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The recently OECD validated H295R steroidogenesis assayprovidesan in vitroalternative to evaluate the potentialinterference of exogenous compounds with endogenous steroid hormonesynthesis. Currently, this assay is used for a simple negative-positivescreening of compounds using testosterone and estradiol levels asend points, measured with specific enzyme immunoassays (EIAs) or targetedliquid chromatography (LC) and gas chromatography (GC)–massspectrometry (MS) methods. However, recent developments in LC-MS andbioinformatics allow for more comprehensive approaches to evaluatechanges in steroid profiles. In the current work, the H295R cell modelwas combined with a metabolomics approach to monitor changes in metaboliteprofiles in both a targeted and untargeted way. H295R cells were exposedfor 48 h to model compounds, i.e., forskolin, abiraterone, prochloraz,ketoconazole, trilostane, formestane, aminoglutethimide, fadrozole,etomidate, and metyrapone, known to affect steroidogenesis. Afterexposure, the levels of 9 natural steroids were determined by a quantitativetargeted GC-MS/MS method and compared to a metabolomics method usingUltra Performance Liquid Chromatography–Time-of-Flight–MassSpectrometry (UPLC-ToF-MS). Like the EIAs, both methods were suitedfor negative-positive screening, but the MS methods also generatedspecific fingerprints, allowing chemical class prediction of the compoundunder investigation. Although the targeted GC-MS/MS was more sensitive,which was an advantage regarding analysis of the estrogens 17β-estradioland estrone, the untargeted UPLC-ToF-MS was able to evaluate effectson the synthesis of the corticosteroids. Moreover, untargeted comparisonof the aligned chemical profiles allowed identification of all m/z-values that are differential betweenexposed and nonexposed H295R cells. In conclusion, application ofa comprehensive metabolite profiling methodology not only providesa tool to screen compounds for steroidogenic modulating properties,but also allows chemical class prediction. As such, steroid profilingmethodologies in conjunction with the H295R assay can contribute tothe prioritization of chemicals for additional safety testing. [ABSTRACT FROM AUTHOR]

Background: Within the European Union the use of growth promoting agents in animal production is prohibited. Illegal use of natural prohormones like dehydroepiandrosterone (DHEA) is hard to prove since prohormones are strongly metabolized in vivo. In the present study, we investigated the feasibility of a novel effect-based approach for monitoring abuse of DHEA. Changes in gene expression profiles were studied in livers of bull calves treated orally (PO) or intramuscularly (IM) with 1000 mg DHEA versus two control groups, using bovine 44K DNA microarrays. In contrast to controlled genomics studies, this work involved bovines purchased at the local market on three different occasions with ages ranging from 6 to 14 months, thereby reflecting the real life inter-animal variability due to differences in age, individual physiology, season and diet. Results: As determined by principal component analysis (PCA), large differences in liver gene expression profiles were observed between treated and control animals as well as between the two control groups. When comparing the gene expression profiles of PO and IM treated animals to that of all control animals, the number of significantly regulated genes (p-value <0.05 and a fold change >1.5) was 23 and 37 respectively. For IM and PO treated calves, gene sets were generated of genes that were significantly regulated compared to one control group and validated versus the other control group using Gene Set Enrichment Analysis (GSEA). This cross validation, showed that 6 out of the 8 gene sets were significantly enriched in DHEA treated animals when compared to an 'independent' control group. Conclusions: This study showed that identification and application of genomic biomarkers for screening of (pro) hormone abuse in livestock production is substantially hampered by biological variation. On the other hand, it is demonstrated that comparison of pre-defined gene sets versus the whole genome expression profile of an animal allows to distinguish DHEA treatment effects from variations in gene expression due to inherent biological variation. Therefore, DNA-microarray expression profiling together with statistical tools like GSEA represent a promising approach to screen for (pro)hormone abuse in livestock production. However, a better insight in the genomic variability of the control population is a prerequisite in order to define growth promoter specific gene sets that can be used as robust biomarkers in daily practice. [ABSTRACT FROM AUTHOR]

