Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30–70 % for pre-adipocyte proliferation; this was 40–80 % for triglyceride accumulation induced by pyraclostrobin, 80–100 % for tributyltin, and 80–100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30–70 % active determinations for pyraclostrobin, 30–50 % for tributyltin, and 20–40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.Over-arching project supported by grants [R01 ES016099 to HMS; R00 ES030405 to CDK] from the National Institute of Environmental Health Sciences (NIEHS); University of Turin; European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489; Brunel University London; NIEHS (1K22ES026208 and R01ES027863); NIEHS (Z0ES102785); Spanish Institute of Health Carlos III (grant FIS-PI16/01812)

Human Umbilical Cord Blood Cells Restore Brain Damage Induced Changes in Rat Somatosensory Cortex

Intraperitoneal transplantation of human umbilical cord blood (hUCB) cells has been shown to reduce sensorimotor deficits after hypoxic ischemic brain injury in neonatal rats. However, the neuronal correlate of the functional recovery and how such a treatment enforces plastic remodelling at the level of neural processing remains elusive. Here we show by in-vivo recordings that hUCB cells have the capability of ameliorating the injury-related impairment of neural processing in primary somatosensory cortex. Intact cortical processing depends on a delicate balance of inhibitory and excitatory transmission, which is disturbed after injury. We found that the dimensions of cortical maps and receptive fields, which are significantly altered after injury, were largely restored. Additionally, the lesion induced hyperexcitability was no longer observed in hUCB treated animals as indicated by a paired-pulse behaviour resembling that observed in control animals. The beneficial effects on cortical processing were reflected in an almost complete recovery of sensorimotor behaviour. Our results demonstrate that hUCB cells reinstall the way central neurons process information by normalizing inhibitory and excitatory processes. We propose that the intermediate level of cortical processing will become relevant as a new stage to investigate efficacy and mechanisms of cell therapy in the treatment of brain injury

Cloning and cortical expression of rat Emx2 and adenovirus-mediated overexpression to assess its regulation of area-specific targeting of thalamocortical axons

A goal of this study was to use recombinant adenovirus (AdV) to ectopically express Emx2 in the embryonic neocortex as a gain-of-function approach to study its role in the area-specific targeting of thalamocortical axons (TCAs), using the rat as a model. First, we cloned the cDNA for the full-length coding region of rat Emx2, a homologue of Drosophila empty spiracles. We also used this sequence to define the full-length coding region of mouse Emx2 from genomic DNA. Our analysis of Emx2 expression shows that in rat, as reported in mouse, Emx2 is expressed in high caudal to low rostral, and high medial to low lateral, gradients across the cortex throughout cortical neurogenesis, and expression is primarily restricted to progenitors in the neuroepithelium. We also carried out an analysis of the distribution of cells infected with a replication defective recombinant type 5 adenovirus (AdV) containing a CAG/LacZ expression construct, following an injection into the lateral ventricle of the cerebral hemisphere at different stages of embryonic cortical neurogenesis. AdV-infected cells are broadly distributed tangentially, but their laminar distribution is differentially restricted and reflects the temporal sequence of generation of cortical neurons. This finding indicates that the AdV predominantly infects progenitors in the ventricular zone, which leads to a preferential labeling of their immediate progeny, and infects cells that have recently become postmitotic and have yet to move far from the ventricular surface. We then show that AdV-mediated ectopic Emx2 expression results in aberrant intracortical pathfinding and areal targeting of TCAs from the dorsal lateral geniculate nucleus. These findings indicate that EMX2 imparts positional information normally associated with caudal cortical areas, such as the primary visual area, that influences the area-specific targeting of TCAs. These results are consistent with a role for EMX2 in areal specification of the neocortex as suggested by recent analyses of Emx2 null mutants

Symbiont microcosm in an ant society and the diversity of interspecific interactions

