A hierarchical approach employing metabolic and gene expression profiles to identify the pathways that confer cytotoxicity in HepG2 cells

<p>Abstract</p> <p>Background</p> <p>Free fatty acids (FFA) and tumor necrosis factor alpha (TNF-α) have been implicated in the pathogenesis of many obesity-related metabolic disorders. When human hepatoblastoma cells (HepG2) were exposed to different types of FFA and TNF-α, saturated fatty acid was found to be cytotoxic and its toxicity was exacerbated by TNF-α. In order to identify the processes associated with the toxicity of saturated FFA and TNF-α, the metabolic and gene expression profiles were measured to characterize the cellular states. A computational model was developed to integrate these disparate data to reveal the underlying pathways and mechanisms involved in saturated fatty acid toxicity.</p> <p>Results</p> <p>A hierarchical framework consisting of three stages was developed to identify the processes and genes that regulate the toxicity. First, discriminant analysis identified that fatty acid oxidation and intracellular triglyceride accumulation were the most relevant in differentiating the cytotoxic phenotype. Second, gene set enrichment analysis (GSEA) was applied to the cDNA microarray data to identify the transcriptionally altered pathways and processes. Finally, the genes and gene sets that regulate the metabolic responses identified in step 1 were identified by integrating the expression of the enriched gene sets and the metabolic profiles with a multi-block partial least squares (MBPLS) regression model.</p> <p>Conclusion</p> <p>The hierarchical approach suggested potential mechanisms involved in mediating the cytotoxic and cytoprotective pathways, as well as identified novel targets, such as NADH dehydrogenases, aldehyde dehydrogenases 1A1 (ALDH1A1) and endothelial membrane protein 3 (EMP3) as modulator of the toxic phenotypes. These predictions, as well as, some specific targets that were suggested by the analysis were experimentally validated.</p

Short time-series microarray analysis: Methods and challenges

The detection and analysis of steady-state gene expression has become routine. Time-series microarrays are of growing interest to systems biologists for deciphering the dynamic nature and complex regulation of biosystems. Most temporal microarray data only contain a limited number of time points, giving rise to short-time-series data, which imposes challenges for traditional methods of extracting meaningful information. To obtain useful information from the wealth of short-time series data requires addressing the problems that arise due to limited sampling. Current efforts have shown promise in improving the analysis of short time-series microarray data, although challenges remain. This commentary addresses recent advances in methods for short-time series analysis including simplification-based approaches and the integration of multi-source information. Nevertheless, further studies and development of computational methods are needed to provide practical solutions to fully exploit the potential of this data

Sodium Propionate and Sodium Butyrate Promote Fatty Acid Oxidation in HepG2 Cells under Oxidative Stress

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disturbances that includes simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and liver cancer. NAFLD affects individuals that consume the typical Western diet consisting of high levels of fats and carbohydrates. The increase in circulating free fatty acids, palmitate and oleate, or lipopolysaccharides (LPS), induce oxidative stress and pro-inflammatory cytokine production in the liver, which all contribute to NAFLD progression. In this study, we are evaluating the mRNA expression of genes associated with fatty acid oxidation (FAO) and the protein expression of pro-inflammatory cytokines related to NAFLD using the HepG2 human liver hepatocellular carcinoma cells exposed to palmitate/oleate or LPS. The treatment of sodium butyrate (NaB) or sodium propionate (NaP) was used to relieve oxidative stress and inflammation in liver cells. The quantitative real-time polymerase chain reaction (qRT-PCR) results show that NaP or NaB, were able to promote FAO, regulate lipolysis, and reduce reactive oxygen species production by significantly increasing the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor alpha (PPARα), adipose triglyceride lipase (ATGL), carnitine palmitoyltransferase 1 alpha (CPT1α), fibroblast growth factor 21 (FGF21), and uncoupling protein 2 (UCP2) in HepG2 cells. Together, NaP and NaB may produce synergistic effects by significantly increasing CPT1α, PPARα, and UCP2 mRNA expression in LPS-induced HepG2 cells and by significantly increasing CPT1α and ATGL mRNA expression in palmitate/oleate-induced HepG2 cells. Only NaP treatment may have the ability to reverse hepatic steatosis and increase whole-body energy expenditure by significantly increasing FGF21 mRNA expression in palmitate/oleate-induced HepG2 cells. The ELISA results reveal that only LPS significantly increased Tumor Necrosis Factor alpha (TNF-α) expression in HepG2 cells. At the same time, NaP alone or in combination with NaB significantly decreased TNF-α expression in LPS-induced HepG2 cells. The expression of IL-8 in both models showed no significant differences in all treatments. The Western blot analysis of CPT1α protein expression increased by NaP alone or in combination with NaB in the palmitate/oleate model. In conclusion, this study shows promising results for the use of NaP and NaB as a potential new therapy in NAFLD