Background: Novel proteins entering the food chain, for example by genetic modification of plants, have to be tested for allergenicity. Allermatch™ http://allermatch.org is a webtool for the efficient and standardized prediction of potential allergenicity of proteins and peptides according to the current recommendations of the FAO/WHO Expert Consultation, as outlined in the Codex alimentarius. Description: A query amino acid sequence is compared with all known allergenic proteins retrieved from the protein databases using a sliding window approach. This identifies stretches of 80 amino acids with more than 35% similarity or small identical stretches of at least six amino acids. The outcome of the analysis is presented in a concise format. The predictive performance of the FAO/WHO criteria is evaluated by screening sets of allergens and non-allergens against the Allermatch databases. Besides correct predictions, both methods are shown to generate false positive and false negative hits and the outcomes should therefore be combined with other methods of allergenicity assessment, as advised by the FAO/WHO. Conclusions: Allermatch™ provides an accessible, efficient, and useful webtool for analysis of potential allergenicity of proteins introduced in genetically modified food prior to market release that complies with current FAO/WHO guidelines. [ABSTRACT FROM AUTHOR]

Background: Transgenic proteins expressed by genetically modified food crops are evaluated for their potential allergenic properties prior to marketing, among others by identification of short identical amino acid sequences that occur both in the transgenic protein and allergenic proteins. A strategy is proposed, in which the positive outcomes of the sequence comparison with a minimal length of six amino acids are further screened for the presence of potential linear IgE-epitopes. This double track approach involves the use of literature data on IgE-epitopes and an antigenicity prediction algorithm. Results: Thirty-three transgenic proteins have been screened for identities of at least six contiguous amino acids shared with allergenic proteins. Twenty-two transgenic proteins showed positive results of six- or seven-contiguous amino acids length. Only a limited number of identical stretches shared by transgenic proteins (papaya ringspot virus coat protein, acetolactate synthase GH50, and glyphosate oxidoreductase) and allergenic proteins could be identified as (part of) potential linear epitopes. Conclusion: Many transgenic proteins have identical stretches of six or seven amino acids in common with allergenic proteins. Most identical stretches are likely to be false positives. As shown in this study, identical stretches can be further screened for relevance by comparison with linear IgE-binding epitopes described in literature. In the absence of literature data on epitopes, antigenicity prediction by computer aids to select potential antibody binding sites that will need verification of IgE binding by sera binding tests. Finally, the positive outcomes of this approach warrant further clinical testing for potential allergenicity. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pyrrolizidine alkaloids (PAs) are secondary metabolites from plants that have been found in substantial amounts in herbal supplements, infusions and teas. Several PAs cause cancer in animal bioassays, mediated via a genotoxic mode of action, but for the majority of the PAs, carcinogenicity data are lacking. It is assumed in the risk assessment that all PAs have the same potency as riddelliine, which is considered to be one of the most potent carcinogenic PAs in rats. This may overestimate the risks, since many PAs are expected to have lower potencies. In this study we determined the concentration-dependent genotoxicity of 37 PAs representing different chemical classes using the γH2AX in cell western assay in HepaRG human liver cells. Based on these in vitro data, PAs were grouped into different potency classes. The group with the highest potency consists particularly of open diester PAs and cyclic diester PAs (including riddelliine). The group of the least potent or non-active PAs includes the monoester PAs, non-esterified necine bases, PA N-oxides, and the unsaturated PA trachelanthamine. This study reveals differences in in vitro genotoxic potencies of PAs, supporting that the assumption that all PAs have a similar potency as riddelliine is rather conservative., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Inclusion of alternative methods that replace, reduce, or refine (3R) animal testing within regulatory safety evaluations of chemicals generally faces many hurdles. The goal of the current work is to i) collect responses from key stakeholders involved in food safety evaluations on what they consider the most relevant factors that influence the acceptance and use of 3R methods and to ii) use these responses to formulate activities needed to increase the acceptance and use of 3R methods, particularly for kinetics. The stakeholders were contacted by e-mail for their opinions, asking the respondents to write down three barriers and/or drivers and scoring these by distributing 5 points over the three factors. The main barriers that obtained the highest aggregated scores were i) uncertain predictability 3R methods/lack of validation, ii) insufficient guidance regulators/industry and iii) insufficient harmonization of legislation. The major driver identified was the possibility of 3R methods to provide more mechanistic information. Based on the results, recommendations are given to enhance the acceptance and application of 3R toxicokinetic methods in food safety evaluations. These include steering of regulatory data requirements as well as creating (funding) opportunities for development and validation of alternative methods for kinetics and development of guidances., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

