Application of toxicogenomics to determine mechanism of tumor modulation by dietary indole phytochemicals in hepatocellular carcinoma

Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM), a primary I3C derivative in vivo, are known chemopreventive agents available as dietary supplements. I3C was found to suppress or enhance tumors in several animal models. Chemoprotection is attributed to the ability of indoles to alter carcinogen metabolism effectively blocking initiation. However, mechanisms for enhancement are unknown. In rainbow trout, I3C promotes hepatocarcinogenesis at concentrations that differentially activated estrogen receptor (ER) or aryl hydrocarbon receptor (AhR)-mediated responses. The relative importance of these pathways was evaluated using toxicogenomics in juvenile trout exposed to I3C and DIM compared to ER and AhR agonists, 17β-estradiol (E2) and β-naphthoflavone. I3C and DIM acted transcriptionally similar to E2 by correlation analysis indicating I3C promotes hepatocarcinogenesis through estrogenic mechanisms in trout and suggesting DIM may be a more potent tumor promoter. The ability of DIM to enhance hepatocarcinogenesis was evaluated in trout initiated with aflatoxin i compared to E2. Tumor incidence was significantly elevated in initiated trout fed 400 ppm DIM. To evaluate the mechanism of promotion, hepatic gene expression profiles were examined in animals on promotional diets during the course of tumorigenesis and in hepatocellular carcinomas (HCCs) using a trout 70-mer oligonucleotide array. DIM altered gene expression profiles similar to E2 at all timepoints measured. Expression profiles in trout HCC were similar to transcriptional changes reported in human and rodent HCC further supporting the validity of the trout tumor model. Further, transcription in HCCs from DIM and E2 treatments indicated decreased invasive potential compared to control HCCs. These findings confirm importance of estrogenic signaling in the mechanism of indoles and indicate a possible dual effect that enhances tumor incidence and decreases potential for metastasis. The estrogenic response of DIM in trout liver was characterized by measuring its in vitro biological activity, its ability to bind to ER and its potential for metabolism to estrogenic metabolites. The results support the role of DIM as a potent estrogen in trout liver that may require metabolism for ligand-dependent activation of ER or activation of extracellular signaling pathways for ligand-independent activation of ER. It is likely that multiple mechanisms are involved for DIM estrogenic activity

Three human cell types respond to multi-walled carbon nanotubes and titanium dioxide nanobelts with cell-specific transcriptomic and proteomic expression patterns

The growing use of engineered nanoparticles (NPs) in commercial and medical applications raises the urgent need for tools that can predict NP toxicity. We conducted global transcriptome and proteome analyses of three human cell types, exposed to two high aspect ratio NP types, to identify patterns of expression that might indicate high vs. low NP toxicity. Three cell types representing the most common routes of human exposure to NPs, including macrophage like (THP-1), small airway epithelial (SAE), and intestinal (Caco-2/HT29-MTX) cells, were exposed to TiO2 nanobelts (TiO2-NB; high toxicity) and multi-walled carbon nanotubes (MWCNT; low toxicity) at low (10 μg/ml) and high (100 μg/ml) concentrations for 1 and 24 h. Unique patterns of gene and protein expressions were identified for each cell type, with no differentially expressed (p<0.05, 1.5-fold change) genes or proteins overlapping across all three cell types. While unique to each cell-type, the early response was primarily independent of NP type, showing similar expression patterns in response to both TiO2-NB and MWCNT. The early response might therefore indicate a general response to insult. In contrast, the 24 h response was unique to each NP type. The most significantly (p<0.05) enriched biological processes in THP-1 cells indicated TiO2-NB regulation of pathways associated with inflammation, apoptosis, cell cycle arrest, DNA replication stress and genomic instability, while MWCNT regulated pathways indicating increased cell proliferation, DNA repair and anti-apoptosis. These two distinct sets of biological pathways might therefore underlie cellular responses to high and low NP toxicity, respectively

