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We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for interpretation of plasma triglyceride (TG) decrease, we extracted 218 probe sets of rat hepatic genes from data of 15 drugs that decreased the plasma TG level but differentially affected food consumption. Pathway and gene ontology analysis revealed that the genes belong to amino acid metabolism, lipid metabolism and xenobiotics metabolism. Principal component analysis (PCA) showed that 12 out of 15 compounds were separated in the direction of PC1, and these 12 were separated in the direction of PC2, according to their hepatic gene expression profiles. It was found that genes with either large or small eigenvector values in principal component PC 2 were those reported to be regulated by peroxisome proliferator-activated receptor (PPAR)alpha or constitutive androstane receptor (CAR), respectively. In fact, WY-14,643, clofibrate, gemfibrozil and benzbromarone, reported to be PPARalpha activators, distributed to the former, whereas propylthiouracil, omeprazole, phenobarbital, thioacetamide, methapyrilene, sulfasalazine and coumarin did to the latter. We conclude that these identified 218 probe sets could be a useful source of biomarkers for classification of plasma TG decrease, based on the mechanisms involving PPARalpha and CAR.

The Toxicogenomics Project is a 5-year collaborative project by the Japanese government and pharmaceutical companies in 2002. Its aim is to construct a large-scale toxicology database of 150 compounds orally administered to rats. The test consists of a single administration test (3, 6, 9 and 24 h) and a repeated administration test (3, 7, 14 and 28 days), and the conventional toxicology data together with the gene expression data in liver as analyzed by using Affymetrix GeneChip are being accumulated. In the project, either methylcellulose or corn oil is employed as vehicle. We examined whether the vehicle itself affects the analysis of gene expression and found that corn oil alone affected the food consumption and biochemical parameters mainly related to lipid metabolism, and this accompanied typical changes in the gene expression. Most of the genes modulated by corn oil were related to cholesterol or fatty acid metabolism (e.g., CYP7A1, CYP8B1, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, squalene epoxidase, angiopoietin-like protein 4, fatty acid synthase, fatty acid binding proteins), suggesting that the response was physiologic to the oil intake. Many of the lipid-related genes showed circadian rhythm within a day, but the expression pattern of general clock genes (e.g., period 2, arylhydrocarbon nuclear receptor translocator-like, D site albumin promoter binding protein) were unaffected by corn oil, suggesting that the effects are specific for lipid metabolism. These results would be useful for usage of the database especially when drugs with different vehicle control are compared.

Human mesenchymal stem cells (hMSCs) are multipotent cells that differentiate into several cell types, and are expected to be a useful tool for cellular therapy. Although the hMSCs differentiate into osteogenic cells during early to middle stages, this differentiation capacity decreases during the late stages of cell culture. To test a hypothesis that there are biomarkers indicating the differentiation potential of hMSCs, we performed microarray analyses and profiled the gene expression in six batches of hMSCs (passages 4–28). At least four genes [necdin homolog (mouse) (NDN), EPH receptor A5 (EPHA5), nephroblastoma overexpressed gene (NOV) and runt-related transcription factor 2 (RUNX2)] were identified correlating with the passage numbers in all six batches. The results showed that the osteogenic differentiation capacity of hMSCs is down-regulated in the late stages of cell culture. It seemed that adipogenic differentiation capacity was also down-regulated in late stage of the culture. The cells in late stage are oligopotent and the genes identified in this study have the potential to act as quality-control markers of the osteogenic differentiation capacity of hMSCs. [ABSTRACT FROM PUBLISHER]

