Comparison of transcriptional responses in liver tissue and primary hepatocyte cell cultures after exposure to hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine

BACKGROUND: Cell culture systems are useful in studying toxicological effects of chemicals such as Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), however little is known as to how accurately isolated cells reflect responses of intact organs. In this work, we compare transcriptional responses in livers of Sprague-Dawley rats and primary hepatocyte cells after exposure to RDX to determine how faithfully the in vitro model system reflects in vivo responses. RESULTS: Expression patterns were found to be markedly different between liver tissue and primary cell cultures before exposure to RDX. Liver gene expression was enriched in processes important in toxicology such as metabolism of amino acids, lipids, aromatic compounds, and drugs when compared to cells. Transcriptional responses in cells exposed to 7.5, 15, or 30 mg/L RDX for 24 and 48 hours were different from those of livers isolated from rats 24 hours after exposure to 12, 24, or 48 mg/Kg RDX. Most of the differentially expressed genes identified across conditions and treatments could be attributed to differences between cells and tissue. Some similarity was observed in RDX effects on gene expression between tissue and cells, but also significant differences that appear to reflect the state of the cell or tissue examined. CONCLUSION: Liver tissue and primary cells express different suites of genes that suggest they have fundamental differences in their cell physiology. Expression effects related to RDX exposure in cells reflected a fraction of liver responses indicating that care must be taken in extrapolating from primary cells to whole animal organ toxicity effects

MicroRNA and messenger RNA profiling reveals new biomarkers and mechanisms for RDX induced neurotoxicity

Background RDX is a well-known pollutant to induce neurotoxicity. MicroRNAs (miRNA) and messenger RNA (mRNA) profiles are useful tools for toxicogenomics studies. It is worthy to integrate MiRNA and mRNA expression data to understand RDX-induced neurotoxicity. Results Rats were treated with or without RDX for 48 h. Both miRNA and mRNA profiles were conducted using brain tissues. Nine miRNAs were significantly regulated by RDX. Of these, 6 and 3 miRNAs were up- and down-regulated respectively. The putative target genes of RDX-regulated miRNAs were highly nervous system function genes and pathways enriched. Fifteen differentially genes altered by RDX from mRNA profiles were the putative targets of regulated miRNAs. The induction of miR-71, miR-27ab, miR-98, and miR-135a expression by RDX, could reduce the expression of the genes POLE4, C5ORF13, SULF1 and ROCK2, and eventually induce neurotoxicity. Over-expression of miR-27ab, or reduction of the expression of unknown miRNAs by RDX, could up-regulate HMGCR expression and contribute to neurotoxicity. RDX regulated immune and inflammation response miRNAs and genes could contribute to RDX- induced neurotoxicity and other toxicities as well as animal defending reaction response to RDX exposure. Conclusions Our results demonstrate that integrating miRNA and mRNA profiles is valuable to indentify novel biomarkers and molecular mechanisms for RDX-induced neurological disorder and neurotoxicity.published_or_final_versio

Development of computations in bioscience and bioinformatics and its application: review of the Symposium of Computations in Bioinformatics and Bioscience (SCBB06)

The first symposium of computations in bioinformatics and bioscience (SCBB06) was held in Hangzhou, China on June 21–22, 2006. Twenty-six peer-reviewed papers were selected for publication in this special issue of BMC Bioinformatics. These papers cover a broad range of topics including bioinformatics theories, algorithms, applications and tool development. The main technical topics contain gene expression analysis, sequence analysis, genome analysis, phylogenetic analysis, gene function prediction, molecular interaction and system biology, genetics and population study, immune strategy, protein structure prediction and proteomics

Identification of Biomarkers That Distinguish Chemical Contaminants Based on Gene Expression Profiles

Background: High throughput transcriptomics profiles such as those generated using microarrays have been useful in identifying biomarkers for different classification and toxicity prediction purposes. Here, we investigated the use of microarrays to predict chemical toxicants and their possible mechanisms of action. Results: In this study, in vitro cultures of primary rat hepatocytes were exposed to 105 chemicals and vehicle controls, representing 14 compound classes. We comprehensively compared various normalization of gene expression profiles, feature selection and classification algorithms for the classification of these 105 chemicals into14 compound classes. We found that normalization had little effect on the averaged classification accuracy. Two support vector machine (SVM) methods, LibSVM and sequential minimal optimization, had better classification performance than other methods. SVM recursive feature selection (SVM-RFE) had the highest overfitting rate when an independent dataset was used for a prediction. Therefore, we developed a new feature selection algorithm called gradient method that had a relatively high training classification as well as prediction accuracy with the lowest overfitting rate of the methods tested. Analysis of biomarkers that distinguished the 14 classes of compounds identified a group of genes principally involved in cell cycle function that were significantly downregulated by metal and inflammatory compounds, but were induced by anti-microbial, cancer related drugs, pesticides, and PXR mediators. Conclusions: Our results indicate that using microarrays and a supervised machine learning approach to predict chemical toxicants, their potential toxicity and mechanisms of action is practical and efficient. Choosing the right feature and classification algorithms for this multiple category classification and prediction is critical