Polycyclic aromatic hydrocarbons as skin carcinogens:Comparison of benzo [a]pyrene, dibenzo[def,p]chrysene and three environmental mixtures in the FVB/N mouse

The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4 nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by (32)P post- labeling, did not correlate with tumor incidence. PAH- dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p<0.05), showed DBC and BaP to cluster distinct from PAH mixtures and each other. BaP and mixtures up-regulated phase 1 and 2 metabolizing enzymes while DBC did not. The carcinogenicity with DBC and two of the mixtures was much greater than would be predicted based on published Relative Potency Factors (RPFs)

Folyóirat vagy gyűjteményes kötet? (Csokonai Diétai Magyar Múzsája)

BACKGROUND: The complex interplay between viral replication and host immune response during infection remains poorly understood. While many viruses are known to employ anti-immune strategies to facilitate their replication, highly pathogenic virus infections can also cause an excessive immune response that exacerbates, rather than reduces pathogenicity. To investigate this dichotomy in severe acute respiratory syndrome coronavirus (SARS-CoV), we developed a transcriptional network model of SARS-CoV infection in mice and used the model to prioritize candidate regulatory targets for further investigation. RESULTS: We validated our predictions in 18 different knockout (KO) mouse strains, showing that network topology provides significant predictive power to identify genes that are important for viral infection. We identified a novel player in the immune response to virus infection, Kepi, an inhibitory subunit of the protein phosphatase 1 (PP1) complex, which protects against SARS-CoV pathogenesis. We also found that receptors for the proinflammatory cytokine tumor necrosis factor alpha (TNFα) promote pathogenesis, presumably through excessive inflammation. CONCLUSIONS: The current study provides validation of network modeling approaches for identifying important players in virus infection pathogenesis, and a step forward in understanding the host response to an important infectious disease. The results presented here suggest the role of Kepi in the host response to SARS-CoV, as well as inflammatory activity driving pathogenesis through TNFα signaling in SARS-CoV infections. Though we have reported the utility of this approach in bacterial and cell culture studies previously, this is the first comprehensive study to confirm that network topology can be used to predict phenotypes in mice with experimental validation

Early life perfluorooctanesulphonic acid (PFOS) exposure impairs zebrafish organogenesis

Additional authors (Zengxin Gai and Xue Ma) appear and the author order is revised on the published version of this article.As a persistent organic contaminant, perfluorooctanesulphonic acid (PFOS) has been widely detected in the environment, wildlife, and humans. The present study revealed that zebrafish embryos exposed to 16 µM PFOS during a sensitive window of 48-96 hour post-fertilization (hpf) disrupted larval morphology at 120 hpf. Malformed zebrafish larvae were characterized by uninflated swim bladder, less developed gut, and curved spine. Histological and ultrastructural examination of PFOS-exposed larvae showed structural alterations in swim bladder and gut. Whole genome microarray was used to identify the early transcripts dysregulated following exposure to 16 µM PFOS at 96 hpf. In total, 1,278 transcripts were significantly misexpressed (p < 0.05) and 211 genes were changed at least two-fold upon PFOS exposure in comparison to the vehicle exposed control group. A PFOS-induced network of perturbed transcripts relating to swim bladder and gut development revealed that misexpression of genes were involved in organogenesis. Taken together, early life stage exposure to PFOS perturbs various molecular pathways potentially resulting in observed defects in swim bladder and gut development.This is an author's peer-reviewed manuscript. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/aquatic-toxicology/.Keywords: Perfluorooctanesulfonic acid, Zebrafish embryo, Developmental toxicity, Swim bladder, GutKeywords: Perfluorooctanesulfonic acid, Zebrafish embryo, Developmental toxicity, Swim bladder, Gu

Release of Severe Acute Respiratory Syndrome Coronavirus Nuclear Import Block Enhances Host Transcription in Human Lung Cells

The severe acute respiratory syndrome coronavirus accessory protein ORF6 antagonizes interferon signaling by blocking karyopherin-mediated nuclear import processes. Viral nuclear import antagonists, expressed by several highly pathogenic RNA viruses, likely mediate pleiotropic effects on host gene expression, presumably interfering with transcription factors, cytokines, hormones, and/or signaling cascades that occur in response to infection. By bioinformatic and systems biology approaches, we evaluated the impact of nuclear import antagonism on host expression networks by using human lung epithelial cells infected with either wild-type virus or a mutant that does not express ORF6 protein. Microarray analysis revealed significant changes in differential gene expression, with approximately twice as many upregulated genes in the mutant virus samples by 48 h postinfection, despite identical viral titers. Our data demonstrated that ORF6 protein expression attenuates the activity of numerous karyopherin-dependent host transcription factors (VDR, CREB1, SMAD4, p53, EpasI, and Oct3/4) that are critical for establishing antiviral responses and regulating key host responses during virus infection. Results were confirmed by proteomic and chromatin immunoprecipitation assay analyses and in parallel microarray studies using infected primary human airway epithelial cell cultures. The data strongly support the hypothesis that viral antagonists of nuclear import actively manipulate host responses in specific hierarchical patterns, contributing to the viral pathogenic potential in vivo. Importantly, these studies and modeling approaches not only provide templates for evaluating virus antagonism of nuclear import processes but also can reveal candidate cellular genes and pathways that may significantly influence disease outcomes following severe acute respiratory syndrome coronavirus infection in vivo

MicroRNAs control neurobehavioral development and function in zebrafish

microRNAs (miRNAs) have emerged as regulators of a broad spectrum of neurodevelopmental processes, including brain morphogenesis, neuronal differentiation, and survival. While the role of miRNAs in establishing and maintaining the developing nervous system is widely appreciated, the developmental neurobehavioral role of miRNAs has yet to be defined. Here we show that transient disruption of brain morphogenesis by ethanol exposure results in behavioral hyperactivity in larval zebrafish challenged with changes in lighting conditions. Aberrations in swimming activity persist in juveniles that were developmentally exposed to ethanol. During early neurogenesis, multiple gene expression profiling studies revealed widespread changes in mRNA and miRNA abundance in ethanol-exposed embryos. Consistent with a role for miRNAs in neurobehavioral development, target prediction analyses identified multiple miRNAs misexpressed in the ethanol-exposed cohorts that were also predicted to target inversely expressed transcripts known to influence brain morphogenesis. In vivo knockdown of miR9/9* or miR-153c persistently phenocopied the effect of ethanol on larval and juvenile swimming behavior. Structural analyses performed on adults showed that repression of miR-153c during development impacts craniofacial skeletal development. Together, these data support an integral role for miRNAs in the establishment of vertebrate neurobehavioral and skeletal systems.-Tal, T. L., Franzosa, J. A., Tilton, S. C., Philbrick, K. A., Iwaniec, U. T., Turner, R. T., Waters, K. M., Tanguay, R. L. MicroRNAs control neurobehavioral development and function in zebrafish. FASEB J. 26, 1452-1461 (2012). www.fasebj.orgKeywords: Brain, Morphogenesis, Alcohol, Conditional loss, Osteoblast differentiation, Gene expression, Microarray data, Neural progenitors, Ethanol exposure, Larval zebrafis

Mechanism-based classification of PAH mixtures to predict carcinogenic potential

We have previously shown that relative potency factors and DNA adduct measurements are inadequate for predicting carcinogenicity of certain polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures, particularly those that function through alternate pathways or exhibit greater promotional activity compared to benzo[a]pyrene (BaP). Therefore, we developed a pathway based approach for classification of tumor outcome after dermal exposure to PAH/mixtures. FVB/N mice were exposed to dibenzo[def,p]chrysene (DBC), BaP or environmental PAH mixtures (Mix 1-3) following a two-stage initiation/promotion skin tumor protocol. Resulting tumor incidence could be categorized by carcinogenic potency as DBC>>BaP=Mix2=Mix3>Mix1=Control, based on statistical significance. Gene expression profiles measured in skin of mice collected 12 h post-initiation were compared to tumor outcome for identification of short-term bioactivity profiles. A Bayesian integration model was utilized to identify biological pathways predictive of PAH carcinogenic potential during initiation. Integration of probability matrices from four enriched pathways (p<0.05) for DNA damage, apoptosis, response to chemical stimulus and interferon gamma signaling resulted in the highest classification accuracy with leave-one-out cross validation. This pathway-driven approach was successfully utilized to distinguish early regulatory events during initiation prognostic for tumor outcome and provides proof-of-concept for using short-term initiation studies to classify carcinogenic potential of environmental PAH mixtures. These data further provide a ‘source-to outcome’ model that could be used to predict PAH interactions during tumorigenesis and provide an example of how mode-of-action based risk assessment could be employed for environmental PAH mixtures.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Oxford University Press and can be found at: http://toxsci.oxfordjournals.org/.Keywords: mixtures, toxicogenomics, skin cancer, polycyclic aromatic hydrocarbons, modelin

A Network Integration Approach to Predict Conserved Regulators Related to Pathogenicity of Influenza and SARS-CoV Respiratory Viruses

Respiratory infections stemming from influenza viruses and the Severe Acute Respiratory Syndrome corona virus (SARS-CoV) represent a serious public health threat as emerging pandemics. Despite efforts to identify the critical interactions of these viruses with host machinery, the key regulatory events that lead to disease pathology remain poorly targeted with therapeutics. Here we implement an integrated network interrogation approach, in which proteome and transcriptome datasets from infection of both viruses in human lung epithelial cells are utilized to predict regulatory genes involved in the host response. We take advantage of a novel "crowd-based" approach to identify and combine ranking metrics that isolate genes/proteins likely related to the pathogenicity of SARS-CoV and influenza virus. Subsequently, a multivariate regression model is used to compare predicted lung epithelial regulatory influences with data derived from other respiratory virus infection models. We predicted a small set of regulatory factors with conserved behavior for consideration as important components of viral pathogenesis that might also serve as therapeutic targets for intervention. Our results demonstrate the utility of integrating diverse 'omic datasets to predict and prioritize regulatory features conserved across multiple pathogen infection models

Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses

ABSTRACT The broad range and diversity of interferon-stimulated genes (ISGs) function to induce an antiviral state within the host, impeding viral pathogenesis. While successful respiratory viruses overcome individual ISG effectors, analysis of the global ISG response and subsequent viral antagonism has yet to be examined. Employing models of the human airway, transcriptomics and proteomics datasets were used to compare ISG response patterns following highly pathogenic H5N1 avian influenza (HPAI) A virus, 2009 pandemic H1N1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV) infection. The results illustrated distinct approaches utilized by each virus to antagonize the global ISG response. In addition, the data revealed that highly virulent HPAI virus and MERS-CoV induce repressive histone modifications, which downregulate expression of ISG subsets. Notably, influenza A virus NS1 appears to play a central role in this histone-mediated downregulation in highly pathogenic influenza strains. Together, the work demonstrates the existence of unique and common viral strategies for controlling the global ISG response and provides a novel avenue for viral antagonism via altered histone modifications.IMPORTANCEThis work combines systems biology and experimental validation to identify and confirm strategies used by viruses to control the immune response. Using a novel screening approach, specific comparison between highly pathogenic influenza viruses and coronaviruses revealed similarities and differences in strategies to control the interferon and innate immune response. These findings were subsequently confirmed and explored, revealing both a common pathway of antagonism via type I interferon (IFN) delay as well as a novel avenue for control by altered histone modification. Together, the data highlight how comparative systems biology analysis can be combined with experimental validation to derive novel insights into viral pathogenesis