Gene Expression Profiling and Network Analysis Reveals Lipid and Steroid Metabolism to Be the Most Favored by TNFα in HepG2 Cells

BACKGROUND: The proinflammatory cytokine, TNFalpha, is a crucial mediator of the pathogenesis of several diseases, more so in cases involving the liver wherein it is critical in maintaining liver homeostasis since it is a major determiner of hepatocyte life and death. Gene expression profiling serves as an appropriate strategy to unravel the underlying signatures to envisage such varied responses and considering this, gene transcription profiling was examined in control and TNFalpha treated HepG2 cells. METHODS AND FINDINGS: Microarray experiments between control and TNFalpha treated HepG2 cells indicated that TNFalpha could significantly alter the expression profiling of 140 genes; among those up-regulated, several GO (Gene Ontology) terms related to lipid and fat metabolism were significantly (p<0.01) overrepresented indicating a global preference of fat metabolism within the hepatocyte and those within the down-regulated dataset included genes involved in several aspects of the immune response like immunoglobulin receptor activity and IgE binding thereby indicating a compromise in the immune defense mechanism(s). Conserved transcription factor binding sites were identified in identically clustered genes within a common GO term and SREBP-1 and FOXJ2 depicted increased occupation of their respective binding elements in the presence of TNFalpha. The interacting network of "lipid metabolism, small molecule biochemistry" was derived to be significantly overrepresented that correlated well with the top canonical pathway of "biosynthesis of steroids". CONCLUSIONS: TNFalpha alters the transcriptome profiling within HepG2 cells with an interesting catalog of genes being affected and those involved in lipid and steroid metabolism to be the most favored. This study represents a composite analysis of the effects of TNFalpha in HepG2 cells that encompasses the altered transcriptome profiling, the functional analysis of the up- and down- regulated genes and the identification of conserved transcription factor binding sites. These could possibly determine TNFalpha mediated alterations mainly the phenotypes of hepatic steatosis and fatty liver associated with several hepatic pathological states

Transcriptomic and metabolomic approaches to investigate molecular responses of human cell lines exposed to flame retardants

With intensive and global usage, flame retardants (FRs) have played critical roles in the prevention of fires for decades. However, there are increasing concerns about the potential adverse effects of these chemicals due to the well documented environmental and human exposures to FRs. To date, relatively little is known about the molecular mechanisms of the potential toxic effects of human exposure to FRs. In this study, microarray-based transcriptomics and direct injection mass spectrometry based metabolomics were employed to investigate the molecular responses of human lung cancer cells (A549) and human hepatoma cells (HepG2/C3A) exposed to a range of sub-lethal concentrations of hexabromocyclododecane (HBCD), tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP) and a mixture of FRs at equivalent concentrations to those found in typical household dust. Combined with the quantification of FRs levels in cells after exposure, this work using the non-targeted capabilities of multi-omics approaches has revealed that at the concentrations investigated, and which are relevant to human exposures, significant molecular perturbations are not induced by exposure to the FRs under study. The results from this thesis are beneficial for both understanding the potential mechanisms of effects of human exposure to FRs and for future risk assessment of these chemicals

Age and Diet Affect Gene Expression Profiles in Canine Liver Tissue

BACKGROUND: The liver plays a central role in nutrient and xenobiotic metabolism, but its functionality declines with age. Senior dogs suffer from many of the chronic hepatic diseases as elderly humans, with age-related alterations in liver function influenced by diet. However, a large-scale molecular analysis of the liver tissue as affected by age and diet has not been reported in dogs. METHODOLOGY/PRINCIPAL FINDINGS: Liver tissue samples were collected from six senior (12-year old) and six young adult (1-year old) female beagles fed an animal protein-based diet (APB) or a plant protein-based diet (PPB) for 12 months. Total RNA in the liver tissue was extracted and hybridized to Affymetrix GeneChip® Canine Genome Arrays. Using a 2.0-fold cutoff and false discovery rate <0.10, our results indicated that expression of 234 genes was altered by age, while 137 genes were differentially expressed by diet. Based on functional classification, genes affected by age and/or diet were involved in cellular development, nutrient metabolism, and signal transduction. In general, gene expression suggested that senior dogs had an increased risk of the progression of liver disease and dysfunction, as observed in aged humans and rodents. In particular for aged liver, genes related to inflammation, oxidative stress, and glycolysis were up-regulated, whereas genes related to regeneration, xenobiotic metabolism, and cholesterol trafficking were down-regulated. Diet-associated changes in gene expression were more common in young adult dogs (33 genes) as compared to senior dogs (3 genes). CONCLUSION: Our results provide molecular insight pertaining to the aged canine liver and its predisposition to disease and abnormalities. Therefore, our data may aid in future research pertaining to age-associated alterations in hepatic function or identification of potential targets for nutritional management as a means to decrease incidence of age-dependent liver dysfunction

Metabolomic profile and Bioassay-guided Phytochemical analysis of the Stems from Cissus trifoliata, evaluation of their Antibacterial and Cytotoxic activity, and determination of the Mechanism of Action of one active compound

Objectives and methods: Bioprospecting the metabolic profile of medicinal plants has provided a reliable resource for drug discovery and advancements in biomedical research. Cissus trifoliata (L.) L belongs to the Vitaceae family and is an important medicinal plant used in Mexico for the management of infectious diseases and tumors. The present study aimed to identify the metabolic profile of the extracts from the stems of C. trifoliata and evaluate their antibacterial and cytotoxic activities. Additionally, to identify the molecules that contribute to their biological activity and to explore the mechanism of action of one active compound. The hexane, CHCl3-MeOH, and aqueous extracts were prepared from the stems of C. trifoliata and their metabolic profile was investigated by column chromatography, NMR, GC-MS, and LC-MS. The antibacterial activity was determined by the broth microdilution method and the cytotoxicity against cancer cell lines using the MTS proliferation assay. A bioassay-guided study of the CHCl3-MeOH extract was performed using WST-1 to identify the active constituents responsible for the antiproliferative effects against cancer cells. Additionally, microarrays were used to identify the mechanism of action of one active compound against prostate cancer cells. Contribution and conclusions: The metabolic profile of C. trifoliata stems was constituted of polyphenols (36%), terpenes (28%), fatty acids (18%), simple phenols (9%) and alkanes (9%). The pathway analysis indicated the high production of stilbenes, flavonoids, and sterols. The extracts showed no antibacterial activity (MIC > 500 µg/ml), but high cytotoxic effects against cancer cells (IC50 ≤ 30 µg/ml). The hexane and aqueous extracts showed high antiproliferative activity against cancer cells from the liver (Hep3B, HepG2) and breast (MCF7). The bioactivity of these extracts was related to the synergistic effect of the triterpenes, sterols, flavonols, and stilbenes with cytotoxic, antiproliferative, and antiestrogenic activities. The bioassay-guided study of the CHCl3-MeOH extract allowed the identification of two active fractions. Both showed significative reduction (p >0.05) of cell viability on PC3 and MCF7 cancer cells at a concentration of 100 µg/ml. The cytotoxic activity was related to the synergistic anticancer effects of the mixture of coumaric acid, kaempferol, apigenin, hydroxyursolic, ursolic, and betulinic acid plus the stilbenes resveratrol, piceatannol, and viniferin. The stilbene that characterizes the Vitaceae plants is the resveratrol, thus was selected for the study of the mechanism of action. To carry out the microarray assay, PC3 cells were exposed to a non-cytotoxic inhibitory concentration (IC25) of resveratrol. Results showed that this phenolic compound induced significative transcriptional changes (2-fold) in 847 genes. The functional analysis suggested that resveratrol influences differentiation and impair cancer stemness by induction of the transcription factors POU4F2, KLF14, Hox-A3, and repression of Nanog. Resveratrol also affected cellular metabolism by upregulation of SIRT5 and repressed genes of the cancer pathways of TGF-β, Notch, PI3K/Akt, insulin/IGF-1, and MDM4/p53. Overall, the metabolic profile and biological evaluation of the stems from C. trifoliata correlate with high anticancer activity and together with the molecular mechanism of the identified bioactive constituents explain its traditional use in the management of tumors

Toxicogenomic Biomarkers Associated with PAH Carcinogenic Potential in a 3D in Vitro Bronchial Epithelial Model

The environmental health science community recognizes polycyclic aromatic hydrocarbons (PAHs) as a re-emerging class of environmental pollutants due to their persistence and prominence in mixtures of concern. Due to their widespread distribution in the environment, exposure to PAHs often occur as complex chemical mixtures. Exposures are linked to numerous adverse health outcomes in humans, with cancer as the greatest concern. Current assessment of cancer risk for PAHs involves testing individual compounds in a two-year rodent bioassay. These studies are time and resource-intensive, and often lack reproducibility or concordance. Furthermore, they require extrapolation of effects to humans, leading to further uncertainties regarding species-specific biology and chemical mode of action (MOA). The primary method for estimating cancer risk of PAH mixtures is the relative potency factor (RPF) approach in which mixtures are evaluated based on a subset of individual component PAHs compared to benzo[a]pyrene (BAP) as a surrogate or reference. However, we and others have found this approach has proved to be inadequate for predicting carcinogenicity of PAH mixtures and certain individual PAHs, particularly those that function through alternate pathways or exhibit greater promotional capacity compared to BAP. Furthermore, the specific mechanisms by which environmental exposures to PAHs may cause cardio-respiratory diseases and increase cancer risk remains poorly understood. In this dissertation, we employed a 3D, organotypic human in vitro bronchial epithelial culture (HBEC) model to address these gaps in knowledge. First, a comparative transcriptomic evaluation was conducted to assess potential differences in mechanism of toxicity for two PAHs, benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC), compared to a complex PAH mixture based on short-term biosignatures identified from global gene expression profiling. Comparison of BAP and DBC gene signatures showed that a majority of genes (~60%) were uniquely regulated by treatment, including those enriched for cell cycle, hypoxia, oxidative stress, and inflammation. Gene networks involved in NRF2-mediated oxidative stress detoxification were upregulated by BAP, while DBC downregulated these same targets, suggesting a chemical-specific pattern in transcriptional regulation involved in antioxidant response, potentially contributing to differences in PAH potency. These findings support research scrutinizing the applicability of the RPF, where assumptions of similar MOA are necessary for quantitative PAH cancer risk assessment. Next, we developed and refined an approach to utilize chemical-specific transcriptional patterns towards accurate classification of carcinogenic potency of PAHs and PAH mixtures. Systems biology information was collected from a human in vitro airway epithelial model exposed to a range of non-carcinogenic and carcinogenic PAHs and PAH mixtures. These transcriptional changes were evaluated for differentially enriched biological functions. Individual pathway-based gene sets were tested for optimal classification performance. Posterior probabilities of best performing gene sets were selected and integrated via Bayesian integration resulting in a 91% accurate classifier with four gene sets, including aryl hydrocarbon receptor signaling, regulation of epithelial mesenchymal transition, regulation of angiogenesis, and cell cycle G2-M. In addition, transcriptional benchmark dose modeling of (BAP) showed that the most sensitive gene sets were largely dissimilar from those that best classified PAH carcinogenicity challenging current assumptions that BAP carcinogenicity (and subsequent mode of action) is reflective of overall PAH carcinogenicity. Lastly, we evaluated molecular mechanisms related to PAH cancer risk through a two-tiered weighted gene co-expression network analysis (WGCNA) two-tiered approach, first to identify gene sets co-modulated to RPF cancer risk and then to link genes to a more comprehensive list of regulatory values, including inhalation-specific risk values. Over 3,000 genes associated with processes of cell cycle regulation, inflammation, DNA damage, and cell adhesion processes were found to be co-modulated with increasing RPF with pathways for cell cycle S phase and cytoskeleton actin identified as the most significantly enriched biological networks correlated to RPF. These gene sets represent potential biomarkers that can be used to evaluate cancer risk associated with PAH mixtures. In this study, the results illustrated the utility of systems toxicology approaches in analyzing global gene expression towards chemical hazard assessment, and information obtained from these analyses could be used towards future predictive model development. This work expanded current understanding of early mechanisms involved in PAH toxicity and provided novel applications utilizing toxicogenomics and organotypic cell culture models for classification, modelling, and biomarker identification. Together, these advances support further development of alternative approaches for use in predictive and mechanistic toxicology towards chemical hazard assessment

A Systems Biology Approach Reveals the Role of a Novel Methyltransferase in Response to Chemical Stress and Lipid Homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors