Transcription profiling in primary mouse hepatocytes discriminates between true and false genotoxic compounds
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Danyel Jennen
PROVIDER: E-MEXP-2539 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The prediction of possible carcinogenicity of chemicals for humans represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk at only limited reliability while simultaneously being expensive and long-lasting. In order to seek for alternatives, in the present study, the ability of a transcriptomics-based primary mouse hepatocyte model to classify carcinogens by their modes of action was evaluated. As it is obvious that exposure will induce a cascade of gene expression modifications, in particular, the influence of exposure time in vitro on discriminating genotoxic (GTX) carcinogens from non-genotoxic (NGTX) carcinogens class prediction was investigated. Primary mouse hepatocytes from male C57Bl6 mice were treated for 12, 24, 36 and 48 h with two GTX and two NGTX carcinogens. For the purpose of validation, two additional GTX compounds were studied after incubation periods of 24 and 48 h. Immunostaining of ?H2AX foci was applied to phenotypically verify DNA damage. Whole genome gene expression modifications were analyzed by means of Affymetrix mouse genome 430 2.0 microarrays. Datasets were normalized using RMA and differentially expressed genes were selected for assessing class prediction. The ?H2AX assay confirmed significant induction of DNA damage after treatment with GTX compounds, whereas NGTX compounds showed no activity beyond background levels. Discrimination of GTX and NGTX carcinogens by Prediction Analysis of Microarray (PAM) was validated and resulted in a perfect classification. The present study shows that gene expression profiling in primary mouse hepatocytes is promising for discriminating between GTX and NGTX compounds and that this classification improves with increasing treatment period.
2010-06-09 | E-MEXP-2209 | biostudies-arrayexpress
Project description:Gene expression profiling in primary mouse hepatocytes discriminates true from false-positive genotoxic compounds with addtional compounds
2011-03-22 | E-MEXP-2636 | biostudies-arrayexpress
Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes expressing HBV to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes infected with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV). Samples were collected either 24h or 48h after infection. cDNA libraries were sequenced two times using the Illumina HiSeq or Illumina NextSeq platform to generate either 1x50bp or 1x75bp reads. Reads from each sequencing run were mapped using the STAR aligner, and output BAMs were merged into a single BAM for each sample. The merged BAM was further analyzed in R using the GenomicAlignments package to quantify number of reads per transcript and DESeq2 to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 24h and 48h post-infection. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of HBV-expressing and non-expressing primary rat hepatocytes were generated, in triplicate, 24h and 48h post-infection using Illumina HiSeq 2500 and NextSeq 500 instruments.
2016-01-15 | E-GEOD-66566 | biostudies-arrayexpress
Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes either expressing HBV or not over a series of time points to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes (PRH) over the course of 72hr. PRH were collected immediately after isolation (0hr), or 24hr, 48hr, or 72hr after plating. In addition, PRH were infected 24hr after plating with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV) and collected at 48hr after plating (24hr after infection) or 72hr after plating (48hr after infection). cDNA libraries were sequenced using the Illumina NextSeq 500 platform to generate either 1x75bp reads. Reads were mapped using the STAR aligner, and output BAMs were further analyzed in R using the GenomicAlignments package, to quantify number of reads per transcript, and DESeq2, to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 48hr and 72hr. In addition, we noted considerable change to the hepatocyte transcriptome as a direct result of the isolation/plating procedure, regardless of the presence of HBV. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of cultured primary rat hepatocytes were generated, in triplicate, using the Illumina NextSeq 500 platform from freshly isolated cells (0hr), 24hr, 48hr, or 72hr after plating, and with or without expression of HBV 48hr or 72hr after plating.
2016-01-20 | E-GEOD-68112 | biostudies-arrayexpress
Project description:Gene expression profiles of sandwich-cutlured primary rat hepatocytes exposed to 5 mM and 10 mM acetaminophen were used in a parallelogram approach in order to compare gene expression responses between rat and human using in vitro cellular models, heaptocytes, and between rat in vitro and in vivo Experiment Overall Design: Samples were retrieved from acetaminophen treated rat hepatocyte cultures from 3 rats (3 biological replicates). Rat hepatocytes from each replicate were cultured in two culture conditions, each treated with 0, 5, and 10 mM acetaminophen for 24 h. This resulted in (3 rats x 2 culture conditions x 3 doses) 18 dual channel arrays on which control (0 mM acetaminophen) and reference samples (0, 5, and 10 mM acetaminophen) were hybridized
2009-01-14 | E-GEOD-13465 | biostudies-arrayexpress
Project description:Drug-induced hepatotoxicity is a leading cause of attrition of candidate drugs in drug development. Therefore new screening methods are necessary which predict these hazards more accurate and earlier in the drug development process. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as 'the gold standard'. However, the use of these hepatocytes is hindered by their scarcity and major inter-individual variation. These limitations may be overcome with use of primary mouse hepatocytes. Within this context changes in protein expressions in primary mouse hepatocytes, after exposure to cyclosporin A were studied using differential gel electrophoresis. Thereafter, the mRNA expression levels of these deregulated proteins from cyclosporin A-treated cells were analyzed. Cyclosporin A induced ER stress and altered the ER-Golgi transport, which may alter vesicle mediated transport and protein secretion. Moreover are the differentially expressed proteins observed upon challenge by cyclosporin A, associated with cholestatic mechanisms. For each biological experiment, one hybridization was conducted and one sample per array. In total, 6 arrays were used for 2 different conditions (Csa or control at 48 hours).
2013-04-01 | E-GEOD-33742 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2016-01-20 | E-GEOD-68113 | biostudies-arrayexpress
Project description:Glucocorticoids are widely used therapeutically to suppress inflammatory/immune responses and most of their effects are produced either by altering transcription of specific genes directly, or by altering the expression of transcription factors that subsequently alter the expression of downstream genes. Relevant data from previous studies indicate that the number of genes regulated by glucocorticoid receptor exceeds 4000 in cells and that 358 different genes are regulated in the liver of adrenalectomized males rats treated with a chronic infusion of methylprednisolone for up to 1 week . However, differences in gene expression between males and females in response to glucocorticoid treatment in isolated hepatocytes are not known. Livers were isolated from adult male and female Sprague-Dawley rats and digested with the collagenase perfusion method developed by Berry and Friend (J Cell Biol 43, 506-520;1969). After collagenase treatment, the liver was excised, minced in balanced salt solution, and centrifuged at 50 g for 3 min. Immediately after isolation, hepatocytes were resuspended in Williams E Medium containing penicillin (100 units/ml), streptomycin (100 M-NM-<g/ml), 2 mM glutamine pH 7.4, and 10% fetal bovine serum and plated on collagen-coated 94 x 16 mm cell culture dishes and maintained for 24 hours in a 95% airM-bM-^@M-^S5% CO2, 37M-BM-0C incubator. The medium was then changed to free serum Williams E Medium and maintained in culture for an additional 24 hours. Hepatocytes isolated from male and rats were treated with vehicle (0.01% ethanol) or dexamethasone (100 nm) for 6 hours. Total RNA from cells were extracted by using the RNeasy Midi Kit (Qiagen) according to the manufacturerM-bM-^@M-^Ys instructions. All samples were treated with the RNase-free DNase set (Qiagen) and were kept at -80M-BM-0C until labeling and hybridization.
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2022-08-02 | PXD032214 | Pride