High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15μM induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein-response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5μM CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress.

The metabolomic profiling of EBC could clearly distinguish different biochemical-metabolic profiles in asthmatic children and enabled the severe asthma phenotype to be fully discriminated. The breathomics approach may therefore be suitable for discriminating between different asthma metabolic phenotypes.

Background: In light of the vulnerability of the developing brain, mixture risk assessment (MRA) for the evaluation of developmental neurotoxicity (DNT) should be implemented, since infants and children are co-exposed to more than one chemical at a time. One possible approach to tackle MRA could be to cluster DNT chemicals in a mixture on the basis of their mode of action (MoA) into 'similar' and 'dissimilar', but still contributing to the same adverse outcome, and anchor DNT assays to common key events (CKEs) identified in DNT-specific adverse outcome pathways (AOPs). Moreover, the use of human in vitro models, such as induced pluripotent stem cell (hiPSC)derived neuronal and glial cultures would enable mechanistic understanding of chemically-induced adverse effects, avoiding species extrapolation. Methods: HiPSC-derived neural progenitors differentiated into mixed cultures of neurons and astrocytes were used to assess the effects of acute (3 days) and repeated dose (14 days) treatments with single chemicals and in mixtures belonging to different classes (i.e., lead(II) chloride and methylmercury chloride (heavy metals), chlorpyrifos (pesticide), bisphenol A (organic compound and endocrine disrupter), valproic acid (drug), and PCB138 (persistent organic pollutant and endocrine disrupter), which are associated with cognitive deficits, including learning and memory impairment in children. Selected chemicals were grouped based on their mode of action (MoA) into 'similar' and 'dissimilar' MoA compounds and their effects on synaptogenesis, neurite outgrowth, and brain derived neurotrophic factor (BDNF) protein levels, identified as CKEs in currently available AOPs relevant to DNT, were evaluated by immunocytochemistry and high content imaging analysis. Results: Chemicals working through similar MoA (i.e., alterations of BDNF levels), at non-cytotoxic (IC 20 /100), very low toxic (IC 5), or moderately toxic (IC 20) concentrations, induce DNT effects in mixtures, as shown by increased number of neurons, impairment of neurite outgrowth and synaptogenesis (the most sensitive endpoint as confirmed by mathematical modelling) and increase of BDNF levels, to a certain extent reproducing autism-like cellular changes observed in the brain of autistic children. Conclusions: Our findings suggest that the use of human iPSC-derived mixed neuronal/glial cultures applied to a battery of assays anchored to key events of an AOP network represents a valuable approach to identify mixtures of chemicals with potential to cause learning and memory impairment in children.

Upon T cell receptor stimulation, CD4 T helper (Th) lymphocytes release extracellular vesicles (EVs) containing microRNAs. However, no data are available on whether human CD4 T cell subsets release EVs containing different pattern of microRNAs. The present work aimed at filling this gap by assessing the microRNA content in EVs released upon T cell receptor stimulation of Th1, Th17, and T regulatory (Treg) cells. Our results indicate that EVs released by Treg cells are significantly different compared with those released by the other subsets. In particular, miR-146a-5p, miR-150-5p, and miR-21-5p are enriched, whereas miR-106a-5p, miR-155-5p, and miR-19a-3p are depleted in Treg-derived EVs. The identified EV-associated microRNA signature was increased in serum of autoimmune patients with psoriasis and returned to healthy levels upon effective treatment with etanercept, a biological drug targeting the TNF pathway and suppressing inflammation. Moreover, Gene Set Enrichment Analysis showed an over-representation of genes relevant for T cell activation, such as CD40L, IRAK1, IRAK2, STAT1, and c-Myb in the list of validated targets of Treg-derived EV miRNAs. At functional level, Treg-derived (but not Th1/Th17-derived) EVs inhibited CD4 T cell proliferation and suppressed two relevant targets of miR-146a-5p: STAT1 and IRAK2. In conclusion, our work identified the miRNAs specifically released by different human CD4 T cell subsets and started to unveil the potential use of their quantity in human serum to mark the pathological elicitation of these cells and their biological effect in cell to cell communication during the adaptive immune response.

One characteristic feature of acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is dramatic interspecies and interstrain variability in sensitivity. This complicates dioxin risk assessment for humans. However, this variability also provides a means of characterizing mechanisms of dioxin toxicity. Long-Evans (Turku/AB) rats are orders of magnitude more susceptible to TCDD lethality than Han/Wistar (Kuopio) rats, and this difference constitutes a very useful model for identifying mechanisms of dioxin toxicity. We adopted a proteomic approach to identify the differential effects of TCDD exposure on liver protein expression in Han/Wistar rats as compared with Long-Evans rats. This allows determination of which, if any, protein markers are indicative of differences in dioxin susceptibility and/or responsible for conferring resistance. Differential protein expression in total liver protein was assessed using two-dimensional gel electrophoresis, computerized gel image analysis, in-gel digestion, and mass spectrometry. We observed significant changes in the abundance of several proteins, which fall into three general classes: (i) TCDD-independent and exclusively strain-specific (e.g. isoforms of the protein-disulfide isomerase A3, regucalcin, and agmatine ureohydrolase); (ii) strain-independent and only dependent on TCDD exposure (e.g. aldehyde dehydrogenase 3A1 and rat selenium-binding protein 2); (iii) dependent on both TCDD exposure and strain (e.g. oxidative stress-related proteins, apoptosis-inducing factor, and MAWD-binding protein). By integrating transcriptomic (microarray) data and genomic data (computational search of regulatory elements), we found that protein expression levels were mainly controlled at the level of transcription. These results reveal, for the first time, a subset of hepatic proteins that are differentially regulated in response to TCDD in a strain-specific manner. Some of these differential responses may play a role in establishing the major differences in TCDD response between these two strains of rats. As such, our work is expected to lead to new insights into the mechanism of TCDD toxicity and resistance. Molecular & Cellular Proteomics 5:882-894, 2006.