Charge of clustered microparticles measured in spatial plasma afterglows follows the smallest enclosing sphere model.

The plasma-induced charge of non-spherical microparticles is a crucial parameter in complex plasma physics, aerosol science and astrophysics. Yet, the literature describes this charge by two competing models, neither of which has been experimentally verified or refuted. Here we offer experimental proof that the charge on a two-particle cluster (doublet) in the spatial afterglow of a low-pressure plasma equals the charge that would be obtained by the smallest enclosing sphere and that it should therefore not be based on its geometrical capacitance but rather on the capacitance of its smallest enclosing sphere. To support this conclusion, the size, mass and charge of single particles (singlets) and doublets are measured with high precision. The measured ratio between the plasma-afterglow-induced charges on doublets and singlets is compared to both models and shows perfect agreement with the predicted ratio using the capacitance of the smallest enclosing sphere, while being significantly dissimilar to the predicted ratio based on the particle's geometrical capacitance.

Development of a QSAR model to predict hepatic steatosis using freely available machine learning tools.

There are various types of hepatic steatosis of which non-alcoholic fatty liver disease, which may be caused by exposure to chemicals and environmental pollutants is the most prevalent, representing a potential major health risk. QSAR modelling has the potential to provide a rapid and cost-effective method to identify compounds which may trigger steatosis. Although models exist to predict key molecular initiating events of steatosis such as nuclear receptor binding, we are aware of no models to predict the apical effect steatosis. In this study, we describe the development of a QSAR model to predict steatosis using freely available machine learning tools. It was built using a dataset of 207 pharmaceuticals and pesticides which were identified as steatotic or non-steatotic from existing data from in vivo human and animal studies. The best performing model developed using the linear discriminant analysis module in TANAGRA, based on four chemical descriptors, had an accuracy of 70%, a sensitivity of 66% and a specificity of 74%. The expansion of the steatosis dataset to other chemical types, to enable the development of further models, would be of benefit in the identification of compounds with a range of mechanisms of action contributing to steatosis.

Predicting estrogen receptor binding of chemicals using a suite of in silico methods - Complementary approaches of (Q)SAR, molecular docking and molecular dynamics.

With the aim of obtaining reliable estimates of Estrogen Receptor (ER) binding for diverse classes of compounds, a weight of evidence approach using estimates from a suite of in silico models was assessed. The predictivity of a simple Majority Consensus of (Q)SAR models was assessed using a test set of compounds with experimental Relative Binding Affinity (RBA) data. Molecular docking was also carried out and the binding energies of these compounds to the ERα receptor were determined. For a few selected compounds, including a known full agonist and antagonist, the intrinsic activity was determined using low-mode molecular dynamics methods. Individual (Q)SAR model predictivity varied, as expected, with some models showing high sensitivity, others higher specificity. However, the Majority Consensus (Q)SAR prediction showed a high accuracy and reasonably balanced sensitivity and specificity. Molecular docking provided quantitative information on strength of binding to the ERα receptor. For the 50 highest binding affinity compounds with positive RBA experimental values, just 5 of them were predicted to be non-binders by the Majority QSAR Consensus. Furthermore, agonist-specific assay experimental values for these 5 compounds were negative, which indicates that they may be ER antagonists. We also showed different scenarios of combining (Q)SAR results with Molecular docking classification of ER binding based on cut-off values of binding energies, providing a rational combined strategy to maximize terms of toxicological interest.

Detection of the mechanism of immunotoxicity of cyclosporine A in murine in vitro and in vivo models.

Transcriptomics in combination with in vitro cell systems is a powerful approach to unravel modes of action of toxicants. An important question is to which extent the modes of action as revealed by transcriptomics depend on cell type, species and study type (in vitro or in vivo). To acquire more insight into this, we assessed the transcriptomic effects of the immunosuppressive drug cyclosporine A (CsA) upon 6 h of exposure of the mouse cytotoxic T cell line CTLL-2, the thymoma EL-4 and primary splenocytes and compared these to the effects in spleens of mice orally treated with CsA for 7 days. EL-4 and CTLL-2 cells showed the highest similarities in response. CsA affected many genes in primary splenocytes that were not affected in EL-4 or CTLL-2. Pathway analysis demonstrated that CsA upregulated the unfolded protein response, endoplasmic reticulum stress and NRF2 activation in EL-4 cells, CTLL-2 cells and primary mouse splenocytes but not in mouse spleen in vivo. As expected, CsA downregulated cell cycle and immune response in splenocytes in vitro, spleens in vivo as well as CTLL-2 in vitro. Genes up- and downregulated in human Jurkat, HepG2 and renal proximal tubular cells were similarly affected in CTLL-2, EL-4 and primary splenocytes in vitro. In conclusion, of the models tested in this study, the known mechanism of immunotoxicity of CsA is best represented in the mouse cytotoxic T cell line CTLL-2. This is likely due to the fact that this cell line is cultured in the presence of a T cell activation stimulant (IL-2) making it more suitable to detect inhibitory effects on T cell activation.

Tailored microarray platform for the detection of marine toxins.

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave clear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.

Bovine liver slices combined with an androgen transcriptional activation assay: an in-vitro model to study the metabolism and bioactivity of steroids.

Previously we described the properties of a rapid and robust yeast androgen bioassay for detection of androgenic anabolic compounds, validated it, and showed its added value for several practical applications. However, biotransformation of potent steroids into inactive metabolites, or vice versa, is not included in this screening assay. Within this context, animal-friendly in-vitro cellular systems resembling species-specific metabolism can be of value. We therefore investigated the metabolic capacity of precision-cut slices of bovine liver using 17beta-testosterone (T) as a model compound, because this is an established standard compound for assessing the metabolic capacity of such cellular systems. However, this is the first time that slice metabolism has been combined with bioactivity measurements. Moreover, this study also involves bioactivation of inactive prohormones, for example dehydroepiandrosterone (DHEA) and esters of T, and although medium extracts are normally analyzed by HPLC, here the metabolites formed were identified with more certainty by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOFMS) with accurate mass measurement. Metabolism of T resulted mainly in the formation of the less potent phase I metabolites 4-androstene-3,17-dione (4-AD), the hydroxy-T metabolites 6alpha, 6beta, 15beta, and 16alpha-OH-T, and the phase II metabolite T-glucuronide. As a consequence the overall androgenic activity, as determined by the yeast androgen bioassay, decreased. In order to address the usefulness of bovine liver slices for activation of inactive steroids, liver slices were exposed to DHEA and two esters of T. This resulted in an increase of androgenic activity, because of the formation of 4-AD and T.

Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay.

Prohormones such as dehydroepiandrosterone (DHEA) are steroid precursors that do not show hormonal activity by themselves. Abuse of these prohormones in cattle fattening is hard to prove because of strong in vivo metabolism and the difficulty to detect metabolites which are not significantly above endogenous levels. The aim of the present work was to develop an in vitro assay capable of detecting the indirect hormonal activity of prohormones that might be present in feed supplements and injection preparations. Sample extracts were incubated with a bovine liver S9 fraction in order to mimic the in vivo metabolic activation. Subsequently incubated extracts were exposed to a highly androgen-specific yeast bioassay to detect hormonal activity. Metabolic activation of DHEA, 4-androstene-3,17-dione (4-adione) and 5-androstene-3,17-diol (5-adiol) resulted in an increased androgenic activity caused by the formation of the active androgen 17beta-testosterone (17beta-T), as shown by ultra-performance liquid chromatography and time-of-flight mass spectrometry with accurate mass measurement. The developed in vitro system successfully mimics the hydroxysteroid dehydrogenase (HSD)- and cytochrome P450-mediated in vivo metabolic transitions, thus allowing assessment of both bioactivity and chemical identification without the use of animal experiments. Screening of unknown supplement samples claimed to contain DHEA resulted in successful bioactivation and positive screening results according to the androgen yeast biosensor.

Ah receptor agonist activity in frequently consumed food items.

The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health perspective. Using the dioxin receptor-chemical-activated luciferase gene expression (DR CALUX) bioassay, extracts from many food items frequently consumed in the Netherlands were screened to estimate the intake of natural AhR agonists (NAhRAs). Using the prototypical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as standard, it was estimated that the daily intake of NAhRAs might be considerably higher than the reported intake of dioxins and dioxin-like polychlorinated biphenyls. Potatoes, cruciferous vegetables, bread, hamburgers, and grapefruit juice contained most NAhRAs. Food preparation and acid treatment can show a significant effect on AhR activation. The interaction of natural and xenobiotic AhR agonists should be taken into account when performing risk-benefit analysis of both types of compounds.

Gene expression profiling in Caco-2 human colon cells exposed to TCDD, benzo[a]pyrene, and natural Ah receptor agonists from cruciferous vegetables and citrus fruits.

Cruciferous vegetables and citrus fruits are reported to possess health-beneficial properties, but also have been shown to contain natural aryl hydrocarbon receptor (AhR) agonists (NAhRAs). Binding to the AhR is widely assumed to activate the main pathway by which dioxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exert their toxicity. To establish whether or not activation of the AhR pathway by NAhRAs and dioxin-like substances results in similar cellular responses, gene expression profiles induced in Caco-2 cells were studied using microarray analysis. Cells were exposed to indolo[3,2-b]carbazole (ICZ), an acid reaction product from cruciferous vegetables, and to extracts of citrus pulp and grapefruit juice. Gene expression profiles induced by these NAhRAs were compared to those of the xenobiotic AhR agonists TCDD and benzo[a]pyrene (B[a]P). Over 20 genes were found more than 1.5 times up- or down-regulated by TCDD, and the expression of most of these genes was modulated in the same direction and to a similar extent by B[a]P and the NAhRAs. Results were confirmed by RT-PCR, and many of these genes may be involved in dioxin-related toxic effects. In conclusion, this in vitro study showed similar effects induced by NAhRAs, TCDD and B[a]P at the transcriptome level in a human intestinal cell line.

Assessment of the safety of foods derived from genetically modified (GM) crops.

This paper provides guidance on how to assess the safety of foods derived from genetically modified crops (GM crops); it summarises conclusions and recommendations of Working Group 1 of the ENTRANSFOOD project. The paper provides an approach for adapting the test strategy to the characteristics of the modified crop and the introduced trait, and assessing potential unintended effects from the genetic modification. The proposed approach to safety assessment starts with the comparison of the new GM crop with a traditional counterpart that is generally accepted as safe based on a history of human food use (the concept of substantial equivalence). This case-focused approach ensures that foods derived from GM crops that have passed this extensive test-regime are as safe and nutritious as currently consumed plant-derived foods. The approach is suitable for current and future GM crops with more complex modifications. First, the paper reviews test methods developed for the risk assessment of chemicals, including food additives and pesticides, discussing which of these methods are suitable for the assessment of recombinant proteins and whole foods. Second, the paper presents a systematic approach to combine test methods for the safety assessment of foods derived from a specific GM crop. Third, the paper provides an overview on developments in this area that may prove of use in the safety assessment of GM crops, and recommendations for research priorities. It is concluded that the combination of existing test methods provides a sound test-regime to assess the safety of GM crops. Advances in our understanding of molecular biology, biochemistry, and nutrition may in future allow further improvement of test methods that will over time render the safety assessment of foods even more effective and informative.

Functional food ingredients against colorectal cancer. An example project integrating functional genomics, nutrition and health.

Functional Food Ingredients Against Colorectal Cancer is one of the first European Union funded Research Projects at the cross-road of functional genomics [comprising transcriptomics, the measurement of the expression of all messengers RNA (mRNAs) and proteomics, the measurement of expression/state of all proteins], nutrition and human health. The goal of Functional Food Ingredients Against Colorectal Cancer is to develop a colon epithelial cell line-based screening assay for nutrients with presumed anti-colorectal carcinogenic properties. Genes involved in colon carcinogenesis are identified at the RNA and protein level, using a variety of methods (subtractive hybridisation, DNA microarray, proteomics) in combination with models for colorectal cancer development (human biopsies, rat model for colorectal carcinogenesis, colorectal cancer epithelial cell lines). Secondly, colorectal cancer epithelial cell lines are selected, in terms of their capacity to undergo gene/protein expression changes representing different phases in the colorectal carcinogenesis. Thirdly, these cell lines are used to determine the effects of nutrients with presumed anti-carcinogenic properties (e.g. resveratrol, flavonoids) on functional genomics-derived endpoints. Once validated against the effects of these nutrients in in vivo animal models and classical biomarkers for colorectal carcinogenesis, these cell line models combined with functional genomics represent useful tools to study colorectal carcinogenesis and screen for nutrients with anti-carcinogenic properties.

The application of DNA microarrays in gene expression analysis.

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

Defective MHC class II expression in an MHC class II deficiency patient is caused by a novel deletion of a splice donor site in the MHC class II transactivator gene.

MHC class II deficiency patients are mutated for transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. Four complementation groups (A-D) are defined and the gene defective in group A has been shown to encode the MHC class II transactivator (CIITA). Here, we report the molecular characterization of a new MHC class II deficiency patient, ATU. Cell fusion experiments indicated that ATU belongs to complementation group A. Subsequent mutation analysis revealed that the CIITA mRNA lacked 84 nucleotides. This deletion was the result of the absence of a splice donor site in the CIITA gene of ATU. As a result of this novel homozygous genomic deletion, ATU CIITA failed to transactivate MHC class II genes. Furthermore, this truncated CIITA of ATU did not display a dominant negative effect on CIITA-mediated transactivation of various isotypic MHC class II promoters.

Discoordinate expression of invariant chain and MHC class II genes in class II transactivator-transfected fibroblasts defective for RFX5.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific transcription factors. In the present study, we show that fibroblasts derived from a recently identified bare lymphocyte syndrome patient, SSI, were mutated for RFX5, one of the DNA-binding components of the RFX complex. Despite the lack of functional RFX5 and resulting MHC class II-deficient phenotype, transfection of exogenous class II transactivator (CIITA) in these fibroblasts can overcome this defect, resulting in the expression of HLA-DR, but not of DP, DQ, and invariant chain. The lack of invariant chain expression correlated with lack of CIITA-mediated transactivation of the invariant chain promoter in transient transfection assays in SSI fibroblast cells. Consequently, these CIITA transfectants lacked Ag-presenting functions.

Incomplete T-cell immune reconstitution in two major histocompatibility complex class II-deficiency/bare lymphocyte syndrome patients after HLA-identical sibling bone marrow transplantation.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II-deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8(+) T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II-expressing CD4(+) and in the CD8(+) T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.