Effect of Dioxin on Bone Cell Proteome

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disrupting environmental pollutant that affects bone tissue, although the mechanistic basis of such effect is far from clear. In this study a proteomic approach has been adopted to investigate the disturbance of the osteogenic process evoked by TCDD in an in vitro osteoblast differentiation model of rat mesenchymal stem cells. Stem cells were isolated from bone marrow of femurs and tibias of rats. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells after 3, 7 and 10 days of culture in presence and absence of TCDD using quantitative RT-PCR. Analysis of differential protein expression in the total cell lysate of untreated and TCDD-treated cells was carried out over the same time points, using two-dimensional gel electrophoresis, computerized gel image, univariate and multivariate statistical analysis, in-gel digestion and tandem mass spectrometry for protein identification. Expression of all measured markers characteristic for various stages of osteoblastic differentiation (Runx2, alkaline phosphatase and osteocalcin) was dramatically reduced only after 10 days of TCDD exposure, indicating that TCDD significantly inhibits osteoblast differentiation in vitro. Similarly, the most significant rearrangement of the proteome during osteoblast differentiation was observed following the 10 days of TCDD exposure, at which time the full progress of osteoblast differentiation should have occurred. Overall, 18 individual proteins showed a statistically significant change in abundance as a result of 10 days of TCDD exposure. These proteins were mostly involved in cytoskeleton organization and biogenesis, actin filament-based processes, protein transport and folding. The alteration in cell architecture and increase in cell adhesion were confirmed by confocal microscopy. The TCDD-induced decrease in the expression of calcium binding proteins may interfere with osteoblast calcium deposition, which was in fact reduced by TCDD. Evidence from proteomics and Western blot analysis indicated a decreased apoptotic capability in TCDD-treated osteoblasts. This is the first study investigating, at the protein expression level, the effect evoked by TCDD during osteoblastic differentiation. Interestingly, MetaCore pathway analysis grouped the majority of these proteins around two principal nodes (c-fos and c-myc) suggesting that they may participate in the transcriptional activation of key pathways in TCDD-driven inhibition of osteoblast differentiation. These findings provide clues to the complex interplay between TCDD's action and the regulatory molecules that alter signal-transduction pathways regulating osteoblast differentiation and indicate new molecular players in the effects of TCDD on bone development

Proteome characterization of a human urothelial cell line resistant to the bladder carcinogen 4-aminobiphenyl

<p>Abstract</p> <p>Background</p> <p>The aromatic amine 4-aminobiphenyl (4-ABP) is an environmental and occupational contaminant known to be a major etiological agent of human bladder cancer. 4-ABP metabolites are able to form DNA adducts that may induce mutations and initiate bladder carcinogenesis. Cells exposed to 4-ABP may develop resistance to the carcinogen. The aim of the present study was to detect and identify proteins whose expression is altered in the bladder carcinoma RT112 sub-lines selected for acquired resistance to 4-ABP, in order to disentangle the mechanisms.</p> <p>Results</p> <p>Differential proteome analysis of cell lysates showed an overall perturbation in cell metabolism and energy pathways in the 4-ABP-resistant human urothelial clones, with over-expression of membrane trafficking proteins such as annexin 2. The resistant clones had altered expression of many proteins linked directly (<it>i.e</it>. lamin A/C, programmed cell death 6 interacting protein) or indirectly (<it>i.e</it>. 94 kDa glucose-regulated protein, fatty acid-binding protein) to decreased apoptosis, suggesting that resistance to 4-ABP might be associated with low apoptotic activity.</p> <p>Conclusion</p> <p>Our data provide evidence that deregulation of apoptosis and membrane trafficking proteins might be strongly implicated in the selection of carcinogen resistant cells. Some of these proteins might have potential as biomarkers of resistance and cancer risk.</p

Differential Expression Profiling of the Hepatic Proteome in a Rat Model of Dioxin Resistance CORRELATION WITH GENOMIC AND TRANSCRIPTOMIC ANALYSES

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

Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress

International audience; 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 14 days. 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 15 µM 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 5 µM 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

A proteomic approach to investigate the modification in the proteome of the cytoplasmatic compartment of Balb/3T3 cells after exposure to gold nanoparticles (AuNPs)

Nanoparticles are widely used in consumer products. However, information about the exposure of the consumer to nanoparticles and their potential health effects is very limited. Nanoparticles may move inside the human body in different manners (via inhalation, ingestion or skin contact), cross the cell membranes and accumulate for long periods of time. Emerging approaches in the area of exposure to nanomaterials and assessment of human health effects combine the use of in vitro cell systems and advanced analytical techniques to study the perturbation of the proteome. The appropriate use of these approaches has the potential to provide information on the possible de-regulation of essential physiological cellular processes. In the present study, we investigated the modification in the proteome of the cytoplasmatic compartment of the Balb/3T3 mouse fibroblast cell line after exposure to 5 and 15 nm gold nanoparticles (AuNPs) for 72 h. Protein separation by two-dimensional gel electrophoresis (2DE) followed by protein identification high-resolution mass spectrometry (MS) allowed us to study the differentially expressed proteome in order to explore underlying cellular mechanisms. Differentially expressed proteins were found to cover a range of functions including stress response, cell metabolism, cell growth and cytoskeleton organization. Remarkably, even small differences in particle size (10 nm) seemed to differently affect biological mechanisms. These findings consolidate existing knowledge and permit to get more insight to the cell mechanisms affected by AuNPs exposure. Our activities in the area of exposure to nanomaterials and potential health effects for the consumer are essential to support EU policy implementation.JRC.I.1-Chemical Assessment and Testin

A proteomic approach to investigate AuNPs effects in Balb/3T3 cells

Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell-nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study AuNPs effects. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. The strength of this investigation resides in combining the high-resolving power of fluorescence two-dimensional differential gel electrophoresis with protein identification by high-resolution mass spectrometry. Of interest was the difference observed in the protein pattern expression of cells exposed to 5 and 15 nm AuNPs. From 2D gel-based proteomic data, it was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15 nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, inflammatory response.JRC.I.4-Nanobioscience

Assessment of developmental neurotoxicity induced by chemical mixtures using an adverse outcome pathway concept

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 concentrations causing no or very low cytotoxicity (IC5), 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.JRC.F.3-Chemicals Safety and Alternative Method

A proteomic approach to investigate AuNPs effects in Balb/3T3 cells.

Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell-nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study AuNPs effects. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. The strength of this investigation resides in combining the high-resolving power of fluorescence two-dimensional differential gel electrophoresis with protein identification by high-resolution mass spectrometry. Of interest was the difference observed in the protein pattern expression of cells exposed to 5 and 15 nm AuNPs. From 2D gel-based proteomic data, it was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15 nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, inflammatory response

ASTHMA SEVERITY IN CHILDHOOD AND METABOLOMIC PROFILING OF BREATH CONDENSATE

Background. Asthma is a heterogeneous disease and its different phenotypes need to be better characterized from a biochemical-inflammatory standpoint. The present study aimed to apply the metabolomic approach to exhaled breath condensate (breathomics) to discriminate different asthma phenotypes, with a particular focus on severe asthma in children. Methods. In this cross sectional study we recruited 42 asthmatic children (age 8-17 years): 31 with non-severe asthma (treated with inhaled steroids or not) and 11 with severe asthma. Fifteen healthy children were enrolled as controls. Children performed exhaled nitric oxide measurement, spirometry, exhaled breath condensate (EBC) collection. Condensate samples were analysed using a metabolomic approach based on mass spectrometry. Results. A robust O2PLS-DA model was found for discriminating both between severe asthma cases and healthy controls (R2=0.93; Q2=0.75) and between severe asthma and non-severe asthma (R2=0.84; Q2=0.47). The metabolomic data analysis leads to a robust model also when the 3 groups of children were considered altogether (R2=0.78, K=0.59), indicating that each group is characterised by a specific metabolomic profile. Compounds related to retinoic acid, adenosine and vitamin D (Human Metabolome Database), were relevant for the discrimination between groups Conclusion. The metabolomic profiling of EBC could clearly distinguish children with different degrees of asthma severity and enabled the severe asthma phenotype to be fully discriminated. The breathomics approach may therefore be suitable for discriminating between different asthma metabolic phenotypes.JRC.I.1-Chemical Assessment and Testin