Colonies of the ponerine army ant Leptogenys distinguenda are regularly inhabited by a highly diverse symbiont fauna including insects, spiders, mites, crustaceans and even molluscs. Each of these myrmecophiles has adapted in a highly specific way to the lifestyle of its host. We studied this diverse myrmecophile fauna of L. distinguenda as a new model for multispecies parasitism to gain a better understanding of fundamental coevolutionary processes. Our study focused on behavioural and on chemical integration and exploitation strategies of the different symbiont species. In addition, we examined potential counterstrategies of the host ant. Myrmecophiles were studied both in large free-living L. distinguenda colonies and in more detail in parts of colonies separated for observation. We found that at least five myrmecophile species imposed cost on their host by exploiting its resources. Their impact varied considerably depending on both the type of resources exploited and their abundance. Myrmecophile species were well integrated into host societies either by chemical mimicry of host cuticular hydrocarbons or by remaining chemically insignificant, lacking most characteristic recognition cues. Despite these chemical integration strategies, host ants were able to recognize and kill the alien intruders to various degrees. This important finding demonstrates that symbiont populations are actively counter-regulated by the host. By constructing a host-parasite interaction network, we finally suggest that host defences can maintain myrmecophile diversity by keeping parasite populations small. This reduces interguild competition, comparable to top-down effects of predators on lower trophic levels in ecological food webs. (C) 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved

Comparative transcriptomic analyses to scrutinize the assumption that genotoxic PAHs exert effects via a common mode of action

In this study, the accuracy of the assumption that genotoxic, carcinogenic polycyclic aromatic hydrocarbons (PAHs) act via similar mechanisms of action as benzo(a)pyrene (BaP), the reference PAH used in the human health risk assessment of PAH-containing complex mixtures, was investigated. Adult male Muta™Mouse were gavaged for 28 days with seven individual, genotoxic PAHs. Global gene expression profiles in forestomach, liver, and lung (target tissues of exposure) were determined at 3 days post-exposure. The results are compared with our previously published results from mice exposed to BaP via the same exposure regimen. Although all PAHs showed enhanced ethoxyresorufin-O-deethylase activity, DNA adduct formation, and lacZ mutant frequency in the lungs, the unsupervised cluster analysis of differentially expressed genes revealed that the transcriptional changes are both PAH- and tissue-specific, with lung showing the most response. Further bioinformatics-/pathway-based analysis revealed that all PAHs induce expression of genes associated with carcinogenic processes, including DNA damage response, immune/inflammatory response, or cell signaling processes; however, the type of pathways and the magnitude of change varied for each PAH and were not the same as those observed for BaP. Benchmark dose modeling showed transcriptomic data closely reflected the known tumor incidence for the individual PAHs in each tissue. Collectively, the results suggest that the underlying mechanisms of PAH-induced toxicity leading to tumorigenesis are tissue-specific and not the same for all PAHs; based on the tissue type considered, use of BaP as a reference chemical may overestimate or underestimate the carcinogenic potential of PAHs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00204-015-1595-5) contains supplementary material, which is available to authorized users

Specific regulation of rat glial cell line-derived neurotrophic factor gene expression by riluzole in C6 glioma cells.

Contrasting with its robust expression during embryogenesis, the glial cell line-derived neurotrophic factor (GDNF) is repressed in the adult organism. However, rapid induction of this neuronal growth factor is observed following diverse neuronal insults and it is now widely accepted that the control of its expression could constitute a powerful target in neuropharmacology. We investigated the effects of the neuroprotective drug, riluzole, on the GDNF gene expression in glial cells. Exposure of C6 glioma cells to riluzole (1 microM) significantly increased GDNF protein and mRNA levels. Using luciferase reporter gene constructs encoding fragments of the 5' untranslated region of the rat GDNF gene, we demonstrated that riluzole mediates its effect at the transcription level. Furthermore, luciferase assays revealed the presence of a negative regulatory region within the +343/+587 region of exon 1. This region was shown to contribute to the high sensitivity and specificity of the induction mediated by riluzole in the C6 glioma cell line at pharmacologically relevant concentrations. The effects of riluzole were inhibited by the mitogen-activated protein kinase extracellular signal-related kinase (MEK) inhibitor PD 98059. Together, these results indicated that the induction of GDNF release by riluzole in the C6 glioma cells results from the activation of its corresponding gene promoter through a signalling pathway involving MEK activity. This study suggests that the regulation of GDNF gene transcription in glial cells could contribute to the pharmacological properties of riluzole and possibly other neuroprotective drugs