An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied <it>in vitro </it>but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on <it>in vitro </it>systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the <it>in vitro </it>model system and model toxicant, respectively.</p> <p>Results</p> <p>The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.</p> <p>Conclusions</p> <p>Untargeted profiling of the polar and apolar metabolites of <it>in vitro </it>cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.</p

Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax's anticancer efficacy

Combining Shapley value and statistics to the analysis of gene expression data in children exposed to air pollution

<p>Abstract</p> <p>Background</p> <p>In gene expression analysis, statistical tests for differential gene expression provide lists of candidate genes having, individually, a sufficiently low <it>p</it>-value. However, the interpretation of each single <it>p</it>-value within complex systems involving several interacting genes is problematic. In parallel, in the last sixty years, <it>game theory </it>has been applied to political and social problems to assess the power of interacting agents in forcing a decision and, more recently, to represent the relevance of genes in response to certain conditions.</p> <p>Results</p> <p>In this paper we introduce a Bootstrap procedure to test the null hypothesis that each gene has the same relevance between two conditions, where the relevance is represented by the Shapley value of a particular coalitional game defined on a microarray data-set. This method, which is called <it>Comparative Analysis of Shapley value </it>(shortly, CASh), is applied to data concerning the gene expression in children differentially exposed to air pollution. The results provided by CASh are compared with the results from a parametric statistical test for testing differential gene expression. Both lists of genes provided by CASh and t-test are informative enough to discriminate exposed subjects on the basis of their gene expression profiles. While many genes are selected in common by CASh and the parametric test, it turns out that the biological interpretation of the differences between these two selections is more interesting, suggesting a different interpretation of the main biological pathways in gene expression regulation for exposed individuals. A simulation study suggests that CASh offers more power than t-test for the detection of differential gene expression variability.</p> <p>Conclusion</p> <p>CASh is successfully applied to gene expression analysis of a data-set where the joint expression behavior of genes may be critical to characterize the expression response to air pollution. We demonstrate a synergistic effect between coalitional games and statistics that resulted in a selection of genes with a potential impact in the regulation of complex pathways.</p

Withaferin a-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak. © 2011 Hahm et al

SMI of Bcl-2 TW-37 is active across a spectrum of B-cell tumors irrespective of their proliferative and differentiation status

The Bcl-2 family of proteins is critical to the life and death of malignant B-lymphocytes. Interfering with their activity using small-molecule inhibitors (SMI) is being explored as a new therapeutic strategy for treating B-cell tumors. We evaluated the efficacy of TW-37, a non-peptidic SMI of Bcl-2 against a range spectrum of human B-cell lines, fresh patient samples and animal xenograft models. Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models. Nanomolar concentrations of TW-37 were able to induce apoptosis in both fresh samples and established cell lines with IC50 in most cases of 165–320 nM. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg × 3 days) via tail vein. TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy regardless of the stage of B-cell differentiation

Primary DNA damage and genetic polymorphisms for CYP1A1, EPHX and GSTM1 in workers at a graphite electrode manufacturing plant

<p>Abstract</p> <p>Background</p> <p>The results of a cross-sectional study aimed to evaluate whether genetic polymorphisms (biomarkers of susceptibility) for <it>CYP1A1</it>, <it>EPHX </it>and <it>GSTM1 </it>genes that affect polycyclic aromatic hydrocarbons (PAH) activation and detoxification might influence the extent of primary DNA damage (biomarker of biologically effective dose) in PAH exposed workers are presented. PAH-exposure of the study populations was assessed by determining the concentration of 1-hydroxypyrene (1OHP) in urine samples (biomarker of exposure dose).</p> <p>Methods</p> <p>The exposed group consisted of workers (n = 109) at a graphite electrode manufacturing plant, occupationally exposed to PAH. Urinary 1OHP was measured by HPLC. Primary DNA damage was evaluated by the alkaline comet assay in peripheral blood leukocytes. Genetic polymorphisms for <it>CYP1A1</it>, <it>EPHX</it> and <it>GSTM1</it> were determined by PCR or PCR/RFLP analysis.</p> <p>Results</p> <p>1OHP and primary DNA damage were significantly higher in electrode workers compared to reference subjects. Moreover, categorization of subjects as normal or outlier highlighted an increased genotoxic risk OR = 2.59 (CI95% 1.32–5.05) associated to exposure to PAH. Polymorphisms in <it>EPHX</it> exons 3 and 4 was associated to higher urinary concentrations of 1OHP, whereas none of the genotypes analyzed (<it>CYP1A1</it>, <it>EPHX</it>, and <it>GSTM1</it>) had any significant influence on primary DNA damage as evaluated by the comet assay.</p> <p>Conclusion</p> <p>The outcomes of the present study show that molecular epidemiology approaches (i.e. cross-sectional studies of genotoxicity biomarkers) can play a role in identifying common genetic risk factors, also attempting to associate the effects with measured exposure data. Moreover, categorization of subjects as normal or outlier allowed the evaluation of the association between occupational exposure to PAH and DNA damage highlighting an increased genotoxic risk.</p

The one dimensional Kondo lattice model at partial band filling

The Kondo lattice model introduced in 1977 describes a lattice of localized magnetic moments interacting with a sea of conduction electrons. It is one of the most important canonical models in the study of a class of rare earth compounds, called heavy fermion systems, and as such has been studied intensively by a wide variety of techniques for more than a quarter of a century. This review focuses on the one dimensional case at partial band filling, in which the number of conduction electrons is less than the number of localized moments. The theoretical understanding, based on the bosonized solution, of the conventional Kondo lattice model is presented in great detail. This review divides naturally into two parts, the first relating to the description of the formalism, and the second to its application. After an all-inclusive description of the bosonization technique, the bosonized form of the Kondo lattice hamiltonian is constructed in detail. Next the double-exchange ordering, Kondo singlet formation, the RKKY interaction and spin polaron formation are described comprehensively. An in-depth analysis of the phase diagram follows, with special emphasis on the destruction of the ferromagnetic phase by spin-flip disorder scattering, and of recent numerical results. The results are shown to hold for both antiferromagnetic and ferromagnetic Kondo lattice. The general exposition is pedagogic in tone.Comment: Review, 258 pages, 19 figure