Background: Transcriptome data from quantitative PCR (Q-PCR) and DNA microarrays are typically obtained from a fixed amount of RNA collected per sample. Therefore, variations in tissue cellularity and RNA yield across samples in an experimental series compromise accurate determination of the absolute level of each mRNA species per cell in any sample. Since mRNAs are copied from genomic DNA, the simplest way to express mRNA level would be as copy number per template DNA, or more practically, as copy number per cell. Results: Here we report a method (designated the "Percellome" method) for normalizing the expression of mRNA values in biological samples. It provides a "per cell" readout in mRNA copy number and is applicable to both quantitative PCR (Q-PCR) and DNA microarray studies. The genomic DNA content of each sample homogenate was measured from a small aliquot to derive the number of cells in the sample. A cocktail of five external spike RNAs admixed in a dose-graded manner (dose-graded spike cocktail; GSC) was prepared and added to each homogenate in proportion to its DNA content. In this way, the spike mRNAs represented absolute copy numbers per cell in the sample. The signals from the five spike mRNAs were used as a dose-response standard curve for each sample, enabling us to convert all the signals measured to copy numbers per cell in an expression profile-independent manner. A series of samples was measured by Q-PCR and Affymetrix GeneChip microarrays using this Percellome method, and the results showed up to 90 % concordance. Conclusion: Percellome data can be compared directly among samples and among different studies, and between different platforms, without further normalization. Therefore, "percellome" normalization can serve as a standard method for exchanging and comparing data across different platforms and among different laboratories. [ABSTRACT FROM AUTHOR]

A large-scale transcriptome database of rat liver (TG-GATEs) has been established by the Toxicogenomics Project in Japan. In the present study, we focused on 8 hepatotoxic compounds within TG-GATEs, i.e., clofibrate, omeprazole, ethionine, thioacetamide, benzbromarone, propylthiouracil, Wy-14,643 and amiodarone, which induced coagulation abnormalities. Aspirin was selected as a reference compound that directly causes coagulation abnormality, but not through liver toxicity. In blood chemical examinations, for all the coagulopathic compounds there was little elevation of aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT), suggesting no severe cell death by treatment with the com- pounds. We extracted 344 probe sets from the data for these 8 typical drugs, which induced this phenotype at any time from 3 to 28 days of repeated administration. Principal component analysis using these probe sets clearly separated dose- and time-dependent clusters of the treated groups from their controls, except aspirin and propylthiouracil, both of which were considered to cause coagulopathy not due to their hepatotoxicity but due to their direct effects on the blood coagulation system. Reviewing the extracted genes, changes in lipid metabolism were found to be dominant. Genes related to blood coagulation were generally down-regulated by these drugs except that vitamin K epoxide reductase complex subunit 1 (Vkorc 1) like 1, a paralogous gene of Vkorc 1, was up-regulated. As expected, expression changes of these genes were least prominent in aspirin or propylthiouracil-treated liver. We concluded that these probe sets could be a good starting point in developing mechanism-based biomarkers for diagnosis or prognosis of hepatotoxicity-related coagulation abnormalities in the early stage of drug development. [ABSTRACT FROM AUTHOR]

Abstract: For assessing carcinogenicity in animals, it is difficult and costly, an alternative strategy has been desired. We explored the possibility of applying a toxicogenomics approach by using comprehensive gene expression data in rat liver treated with various compounds. As prototypic non-genotoxic hepatocarcinogens, thioacetamide (TAA) and methapyrilene (MP) were selected and 349 commonly changed genes were extracted by statistical analysis. Taking both compounds as positive with six compounds, acetaminophen, aspirin, phenylbutazone, rifampicin, alpha-naphthylisothiocyanate, and amiodarone as negative, prediction analysis of microarray (PAM) was performed. By training and 10-fold cross validation, a classifier containing 112 probe sets that gave an overall success rate of 95% was obtained. The validity of the present discriminator was checked for 30 chemicals. The PAM score showed characteristic time-dependent increases by treatment with several non-genotoxic hepatocarcinogens, including TAA, MP, coumarin, ethionine and WY-14643, while almost all of the non-carcinogenic samples were correctly predicted. Measurement of hepatic glutathione content suggested that MP and TAA cause glutathione depletion followed by a protective increase, but the protective response is exhausted during repeated administration. Therefore, the presently obtained PAM classifier could predict potential non-genotoxic hepatocarcinogenesis within 24h after single dose and the inevitable pseudo-positives could be eliminated by checking data of repeated administrations up to 28 days. Tests for carcinogenicity using rats takes at least 2 years, while the present work suggests the possibility of lowering the time to 28 days with high precision, at least for a category of non-genotoxic hepatocarcinogens causing oxidative stress. [Copyright &y& Elsevier]

Abstract: We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for diagnosis of hepatic phospholipidosis, we extracted 78 probe sets of rat hepatic genes from data of 5 drugs, amiodarone, amitriptyline, clomipramine, imipramine, and ketoconazole, which actually induced this phenotype. Principal component analysis (PCA) using these probes clearly separated dose- and time-dependent clusters of treated groups from their controls. Moreover, 6 drugs (chloramphenicol, chlorpromazine, gentamicin, perhexiline, promethazine, and tamoxifen), which were reported to cause phospholipidosis but judged as negative by histopathological examination, were designated as positive by PCA using these probe sets. Eight drugs (carbon tetrachloride, coumarin, tetracycline, metformin, hydroxyzine, diltiazem, 2-bromoethylamine, and ethionamide), which showed phospholipidosis-like vacuolar formation in the histopathology, could be distinguished from the typical drugs causing phospholipidosis. Moreover, the possible induction of phospholipidosis was predictable by the expression of these genes 24 h after single administration in some of the drugs. We conclude that these identified 78 probe sets could be useful for diagnosis of phospholipidosis, and that toxicogenomics would be a promising approach for prediction of this type of toxicity. [Copyright &y& Elsevier]

To identify candidate biomarker gene sets to evaluate the potential risk of chemical-induced glutathione depletion in livers, we conducted microarray analysis on rat livers administered with phorone (40, 120 and 400 mg/kg), a prototypical glutathione depletor. Hepatic glutathione content was measured and glutathione depletion-responsive gene probe sets (GSH probe sets) were identified using Affymetrix Rat Genome 230 2.0 GeneChip by the following procedure. First, probe sets, whose signal values were inversely correlated with hepatic glutathione content throughout the experimental period, were statistically identified. Next, probe sets, whose average signal values were greater than 1.5-fold compared to those of controls 3 hr after phorone treatment, were selected. Finally, probe sets without unique Entrez Gene ID were removed, ending up with 161 probe sets in total. The usefulness of the identified GSH probe sets was verified by a toxicogenomics database. It was shown that signal profiles of the GSH probe sets in rats treated with bromobenzene were strongly altered compared with other chemicals. Focusing on bromobenzene, time-course profiles of hepatic glutathione content and gene expression revealed that the change in gene expression profile was marked after the bromobenzene treatment, whereas hepatic glutathione content had recovered after initial acute depletion, suggesting that the gene expression profile did not reflect the hepatic glutathione content itself, but rather reflects a perturbation of glutathione homeostasis. The identified GSH probe sets would be useful for detecting glutathione-depleting risk of chemicals from microarray data. [ABSTRACT FROM AUTHOR]

ABC transporter A1 (ABCA1) mediates and rate-limits biogenesis of high density lipoprotein (HDL), and hepatic ABCA1 plays a major role in regulating plasma HDL levels. HDL generation is also responsible for release of cellular cholesterol. In peripheral cells ABCA1 is up-regulated by the liver X receptor (LXR) system when cell cholesterol increases. However, cholesterol feeding has failed to show a significant increase in hepatic ABCA1 gene expression, and its expression is up-regulated by statins (3-hydroy-3-methylglutaryl-CoA reductase inhibitors), suggesting distinct regulation. In this study we investigated the mechanism of regulation of the rat hepatic ABCA1 gene and identified two major ABCA1 transcripts and two corresponding promoter regions. Compactin activated the novel liver-type promoter in rat hepatoma McARH7777 cells by binding the sterol regulatory element-binding protein-2 (SREBP-2). In contrast, compactin repressed the previously identified peripheral-type promoter in an LXR-responsive element-dependent but not E-box-dependent manner. Thus, compactin increased the liver-type transcript and decreased the peripheral-type transcript. The same two transcripts were also dominant in human and mouse livers, whereas the intestine contains only the peripheral-type transcript. Treatment of rats with pravastatin and a bile acid binding resin (colestimide), which is known to activate SREBP-2 in the liver, caused a reduction in the hepatic cholesterol level and the same differential responses in vivo, leading to increases in hepatic ABCA1 mRNA and protein and plasma HDL levels. We conclude that the dual promoter system driven by SREBP-2 and LXR regulates hepatic ABCA1 expression and may mediate the unique response of hepatic ABCA1 gene expression to cellular cholesterol status. [ABSTRACT FROM AUTHOR]