It has previously been demonstrated by others that acetone extracts of Senecio jacobaea (syn. Jacobaea vulgaris, common or tansy ragwort) test positive in the Salmonella/microsome mutagenicity test (Ames test). Pyrrolizidine alkaloids (PAs) are thought to be responsible for these mutagenic effects. However, it was also observed that the major PA present in common ragwort, jacobine, produced a negative response (with and without the addition of rat liver S9) in Salmonella test strains TA98, TA100, TA1535 and TA1537. To investigate which compounds in the plant extracts were responsible for the positive outcome, the present study investigated the contents and mutagenic effects of methanol and acetone extracts prepared from dried ground S. jacobaea and Senecio inaequidens (narrow-leafed ragwort). Subsequently, a fractionation approach was set up in combination with LC-MS/MS analysis of the fractions. It was shown that the positive Ames test outcomes of S. jacobaea extracts are unlikely to be caused by PAs, but rather by the flavonoid quercetin. This study also demonstrates the importance of identifying compounds responsible for positive test results in bioassays., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity., (Copyright © 2014 John Wiley & Sons, Ltd.)

In the present study, a previously established integrated testing strategy (ITS) for in vitro estrogenicity testing was extended with additional in vitro assays in order to broaden its sensitivity to different modes of action resulting in apparent estrogenicity, i.e., other than estrogen receptor (ER) binding. To this end, an extra set of 10 estrogenic compounds with modes of action in part different from ER binding, were tested in the previously defined ITS, consisting of a yeast estrogen reporter gene assay, an U2OS ERα CALUX reporter gene assay and a cell-free coregulator binding assay. Two androgen reporter gene assays and the enhanced H295R steroidogenesis assay were added to that previous defined ITS. These assays had added value, as several estrogenic model compounds also elicited clear and potent antiandrogenic properties and in addition also showed effects on steroidogenesis that might potentiate their apparent estrogenic effects in vivo. Adding these assays, examining mechanisms of action for estrogenicity apart from ERα binding, gives a more complete and comprehensive assessment of the ability of test compounds to interfere with endocrine signaling. It was concluded that the extended ITS will go beyond in vivo estrogenicity testing by the uterotrophic assay, thereby contributing to the 3R-principles., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Previously, we studied the effects of deoxynivalenol (DON) and tributyltin oxide (TBTO) on whole genome mRNA expression profiles of human T lymphocyte Jurkat cells. These studies indicated that DON induces ribotoxic stress and both DON and TBTO induced ER stress which resulted into T-cell activation and apoptosis. The first goal of the present study was to provide final proof for these mode of actions by comparing the effects of 6 h exposure to DON and TBTO on mRNA expression to those of positive controls of ribotoxic stress (anisomycin), ER stress (thapsigargin) and T cell activation (ionomycin). Genes affected by anisomycin and the majority of genes affected by thapsigargin were affected in the same direction by DON and TBTO, respectively, confirming the expected modes of action. Pathway analysis further sustained that DON induces ribotoxic stress and both DON and TBTO induce unfolded protein response (UPR), ER stress, T cell activation and apoptosis. The second goal was to assess whether DON and/or TBTO affect other pathways above those detected before. TBTO induced groups of genes that are involved in DNA packaging and heat shock response that were not affected by thapsigargin. DON did not affect other genes than anisomycin indicating the effect of DON to be restricted to ribotoxic stress. This study also demonstrates that comparative gene expression analysis is a very promising tool for the identification of modes of action of immunotoxic compounds., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Precision cut liver slices (PCLSs) are widely used as a model to study hepatotoxicity. For culturing of PCLS diverse protocols are used which could affect slices viability and results. We aimed to identify the most optimal culture protocol for mouse PCLS. Slices were cultured for 24h under different concentrations of serum, glucose, insulin, and oxygen. Thereafter, slices viability was assessed by biochemical methods. Transcriptome analysis was performed to identify changes introduced by culture at different oxygen concentrations (20%, 40%, 60%, and 80% of oxygen). Medium composition did not affect the slices viability. Although metabolic competence was unaffected by oxygen concentrations, culturing at 80% of oxygen yielded slices with the best biochemical characteristics. The comparison of uncultured vs. cultured slices revealed 2524 genes to be differentially expressed. Genes involved in drug metabolism, peroxisomal and mitochondrial functions were down-regulated while several adaptive/stress response processes were up-regulated. Moreover, 80% of oxygen was the most favorable condition with respect to maintenance of expression of genes involved in drug and energy metabolism. The outcome of this study indicates that mouse PCLS are a valuable tool in research on hepatic functions and toxicity, particularly if they are cultured under a controlled oxygen concentration of 80%., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the current study, a set of 12 reference compounds was tested in a low-density DNA microchip that contains probes for 11 different estrogen-responsive marker genes. Our results show that the seven most informative marker genes on the chip resulted in fingerprints that correctly predicted the (anti-)estrogenic activity of the model compounds except that of the negative control testosterone. Two marker genes, myeloid leukemia factor-1 interacting protein and ubiquitin-conjugating enzyme E2C, were even capable of correctly predicting the estrogenic potency of all five estrogen receptor (ER) agonists tested and correlated well with the potencies as determined in the MCF-7/BOS proliferation assay and the in vivo uterotrophic assay. In addition, it was demonstrated that the estrogenic responses of testosterone, both in the array tube assay and in the proliferation assay, were partially due to the conversion of testosterone into 17β-estradiol by aromatase but also due to formation of other estrogenic metabolites, the presence and estrogenic potency of which were confirmed by gas chromatography-tandem mass spectrometry analysis and a yeast-based reporter gene assay, respectively. It is concluded that low-density DNA microchip-based fingerprinting in MCF-7/BOS cells for estrogenicity marker genes provides a faster in vitro alternative to the current MCF-7/BOS cell proliferation assay (E-screen)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A toxicogenomics approach was applied to assess the usefulness of the mouse cytotoxic T cell line CTLL-2 for in vitro immunotoxicity testing. CTLL-2 cells were exposed for 6 h to two model immunotoxic compounds: (1) the mycotoxin deoxynivalenol (DON, 1 and 2 μM), a ribotoxic stress inducer, and (2) the organotin compound tributyltin oxide (TBTO, 100 and 200 nM), an endoplasmic reticulum (ER) stress inducer. Effects on whole-genome mRNA expression were assessed by microarray analysis. The biological interpretation of the microarray data indicated that TBTO (200 nM) induced genes involved in T cell activation, ER stress, NFκB activation and apoptosis, which agreed very well with results obtained before on TBTO exposed Jurkat cells and mouse primary thymocytes. Remarkably, DON (2 μM) downregulated genes involved in T cell activation, ER stress and apoptosis, which is opposite to results obtained before for DON-exposed Jurkat cells and mouse primary thymocytes. Furthermore, the results for DON in CTLL-2 cells are also opposite to the results obtained for TBTO in CTLL-2 cells. In agreement with the lack of induction of ER stress and apoptosis, viability assays showed that CTLL-2 cells are much more resistant to the toxicity of DON than Jurkat cells and primary thymocytes. We propose that CTLL-2 cells lack the signal transduction that induces ER stress and apoptosis in response to ribotoxic stress. Based on the results for TBTO and DON, the CTLL-2 cell line does not yield an added value for immunotoxicity compared to the human Jurkat T cell line., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Biotransformation of inactive prohormones like dehydroepiandrosterone (DHEA) can lead to the formation of potent androgens and subsequent androgenic responses in target tissues. In the present study, precision-cut bovine liver slices were used to study the effects of DHEA on the metabolite, transcript and androgenic activity level. Bovine liver slices were exposed for 6h to various concentrations of DHEA. Changes in androgenic activity of the DHEA containing cell culture media were measured using a yeast androgen bioassay and metabolites were identified using ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOFMS), while gene expression in the DHEA-treated liver slices was examined using bovine microarrays and compared with the profile as obtained with 17ß-testosterone (17ß-T). An increase in androgenic activity was observed in the bioassay upon testing of samples from incubations of DHEA with liver slices and the formation of 4-androstenedione (4-AD), 5-androstene-3ß,17ß-diol, 17ß-T, 7α-hydroxy-DHEA, 7-keto-DHEA and 17α-T could be confirmed by UPLC-TOFMS analysis. Exposure of liver slices to DHEA and the strong androgen 17ß-T resulted in the identification of significantly up- and down-regulated genes and revealed similar gene expression profiles for both compounds. The results indicate that DHEA itself is biologically not very active, but is rapidly converted by the liver slices into the more androgen active compounds 4-AD and 17ß-T. Moreover, the present data highlight the multi-functionality of bovine liver slices as an in vitro bioactivation model, allowing the assessment of androgen activity or gene expression as effect-based endpoints for prohormone exposure., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Acetaminophen is the primary cause of acute liver toxicity in Europe/USA, which led the FDA to reconsider recommendations concerning safe acetaminophen dosage/use. Unfortunately, the current tests for liver toxicity are no ideal predictive markers for liver injury, i.e. they only measure acetaminophen exposure after profound liver toxicity has already occurred. Furthermore, these tests do not provide mechanistic information. Here, 'omics techniques (global analysis of metabolomic/gene-expression responses) may provide additional insight. To better understand acetaminophen-induced responses at low doses, we evaluated the effects of (sub-)therapeutic acetaminophen doses on metabolite formation and global gene-expression changes (including, for the first time, full-genome human miRNA expression changes) in blood/urine samples from healthy human volunteers. Many known and several new acetaminophen-metabolites were detected, in particular in relation to hepatotoxicity-linked, oxidative metabolism of acetaminophen. Transcriptomic changes indicated immune-modulating effects (2g dose) and oxidative stress responses (4g dose). For the first time, effects of acetaminophen on full-genome human miRNA expression have been considered and confirmed the findings on mRNA level. 'Omics techniques outperformed clinical chemistry tests and revealed novel response pathways to acetaminophen in humans. Although no definitive conclusion about potential immunotoxic effects of acetaminophen can be drawn from this study, there are clear indications that the immune system is triggered even after intake of low doses of acetaminophen. Also, oxidative stress-related gene responses, similar to those seen after high dose acetaminophen exposure, suggest the occurrence of possible pre-toxic effects of therapeutic acetaminophen doses. Possibly, these effects are related to dose-dependent increases in levels of hepatotoxicity-related metabolites., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. In the present study four different human cell lines derived from three different female estrogen-sensitive tissues, i.e. breast (MCF-7/BOS and T47D), endometrial (ECC-1) and ovarian (BG-1) cells, were characterised by investigating their relative ERα and ERβ amounts, as the ERα/ERβ ratio is a dominant factor determining their estrogen-dependent proliferative responses. All four cell lines clearly expressed the ERα type and a very low but detectable amount of ERβ on both the mRNA and protein level, with the T47D cell line expressing the highest level of the ERβ type. Subsequently, a set of reference compounds representing different modes of estrogen action and estrogenic potency were used to investigate the proliferative response in the four cell lines, to determine which cell line most accurately predicts the effect observed in vivo. All four cell lines revealed a reasonable to good correlation with the in vivo uterotrophic effect, with the correlation being highest for the MCF-7/BOS cell line (R²=0.85). The main differences between the in vivo uterotrophic assay and the in vitro proliferation assays were observed for tamoxifen and testosterone. The proliferative response of the MCF-7/BOS cells to testosterone was partially caused by its conversion to estradiol by aromatase or via androstenedione to estrone. It is concluded that of the four cell lines tested, the best assay to include in an integrated testing strategy for replacement of the in vivo uterotrophic assay is the human MCF-7/BOS breast cancer cell line., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hepatic systems toxicology is the integrative analysis of toxicogenomic technologies, e.g., transcriptomics, proteomics, and metabolomics, in combination with traditional toxicology measures to improve the understanding of mechanisms of hepatotoxic action. Hepatic toxicology studies that have employed toxicogenomic technologies to date have already provided a proof of principle for the value of hepatic systems toxicology in hazard identification. In the present review, acetaminophen is used as a model compound to discuss the application of toxicogenomics in hepatic systems toxicology for its potential role in the risk assessment process, to progress from hazard identification towards hazard characterization. The toxicogenomics-based parallelogram is used to identify current achievements and limitations of acetaminophen toxicogenomic in vivo and in vitro studies for in vitro-to-in vivo and interspecies comparisons, with the ultimate aim to extrapolate animal studies to humans in vivo. This article provides a model for comparison of more species and more in vitro models enhancing the robustness of common toxicogenomic responses and their relevance to human risk assessment. To progress to quantitative dose-response analysis needed for hazard characterization, in hepatic systems toxicology studies, generation of toxicogenomic data of multiple doses/concentrations and time points is required. Newly developed bioinformatics tools for quantitative analysis of toxicogenomic data can aid in the elucidation of dose-responsive effects. The challenge herein is to assess which toxicogenomic responses are relevant for induction of the apical effect and whether perturbations are sufficient for the induction of downstream events, eventually causing toxicity., (© 2010 Elsevier Inc. All rights reserved.)

Selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) are compounds that activate their cognate receptor in particular target tissues without affecting other organs. Many of these compounds will find their use in therapeutic treatments. However, they also will have a high potential for misuse in veterinary practice and the sporting world. Here we demonstrate that yeast estrogen and androgen bioassays can be used to detect SERMs and SARMs, and are also useful screening tools to investigate their mode of action. Six steroidal 11beta-substituents of E2 (SERMs) and some arylpropionamide- and quinoline-based SARMs were tested. In addition, 7 compounds previously tested on AR agonism and determined as inactive in the yeast androgen bioassay, while QSAR modelling revealed strong binding to the human androgen receptor, are now shown to act as AR antagonists., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Binding and activation of the aryl hydrocarbon receptor (AhR) is thought to be an essential step in the toxicity of the environmental pollutants dioxins and dioxin-like PCBs. However, also a number of natural compounds, referred to as NAhRAs (natural Ah-receptor agonists), which are present in, for example, fruits and vegetables, can bind and activate this receptor. To study their potential effects in humans, we first investigated the effect of the prototypical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gene expression in ex vivo exposed freshly isolated human lymphocytes, and compared the resulting gene expression profile with those caused by the well-known NAhRA indolo[3,2-b]carbazole (ICZ), originating from cruciferous vegetables, and by a hexane extract of NAhRA-containing grapefruit juice (GJE). Only ICZ induced a gene expression profile similar to TCDD in the lymphocytes, and both significantly up-regulated CYP1B1 and TIPARP (TCDD-inducible poly (ADP-ribose) polymerase) mRNA. Next, we performed a human intervention study with NAhRA-containing cruciferous vegetables and grapefruit juice. The expression of the prototypical AhR-responsive genes CYP1A1, CYP1B1 and NQO1 in whole blood cells and in freshly isolated lymphocytes was not significantly affected. Also enzyme activities of CYP1A2, CYP2A6, N-acetyltransferase 2 (NAT2) and xanthine oxidase (XO), as judged by caffeine metabolites in urine, were unaffected, except for a small down-regulation of NAT2 activity by grapefruit juice. Examination of blood plasma with DR CALUX showed a 12% increased AhR agonist activity 3 and 24 h after consumption of cruciferous vegetables, but did not show a significant effect of grapefruit juice consumption. We conclude that intake of NAhRAs from food may result in minor AhR-related effects measurable in human blood and urine.

Hexabromoyclododecane (HBCD), used as flame retardant (FR) mainly in textile industry and in polystyrene foam manufacture, has been identified as a contaminant at levels comparable to other brominated FRs (BFRs). HBCD levels in biota are increasing slowly and seem to reflect the local market demand. The toxicological database of HBCD is too limited to perform at present a solid risk assessment, combining data from exposure and effect studies. In order to fill in some gaps, a 28-day HBCD repeated dose study (OECD407) was done in Wistar rats. In the present work liver tissues from these animals were used for gene expression profile analysis. Results show clear gender specificity with females having a higher number of regulated genes and therefore being more sensitive to HBCD than males. Several specific pathways were found to be affected by HBCD exposure, like PPAR-mediated regulation of lipid metabolism, triacylglycerol metabolism, cholesterol biosynthesis, and phase I and II pathways. These results were corroborated with quantitative RT-PCR analysis. Cholesterol biosynthesis and lipid metabolism were especially down-regulated in females. Genes involved in phase I and II metabolism were up-regulated predominantly in males, which could explain the observed lower HBCD hepatic disposition in male rats in this 28-day study. These sex-specific differences in gene expression profiles could also underlie sex-specific differences in toxicity (e.g. decreased thyroid hormone or increased serum cholesterol levels). To our knowledge, this is the fist study that describes the changes in rat hepatic gene profiles caused by this commonly used flame retardant.

Recently we constructed recombinant yeast cells that express the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. When exposed to 17beta-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. Relative androgenic potencies (RAP), defined as the ratio between the EC50 of 17beta-testosterone and the EC50 of the compound, were 1.7, 1.2 and 0.008 for 19-nortestosterone, tetrahydrogestrinone and 17beta-estradiol respectively. Steroids representative for other hormone receptors, like estrone, 17alpha-ethynylestradiol, and diethylstilbestrol for the estrogen receptor and corticosterone and dexamethasone for the glucocorticoid receptor, showed no agonistic response. Only compounds known to exert androgenic effects give a response. Determined RAPs were in line with results obtained from optimised QSAR model calculations and demonstrated that Saccharomyces cerevisiae showed no metabolism of test compounds and displayed no crosstalk from endogenous hormone receptors. The suitability of this bioassay to verify the outcomes of (Q)SAR models to predict the activities of different steroids was further examined by studies with steroid isomers and a number of designer steroids, confirming that the 17beta-hydroxyl group, 3-keto group and 5alpha-steroidal framework are extremely important for the activity of the androgenic steroid.

Lactic acid bacteria are claimed to have immunomodulating effects. Stimulation as well as suppression of T helper (Th)1 mediated immune responses, have been described for various strains. Experiments involving Lactobacillus casei Shirota (LcS) detected mainly enhancement of innate immune responses and promotion of Th1 mediated immune reactivity. To confirm and further investigate modulation of Th1 responses and development of autoimmune disease by LcS, the consequences of oral administration of LcS were assessed in several experiments. The effect of LcS varied between the different models. No modulation was found in the mitogen-induced cell proliferation and cytokine release assays in mesenteric lymph nodes of Wistar rats. LcS inhibited the Th1 mediated immune response in an adapted murine Local Lymph Node Assay (LLNA) in BALB/c mice, whereas experimental autoimmune encephalomyelitis (EAE) in Lewis rats was aggravated. These varying effects on Th1 responses indicate that beneficial as well as harmful effects on immune related disorders could occur after LcS consumption. Since microarray analysis is suggested to be more sensitive and predictive than functional tests, gene expression profiling was included as an alternative endpoint in the testing of immunomodulation. The detected gene expression profiles did not reflect the effects of LcS on the immune system. Microarray analysis may therefore have no more predictive value than immune function assays when investigating immunomodulation by probiotics. To gain further insight into effects of probiotics on immune function, experiments including cytokine assays and gene expression analysis combined with disease models could be useful.

Previous studies suggest that vitamin A deficiency may induce or intensify inflammatory changes in the rat gastrointestinal system. The present study was designed to compare the expression profiles of rat models of vitamin A deficiency and induced colitis. cDNA-microarray technology was used to determine the genes involved in the inflammatory processes in the two models. mRNA was extracted from colons of rats that were vitamin A deficient, vitamin A supplemented (control), or had 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, reverse-transcribed into fluorescence-labeled cDNA and hybridized onto microarrays containing a duplicate set of 1152 cDNAs, derived mainly from the colon carcinoma cell line Caco-2. Differential gene expression was detected in vitamin A deficiency and in TNBS-induced colitis vs. control. beta-Actin, translation initiation factor A4 and translation elongation factor 1, ornithine decarboxylase (ODC) and keratin 19 were markedly down-regulated, whereas spermidine/spermine N1-acetyltransferase (SSAT) and polyubiquitin (UbC) were up-regulated in both vitamin A-deficient rats and those with TNBS-induced colitis. The strong association between the differential gene expression in the two animal models, compared with the control, suggests that deficiency of vitamin A causes inflammatory changes in the rat colon that are similar to processes occurring in colitis. Further investigation is required to elucidate the importance of each of the regulated genes to the pathology of colitis and vitamin A inadequacy.