Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex ...reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs.
While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics ...provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography–mass spectrometry (LC–MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is
not
detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.
The number of candidate chemicals or drugs for registration and authorization is increasing at a fast rate and only few of the existing substances have been tested for teratogenicity to date. ...Therefore, there is high pressure on authorities to accept models like the whole embryo culture as a screening system for safety evaluation procedures. In view of this background the gradual development of the whole embryo culture into a standardized, scientifically validated tool for developmental toxicology during the last 70 years is summarized. The methodological development of the culture technique is described with the completion, improvement and refinement of the basic culture method as main intention. Special attention was paid to different culture techniques, culture media, gassing schedules, and evaluation strategies. Furthermore the importance of taking "in vitro pharmacokinetics" into consideration when a comparison of in vitro/in vivo results from embryotoxicity testing is intended, is stressed. Additionally, the demonstration of the broad spectrum of useful scientific applications when using this culture system in combination with sophisticated analytical techniques is demonstrated. Finally, an overview on different strategies for the validation of this culture system as an in vitro embryo toxicity test is provided and the officially accepted formal validation process for this application is summarized. The successful validation makes the whole embryo culture a complex in vitro embryotoxicity test with high accuracy and predictability. This robust in vitro system modelling the main phase of rodent organogenesis with a high reproducibility is valuable enough to attract special attention in related scientific fields.
Considerable attention has focused on regulation of central tryptophan hydroxylase (TPH) activity and protein expression. At the time of these earlier studies, it was thought that there was a single ...central TPH isoform. However, with the recent identification of TPH2, it becomes important to distinguish between regulatory effects on the protein expression and activity of the two isoforms. We have generated a TPH2-specific polyclonal antiserum (TPH2-6361) to study regulation of TPH2 at the protein level and to examine the distribution of TPH2 expression in rodent and human brain. TPH2 immunoreactivity (IR) was detected throughout the raphe nuclei, in lateral hypothalamic nuclei and in the pineal body of rodent and human brain. In addition, a prominent TPH2-IR fiber network was found in the human median eminence. We recently reported that glucocorticoid treatment of C57/Bl6 mice for 4 days markedly decreased TPH2 messenger RNA levels in the raphe nuclei, whereas TPH1 mRNA was unaffected. The glucocorticoid-elicited inhibition of TPH2 gene expression was blocked by co-administration of the glucocorticoid receptor antagonist mifepristone (RU-486). Using TPH2-6361, we have extended these findings to show a dose-dependent decrease in raphe TPH2 protein levels in response to 4 days of treatment with dexamethasone; this effect was blocked by co-administration of mifepristone. Moreover, the glucocorticoid-elicited inhibition of TPH2 was functionally significant: serotonin synthesis was significantly reduced in the frontal cortex of glucocorticoid-treated mice, an effect that was blocked by mifepristone co-administration. This study provides further evidence for the glucocorticoid regulation of serotonin biosynthesis via inhibition of TPH2 expression, and suggest that elevated glucocorticoid levels may be relevant to the etiology of psychiatric diseases, such as depression, where hypothalamic-pituitary-adrenal axis dysregulation has been documented.
Colony-stimulating factor-1 receptor (CSF-1R) is the major regulator of macrophage development and is associated with epithelial cancers of the breast and ovary. Immunohistochemistry analysis of ...murine prostate development demonstrated epithelial expression of CSF-1R during the protrusion of prostatic buds from the urogenital sinus, during the prepubertal and androgen-driven proliferative expansion and branching of the gland, with a decline in older animals. Models of murine prostate cancer showed CSF-1R expression in areas of carcinoma- and tumor-associated macrophages. Several human prostate cancer cell lines and primary cultures of human prostate epithelial cells had low but detectable levels of CSF-1R. Human prostatectomy samples showed low or undetectable levels of receptor in normal glands or benign prostatic hypertrophy specimens. Staining was strongest in areas of prostatic intraepithelial neoplasia or carcinoma of Gleason histological grade 3 or 4. The activated form of the receptor reactive with antibodies specific for phosphotyrosine modified peptide sequences was observed in samples of metastatic prostate cancer. Immunohistochemistry showed strong expression of CSF-1R by macrophage lineage cells, including villous macrophages and the syncytiotrophoblast layer of placenta, Kupper cells in the liver, and histiocytes infiltrating near prostate cancers. These observations correlate CSF-1R expression with changes in the growth and development of the normal and neoplastic prostate.
► Fifth percentile of 104 NOAELs of rabbit developmental tox studies is 2mg/kgbw. ► A TTC value for rabbit developmental toxicity of 4μg/kgbw/d was calculated. ► Rabbits are not overtly more ...sensitive for developmental toxicity than rats.
The Threshold Toxicological Concern (TTC) is based on the concept that reasonable assurance of safety can be given if exposure is sufficiently low. We report on the evaluation of BASF’s data for oral developmental toxicity studies in rabbits with 48 NOAEL values for maternal and developmental toxicity. The 5th percentile of the NOAEL distributions was calculated to be 5mg/kgbw/d for both maternal and developmental toxicity. From literature 56 compounds tested in rabbits were taken and combined with values from BASF’s studies. The 5th percentile value for developmental toxicity of these 104 studies (mostly active ingredients) was 2mg/kgbw/d. Thus, a TTC value of 4μg/kgbw/d was calculated using a safety factor of 500 to account for relatively small database. This value is in the same range as the TTC value for developmental toxicity in rats of 8μg/kgbw/d. The lower value may serve as guidance to determine whether further evaluation is needed or whether to rely on a TTC value for industrial chemicals or low concentration (environmental) contaminants if exposure is sufficiently low. A comparison of 30 compounds tested at BASF in both species, suggests that rabbits are not more sensitive than rats. We encourage others to publish data on rabbit developmental toxicity.
