chemicals. Results from microarrays indicate that atrazine alone didn\u2019t stimulate the expression of any of the cancer genes analyzed in both male and female. Arsenic induced gene expression modulation only in female and had no effects on male. Major significant changes on the gene expression in bone marrow cells resulted following the co-exposure to arsenic and atrazine in both male and female. These results indicate that co-exposure of mice to atrazine and arsenate induces significant effects at the level of transcriptional activation of genes in bone marrow cells, as well as stimulating the myeloid progenitors to proliferate, particularly when co-administered in drinking water to female mice

Combined in-utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates the gene expression and clonogenicity of myeloid progenitors in bone marrow.

Increasing evidence proves that human fetuses are exposed to multiple risk factors and major concerns have been expressed towards exposure to potential endocrine modulating chemicals at early stage of life and during growth. Understanding that exposures occur as mixture of chemicals and that they converge on other inherent and environmental risk-modulating factors, there is a need to develop experimental models to assess the effects of exposure to multiple chemicals during different stage of life. In the present study, the clonogenicity of myeloid progenitors (CFU-GM) and the modulation of gene expression of 1197 cancer-related genes (DNA macroarrays) in bone marrow were used to investigate in male and female young mice the combined effects of continuous exposure to arsenate and atrazine in drinking water. Female adult mice were treated with arsenate in drinking water (1 mg As/L) for 10 days before mating and during the gestation. Offspring were randomly put into separate groups of males and females. One group of arsenic exposed offspring were exposed for 4 months to atrazine (1mg Atr/L) and arsenate (1 mg As/L) in drinking water (As+Atr). One group of each of arsenic unexposed offspring were exposed for 4 months to atrazine (1mg Atr/L) in drinking water (Atr). Additional arsenate (1 mg As/L) was given to one group of arsenic exposed offspring (As). Control mice without any treatment were also analysed (Ctrl). In male mice the exposure to arsenate or to atrazine alone did not result in significant changes on the gene expression in bone marrow cells, whereas, co-exposure to arsenic and atrazine (As+Atr) resulted in a significant up-modulation of gene expression. The percentage of CFU-GM weakly decreased after exposure to individual compounds, while the co-exposure did not change the clonogenicity of the progenitors. In female mice, the co-exposure to both chemicals resulted in a drastic up-modulation of gene expression, while in these cells the single treatments showed a up-modulation of few genes as well. The percentage of CFU-GM decreased significantly after atrazine exposure, did not change with arsenic treatment, but dramatically increased after the combined administration. These results indicate that in-utero and juvenile co-exposure of mice to atrazine and arsenate induce significant effects at the level of transcriptional activation of genes in bone marrow cells, as well as stimulating the myeloid progenitors to proliferate, particularly when co-administered in drinking water to female mice

Toxicogenomic Analysis on Urinary Bladder of Mice Chronically Exposed to Sodium Arsenate and Dimethylarsinic Acid in Drinking Water

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra