1,721,151 research outputs found
Evaluation of a multiwell perfused bioreactor system for the long-term culture of mouse hepatocytes
Get PDFLiver is the primary organ for xenobiotic metabolism and in vitro models that faithfully recapitulate its function are needed for medium/high-throughput toxicity studies. We evaluated a three-dimensional multiwell perfused bioreactor system as a model of murine liver. System parameters (flow rate, seeding procedure and cell density, scaffold matrix, and media composition) were varied to identify optimal conditions for mouse hepatocytes. Biochemical assays (pyruvate, lactate, urea, albumin, and intracellular ATP) indicated that the three-dimensional system improves functionality of hepatocytes cultured for 3-5 days. We also tested hepatocyte's basal and inducible metabolism using model toxicants acetaminophen and WY-14,643. Finally, we cultured non-parenchymal cells with mouse hepatocytes to determine whether it may improve the functionality of cells. This work demonstrates that the three-dimensional multiwell perfused bioreactor seeded with mouse hepatocytes is a useful model for short-term studies; however, improvements in the system are needed for it to become a promising sub-chronic model
Genome-level analysis of genetic regulation of liver gene expression networks
Get PDFThe liver is the primary site for metabolism of nutrients, drugs and chemical agents. While metabolic pathways are complex and tightly regulated, genetic variation among individuals, reflected in variation in gene expression levels, introduces complexity into research on liver disease. This study aimed to dissect genetic networks that control liver gene expression by combining large-scale quantitative mRNA expression analysis with genetic mapping in a reference population of BXD recombinant inbred mouse strains for which extensive SNP, haplotype and phenotypic data is publicly available. We profiled gene expression in livers of naive mice of both sexes from C57BL/6J, DBA/2J, B6D2F1, and 37 BXD strains using Agilent oligonucleotide microarrays. This data was used to map quantitative trait loci (QTLs) responsible for variation in expression of about 19,000 transcripts. We identified polymorphic cis- and trans-acting loci, including several loci that control expression of large numbers of genes in liver
Genetic regulation of sex-specific gene expression in mouse liver
Get PDFSexual dimorphism in the expression of many genes is thought to play an important role in disease susceptibility, drug metabolism, and xenobiotic response in both humans and other species. While previous research has explored the relationship between phenotypes and sex-dependent differences in expression of individual genes, this study dissected the genetic underpinnings that control sex-specific gene expression in mouse liver. We performed genetic mapping of genome-wide liver mRNA expression data in naïve male and female mice from C57BL/6J, DBA/2J, B6D2F1, and 37 BXD strains. Thousands of liver transcripts exhibited considerable differences in expression between females and males. An array permutation based functional analysis identified several xenobiotic metabolism pathways, which are strongly dependent on subject's sex. Furthermore, expression quantitative trait locus (eQTL) mapping identified several eQTLs that are major sex-specific regulators of gene expression in mouse liver and the candidate genes that are likely to be the regulators for these loci were revealed. Co-expressed genes were shown to be more likely to be involved in similar functions, supporting the hypothesis of "guilt by association". Conclusion: This study provided more evidence in the sexually dimorphic gene expression in the liver, which can convey important implications to toxicological and pharmaceutical studies
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Endocrine disrupting potential of environmental chemicals characterized by high-throughput screening
Get PDFOver the past 20 years, an increased focus on detecting environmental chemicals that pose a risk of endocrine disruption and congressional legislation have driven the creation of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). Several thousand chemicals are subject to the EDSP, which will require millions of dollars and decades to process using current test batteries. In order to identify opportunities for increased chemical throughput, we initially investigated how well EPA ToxCast in vitro high-throughput screening (HTS) assays relevant for estrogen, androgen, steroidogenic, and thyroid disrupting mechanisms could identify compounds relative to in vitro and in vivo data collected from studies related to the EDSP Tier 1 screen. An iterative, balanced optimization model was implemented and indicated that ToxCast HTS assays measuring estrogen receptor (ER) and androgen receptor (AR) activation classify compounds with estrogenic and androgenic activity in guideline studies with a high degree of accuracy, respectively. The ER signaling pathway involves a wide array of molecular initiating events and cellular processes. This dissertation examined whether active chemicals in ToxCast ER transactivation assays could indicate chemical-induced upregulation of the ER pathway through ligand binding leading to induced changes in T47D growth kinetics. Considering the complex set of ER in vitro assays in toto increased the overall sensitivity of detection for ER reference chemicals. In addition, this research highlighted important aspects of the biological response such as non-ER specificity in the cell growth assay. These nuances are likely important considerations for the construction of a predictive model. In effort to accurately predict the estrogenic potential of environmental chemicals in a high-throughput format, multiple orthogonal in vitro ER assays were used to develop a predictive model for ~2000 chemicals. Model results indicate a high degree of predictivity for both the uterotrophic in vivo assay and ER reference chemicals. The information provided by the model will aid in understanding how environmental chemicals contribute to endocrine-related human health consequences and predict the estrogenic potential of chemicals through the use of in vitro assays, limiting the need to run more costly and animal-intensive in vivo bioassays.Doctor of Philosoph
Genome-wide analysis of transcriptional regulation in the murine liver
Get PDFThe liver is a primary organ for toxicant metabolism in the body. This metabolism in influenced by genetic polymorphisms that can alter protein structure and influence gene expression levels in an allele specific manner. These polymorphisms produce significant differences in toxicant metabolism across human populations and an understanding of their influence on gene expression will improve our ability to predict toxic outcomes from chemical exposures. In order to understand the influence of polymorphisms on gene expression across populations, we studied liver gene expression in a model system consisting of two panels of laboratory inbred mice. We performed gene expression quantitative trait locus mapping in both panels of mice to discover polymorphisms that influence gene expression through both local and distant mechanisms and found several regions of the genome that regulate large numbers of genes in the liver. We examined the effect of sex of liver gene expression in one of the mouse panels and found significant differences between the sexes in many genes involved in xenobiotic metabolism. We also found that while gene expression level differences between sexes are important, there are also important differences in correlation between sets of genes in each sex. Finally, we studied the effect miRNAs on gene expression levels in the mouse liver and found, surprisingly, that their effect is mild. This report adds several new discoveries to the literature on transcriptional regulation in the liver and improves our understanding of the complex factors that control constitutive gene expression
Evaluation of in vitro Toxicogenetic Models for Hepatotoxicity
Get PDFNumerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether high-throughput in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to: (1) determine whether the near-physiological maintenance of the cells isolated from different mouse inbred strains can be achieved, (2) evaluate whether viability and reproducibility of functionality be attained over subsequent isolations and (3) assess the utility of the model for toxicity screening. Our data suggest that cell function and expression of key liver specific genes of hepatocytes isolated from different strains is comparable. These experiments open new opportunities for high-throughput and low-cost in vitro assays that may be used for studies of toxicity in a genetically diverse population.Master of Science in Public Healt
Role of nuclear receptor-independent pathways in the mechanism of action of peroxisome proliferators
Get PDFPeroxisome proliferators are a structurally diverse group of chemicals that are non-genotoxic hepatocarcinogens in rodents. For decades there has been controversy surrounding these compounds because of the uncertainty of human risk, high potential for exposure and insufficient understanding of their mechanism of action in rodents. Two key molecular pathways are thought to be important in the mode of action: activation of the nuclear receptor PPARα in liver parenchymal cells, and activation of Kupffer cells, which do not express PPARα. In hepatocytes, PPARα mediates peroxisome induction, increased fatty acid metabolism and alterations in gene expression. Furthermore, activation of the PPARα is required for peroxisome proliferator-induced carcinogenesis. In Kupffer cells, acute administration of peroxisome proliferators stimulates oxidant production and mitogenic cytokine release, as well as activation of NFκB, a transcription factor implicated in cell proliferation and apoptosis. The role that Kupffer cells play in chronic effects of peroxisome proliferators is not yet known. We hypothesized that peroxisome proliferators activate Kupffer cells to produce oxidants that may be involved in oxidative cellular damage, and that mediate cytokine production. The cytokines stimulate proliferative and anti-apoptotic effects of these chemical agents. To test this hypothesis, we first evaluated whether peroxisome proliferators cause a sustained increase in reactive oxygen species (ROS) in rodent liver. In vivo measurements of ROS in PPARα -null or NADPH oxidase-deficient (p47phox-null) mice following sub-acute treatment with di-(2-ethylhexyl) phthalate (DEHP) or 4-chloro-6-(2,3- xylidino)-2-pyrimidinylthio acetic acid (WY-14,643), both model peroxisome proliferators revealed a persistent elevation in oxidant production with parenchymal cells, not Kupffer cells as the primary molecular source. Next, the role of Kupffer cell oxidants and PPARα in mediating proliferative, apoptotic and oxidative stress responses was assessed. Findings from a five month WY-14,643 feeding study suggest that NADPH oxidase is not required for increased hepatocellularl proliferation or DNA damage, but may be important to antiapoptotic effects. Finally, gene expression profiling revealed a temporal shift from Kupffer cell to PPARα-dependence of peroxisome proliferator-induced changes. Collectively, our findings demonstrate that Kupffer cell-mediated events play an important role in early responses, but are short-lived and likely not required for chronic effects of peroxisome proliferators, including hepatocarcinogenesis
Comparative Analysis of Epigenetic and Gene Expression Endpoints Between Tumorous and Non-tumorous Tissues from HCV-positive Patients with Hepatocellular Carcinoma
Get PDFTranscriptional silencing induced by promoter CpG island hypermethylation is an important epigenetic mechanism of hepatocarcinogenesis. The goals of our study were to examine promoter methylation and mRNA levels of candidate genes, as well as global changes in DNA methylation, in a cohort of HCV-positive HCC patients from Japan. Methylation-specific PCR was used to assess the methylation status of seven cancer-related genes, while the methylation status of long interspersed nuclear elements was used as marker of global genomic methylation, in tissues obtained from patients who underwent tumor resection surgery. Methylation frequencies for most of the genes were significantly higher in tumorous versus non-tumorous tissues. The methylation status of only three genes correlated with reduced mRNA levels. Genomic DNA was significantly more hypomethylated in tumorous tissues, and was associated with shorter recurrence but not with clinicopathological variables. In summary, this study establishes an aberrant gene-specific and global methylation profile in HCV-associated HCCs
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