The origins of the “endocrine disrupter hypothesis” may be traced to reports on adolescent daughters born to women who had taken the highly potent synthetic estrogen, diethylstilbestrol, while ...pregnant, and who developed a rare form of vaginal cancer and adenocarcinoma. Bisphenol A (BPA) is an estrogenic chemical that is highly employed in the manufacture of a wide range of consumer products. Some observational studies have suggested that the amounts of BPA to which we are exposed could alter the reproductive organs of developing rodents. We examined the influence of BPA at low doses to address the questions of (a) whether in utero exposure affects the vagina of the offspring and (b) which mechanisms cause the toxic effects. Gravid Sprague-Dawley dams were administered either 0.1 (low dose) or 50 mg/kg per day BPA, the no observed effect level, or 0.2 mg/kg per day 17αethinyl estradiol by gavage. Striking morphological changes were observed in the vagina of postpubertal offspring leading us to examine vaginal estrogen receptor (ER) expression because BPA binds to the ERα, which is important for growth of the vaginal epithelium. We show that the full-length ERα is not expressed during estrus in the vagina of female offspring exposed to either dose of BPA when compared to the control group, whereas ERα expression does not differ from the control group during the diestrus stage. ERa downregulation seems to be responsible for the observed altered vaginal morphology.
This study examined middle school students' knowledge and beliefs about earthquake and tsunami through a multidimensional perspective of conceptual change theory. Four related constructs of ...conceptual change were examined including students' science knowledge, preparedness knowledge, ontological beliefs, and epistemic beliefs. Students responded textually and graphically to prompts in workbooks and verbally in semi-structured interviews. Four prominent themes were identified in the students' responses: (1) supergiant tsunami wave with near absolute and unavoidable catastrophic effects, (2) diminished effect of earthquakes as compared to tsunamis, (3) earthquakes and tsunamis could occur at any time in the area, and (4) strong intent to protect self and family from harm. Study results indicate that all four conceptual change constructs contributed to the students' conceptions represented by these prominent themes. Students hold parallel ontologies of the nature of earthquake and tsunami geophysical phenomena and preparedness phenomena. Earthquakes and tsunamis are emotionally charged topics for students living in a region subject to a megathrust earthquake and resulting tsunami. Students seek information about these hazards, often through online sources which may foster naïve conceptions. Educational theory for earthquake and tsunami instruction can be further developed using the methods and initial results of this study to examine in greater detail the relationships between students' geoscience and preparedness knowledge and their ontological and epistemic beliefs. Implications for instruction include formative assessment that makes visible students' knowledge and beliefs in order to develop metacognition of their own conceptions and to inform subsequent instruction. Results indicate that geoscience instruction should be accompanied by instruction on preparedness actions that are locally relevant to students' lives in order to reduce fear and anxiety and to develop students' perceptions of themselves as active agents who can have a positive influence on the outcome of these events.
In the search for strategies to treat schizophrenia, attention has focused on enhancing NMDA receptor function.
In vitro experiments show that metabotropic glutamate 5 receptor (mGluR5) activation ...enhances NMDA receptor activity, and
in vivo experiments indicate that mGluR5 positive allosteric modulators (PAMs) are effective in preclinical assays measuring antipsychotic potential and cognition. Here we characterized the dose–effect function of CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide), an mGluR5 PAM, on novel object recognition memory in unimpaired Wistar Hannover rats (0, 10 or 30 mg/kg CDPPB) and animals with an MK-801-induced deficit (0, 3, 10, or 30 mg/kg CDPPB). In each experiment compound was given 30 min prior to the first exposure in order to affect acquisition/consolidation of the memory. In both cases, an inverted-U-shaped dose–effect function was observed, with lower doses improving recognition but higher doses having no effect. We then examined the effects of CDPPB (0, 3, 10, or 30 mg/kg) on markers of synaptic plasticity in prefrontal cortex and hippocampus, focusing on the expression and phosphorylation status of proteins involved in NMDA related signaling, including the NMDA receptor subunits NR1 and NR2B, the AMPA receptor subunit GluR1, αCa
(2+)/CaM dependent Ser–Thr kinases II (αCaMKII), and the transcription factor CREB. Expression and phosphorylation of many of these proteins, particularly in the prefrontal cortex, were also characterized by an inverted-U-shaped dose–effect function. Taken together, these findings show that mGluR5 activation enhances NMDA receptor function and markers of neuronal plasticity commensurate with improvements in recognition memory. However, the effects of CDPPB are heavily dependent on dose, with higher doses being ineffective in improving recognition memory and producing downstream effects consistent with heightened NMDA receptor activation. These findings may have important implications for the development of mGluR5 PAMs to treat schizophrenia.
► MDMA has concentration-dependent embryotoxic effects in cultured rat embryos. ► Two MDMA metabolites from different metabolic pathways produced embryotoxic effects. ► The embryotoxic potential of ...MDMA, MDA and HHMA was comparable in vitro.
MDMA consumers are young people of childbearing age. Consequently, developmental exposure to this drug is a potential public health concern. Several studies have addressed MDMA neurotoxicity in adults; however, knowledge of the effects of MDMA on developing embryos is limited. After administration, MDMA is metabolized species specifically via two main pathways. One leads to the formation of MDA and the other to the formation of HHMA. Here we evaluated the embryotoxic effects of MDMA, and also those of MDA, a main metabolite of MDMA in rats, and HHMA, a main metabolite in humans. For this purpose, we used the whole embryo culture (WEC). Our results show a concentration-dependent embryotoxic effect of MDMA, MDA and HHMA at a concentration range of 25–50μg/ml. The embryotoxic potential of the parent compound and the two metabolites was comparable in vitro.
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