Reverse Engineering of Gene Regulatory Networks for Discovery of Novel Interactions in Pathways Using Gene Expression Data

A variety of chemicals in the environment have the potential to adversely affect the biological systems. We examined the responses of Rat (Rattus norvegicus) to the RDX exposure and female fathead minnows (FHM, Pimephales promelas) to a model aromatase inhibitor, fadrozole, using a transcriptional network inference approach. Rats were exposed to RDX and fish were exposed to 0 or 30mg/L fadrozole for 8 days. We analyzed gene expression changes using 8000 probes microarrays for rat experiment and 15,000 probe microarrays for fish. We used these changes to infer a transcriptional network. The central nervous system is remarkably plastic in its ability to recover from trauma. We examined recovery from chemicals in rats and fish through changes in transcriptional networks. Transcriptional networks from time series experiments provide a good basis for organizing and studying the dynamic behavior of biological processes. The goal of this work was to identify networks affected by chemical exposure and track changes in these networks as animals recover. The top 1254 significantly changed genes based upon 1.5-fold change and P\u3c 0.05 across all the time points from the fish data and 937 significantly changed genes from rat data were chosen for network modeling using either a Mutual Information network (MIN) or a Graphical Gaussian Model (GGM) or a Dynamic Bayesian Network (DBN) approach. The top interacting genes were queried to find sub-networks, possible biological networks, biochemical pathways, and network topologies impacted after exposure to fadrozole. The methods were able to reconstruct transcriptional networks with few hub structures, some of which were found to be involved in major biological process and molecular function. The resulting network from rat experiment exhibited a clear hub (central in terms of connections and direction) connectivity structure. Genes such as Ania-7, Hnrpdl, Alad, Gapdh, etc. (all CNS related), GAT-2, Gabra6, Gabbrl, Gabbr2 (GABA, neurotransmitter transporters and receptors), SLC2A1 (glucose transporter), NCX3 (Na-Ca exchanger), Gnal (Olfactory related), skn-la were showed up in our network as the \u27hub\u27 genes while some of the known transcription factors Msx3, Cacngl, Brs3, NGF1 etc. were also matched with our network model. Aromatase in the fish experiment was a highly connected gene in a sub-network along with other genes involved in steroidogenesis. Many of the sub-networks were involved in fatty acid metabolism, gamma-hexachlorocyclohexane degradation, and phospholipase activating pathways. Aromatase was a highly connected gene in a sub-network along with the genes LDLR, StAR, KRT18, HER1, CEBPB, ESR2A, and ACVRL1. Many of the subnetworks were involved in fatty acid metabolism, gamma-hexachlorocyclohexane degradation, and phospholipase activating pathways. A credible transcriptional network was recovered from both the time series data and the static data. The network included transcription factors and genes with roles in brain function, neurotransmission and sex hormone synthesis. Examination of the dynamic changes in expression within this network over time provided insight into recovery from traumas and chemical exposures

METABOLIC REPROGRAMMING IN ONCOGENE-DRIVEN HEPATOCELLULAR CARCINOMA: EFFECT OF LDHA KNOCK-DOWN

Il carcinoma epatocellulare (HCC), così come altri tumori solidi, è un processo multi- fasico nel quale le cellule neoplastiche vanno incontro ad una consistente riprogrammazione metabolica. Numerose evidenze suggeriscono che le differenze nelle alterazioni metaboliche che caratterizzano l’HCC siano attribuibili all’attivazione di specifici oncogeni spiegando quindi, almeno parzialmente, l’eterogeneità propria dell’HCC. A tal proposito, l’iper-espressione dell’oncogene c-Myc, frequentemente osservata nell’HCC umano, determina una riprogrammazione metabolica verso un fenotipo Warburg, tramite il quale le cellule tumorali prendono avidamente il glucosio e generano grandi quantità di lattato, mediante l’azione dell’enzima lattato deidrogenasi A (Ldha), anche in presenza di ossigeno. L’iper- espressione della Ldha, causata da una aumentata glicolisi, è associata a prognosi infausta dal momento che porta ad un rilascio eccessivo di lattato da parte delle cellule tumorali, inducendo l’acidificazione dello spazio extracellulare e favorendo l’emergenza di un microambiente tumorale (TME) immuno-permissivo. La presente tesi ha avuto come primo obiettivo lo studio della riprogrammazione metabolica in tumori indotti dalla concomitante espressione di c-Myc e h-Ras, due tra gli oncogeni più frequentemente iper-espressi nell’HCC umano. Usando lo stesso modello sperimentale, abbiamo anche valutato l’effetto dell’inibizione della Ldha - ottenuto tramite una tecnica di “Ldha knock-down inducibile ed epatocita-specifico” - sulla riprogrammazione metabolica delle cellule tumorali e sulle alterazioni del TME. I risultati ottenuti hanno rivelato che i tumori c-Myc/h-Ras sono caratterizzati da un forte metabolismo glicolitico indirizzato verso il fenotipo Warburg, e da un’aumentata attività della via del pentosio fosfato (PPP). Tuttavia, al contrario di quando osservato negli HCC generati dalla sola iper-espressione di c-Myc, la glutammina non viene utilizzata per rifornire il ciclo degli acidi tricarbossilici (TCA) ma per sostenere la sintesi di glutatione. Parallelamente, si assiste ad una parziale inibizione della fosforilazione ossidativa (OXPHOS). I nostri risultati hanno anche dimostrato che l’abrogazione della Ldha ostacola lo sviluppo e la progressione tumorale, tramite la parziale inibizione del metabolismo Warburg. Inoltre, la perdita della Ldha causa un notevole incremento dei linfociti Cd4+ nel TME, rispetto a quanto osservato negli Ldha WT, preservando così l’effetto anti-neoplastico esercitato da questa popolazione linfocitaria Nel complesso, questi risultati suggeriscono che la combinazione dell’immunoterapia con l’inibizione farmacologica della Ldha possa contribuire ad aumentare l’efficacia delle opzioni terapeutiche attualmente disponibili

Einfluss von Dexamethason auf die CYP-Induktion durch den Modellinduktor Phenobarbital an Rattenleberschnitten in vitro

Präzisionsleberschnitte haben sich als geeignetes In-vitro-Modell für die Induktion verschiedener Cytochrom-P450 (CYP)-Formen bewährt. Der Zweck der Untersuchungen bestand darin zu prüfen, inwiefern Schwankungen im hepatischen CYP-Expressionsmuster, z.B. ausgelöst durch CYP-Induktoren in vivo, Auswirkungen auf In-vitro-Testungen der CYP-Induktion haben können. Bei der angestrebten Nutzung von humanem Lebergewebe ist für solche Tests mit großen Unterschieden ohne Einflussmöglichkeiten bezüglich Gewebeeigenschaften zu rechnen. In dieser Arbeit wurde der Teilaspekt der Phenobarbital (PB)-abhängigen Induktion des CYP2B1 unter dem Einfluss des CYP3A-Modellinduktors Dexamethason (DEXA) an Leberschnitten weiblicher Ratten untersucht. Im Mittelpunkt stand dabei die In-vitro-Induktion des CYP2B1-Isoenzyms durch PB in Leberschnitten nach Vorbehandlung der Ratten mit DEXA in vivo (4mg/kg Körpermasse (KM) an 3 aufeinander folgenden Tagen). Neben diesem In-vivo-Versuch wurde in weiteren In-vitro-Versuchsreihen die CYP2B1-Induktion an Rattenleberschnitten nach alleiniger PB-Exposition, nach simultaner DEXA- und PB-Exposition und nach sukzessiver DEXA- und PB-Exposition geprüft. PB wurde in den Konzentrationen von 5, 10, 50 und 100 µM eingesetzt. Für DEXA wurden die Konzentrationen 0,01 µM und 1 µM gewählt, um die Interaktion mit einem schwachen oder starken CYP3A-Induktor zu simulieren. Das Ausmaß der PB-abhängigen CYP2B1-Induktion wurde auf Enzymaktivitätsebene mit Hilfe der Modellreaktionen Pentoxyresorufin-O-Depentylierung (PROD) und 16β-Testosteronhydroxylierung (16β-TH) bestimmt. Desweiteren erfolgten Bestimmungen der CYP2B1-mRNA-Expression. Ergänzend wurde die Induktion des CYP3A1-Isoenzyms durch DEXA anhand der 2β-Testosteronhydroxylierung (2β-TH) kontrolliert

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds