Yuji Morikawa

Prediction model of potential hepatocarcinogenicity of rat hepatocarcinogens using a large-scale toxicogenomics database.

The present study was performed to develop a robust gene-based prediction model for early assessment of potential hepatocarcinogenicity of chemicals in rats by using our toxicogenomics database, TG-GATEs (Genomics-Assisted Toxicity Evaluation System developed by the Toxicogenomics Project in Japan). The positive training set consisted of high- or middle-dose groups that received 6 different non-genotoxic hepatocarcinogens during a 28-day period. The negative training set consisted of high- or middle-dose groups of 54 non-carcinogens. Support vector machine combined with wrapper-type gene selection algorithms was used for modeling. Consequently, our best classifier yielded prediction accuracies for hepatocarcinogenicity of 99% sensitivity and 97% specificity in the training data set, and false positive prediction was almost completely eliminated. Pathway analysis of feature genes revealed that the mitogen-activated protein kinase p38- and phosphatidylinositol-3-kinase-centered interactome and the v-myc myelocytomatosis viral oncogene homolog-centered interactome were the 2 most significant networks. The usefulness and robustness of our predictor were further confirmed in an independent validation data set obtained from the public database. Interestingly, similar positive predictions were obtained in several genotoxic hepatocarcinogens as well as non-genotoxic hepatocarcinogens. These results indicate that the expression profiles of our newly selected candidate biomarker genes might be common characteristics in the early stage of carcinogenesis for both genotoxic and non-genotoxic carcinogens in the rat liver. Our toxicogenomic model might be useful for the prospective screening of hepatocarcinogenicity of compounds and prioritization of compounds for carcinogenicity testing.

Plasma miR‐208 as a useful biomarker for drug‐induced cardiotoxicity in rats

Cardiotoxicity is one of the major safety concerns in drug development. Therefore, detecting and monitoring cardiotoxicity throughout preclinical and clinical studies is important for pharmaceutical companies. The present study was conducted in order to explore a plasma miRNA biomarker for cardiotoxicity in rats. As organ specificity is an important factor for a biomarker, we analyzed the miRNA microarray dataset in 55 organs/tissues in normal male rats. Based on this analysis, 5 miRNAs consisting of miR‐208 (heart‐specific), miR‐1, miR‐133a, miR‐133b (heart and skeletal muscle‐specific) and miR‐206 (skeletal muscle‐specific) were selected. Next, we evaluated the usefulness of those 5 miRNAs as circulating biomarkers in rats administered with single‐dose isoproterenol or doxorubicin. Plasma miR‐208 was consistently increased through 24 h after dosing in rats administered with isoproterenol, whereas plasma concentrations of cardiac troponin (cTn) showed transient elevation. In contrast, the plasma levels of miR‐1, miR‐133a, miR‐133a and miR‐206 were elevated after treatment with doxorubicin, probably as a result of skeletal muscle toxicity. Additionally, the plasma miR‐208 level was elevated even after repeat‐dose administration (once daily for 7 days) of isoproterenol under which the pathological condition proceeded to the sub‐chronic phase such as fibrosis. Thus, our data suggest that miR‐208 is a promising plasma biomarker for cardiotoxicity in rats. Monitoring of plasma miR‐208 levels in rats may lead to more accurate evaluation of cardiotoxicity in preclinical studies. Copyright

Toxicogenomic multigene biomarker for predicting the future onset of proximal tubular injury in rats.

Drug-induced renal tubular injury is a major concern in the preclinical safety evaluation of drug candidates. Toxicogenomics is now a generally accepted tool for identifying chemicals with potential safety problems. The specific aim of the present study was to develop a model for use in predicting the future onset of drug-induced proximal tubular injury following repeated dosing with various nephrotoxicants. In total, 41 nephrotoxic and nonnephrotoxic compounds were used for the present analysis. Male Sprague-Dawley rats were dosed orally or intravenously once daily. Animals were exposed to three different doses (low, middle, and high) of each compound, and kidney tissue was collected at 3, 6, 9, and 24 h after single dosing, and on days 4, 8, 15, and 29 after repeated dosing. Gene expression profiles were generated from kidney total RNA using Affymetrix DNA microarrays. Filter-type gene selection and linear classification algorithms were employed to discriminate future onset of proximal tubular injury. We identified genomic biomarkers for use in future onset prediction using the gene expression profiles determined on day 1, when most of the nephrotoxicants had yet to produce detectable histopathological changes. The model was evaluated using a five-fold cross validation, and achieved a sensitivity of 93% and selectivity of 90% with 19 probes. We also found that the prediction accuracy of the optimized model was substantially higher than that produced by any of the single genomic biomarkers or histopathology. The genes included in our model were primarily involved in DNA replication, cell cycle control, apoptosis, and responses to oxidative stress and chemical stimuli. In summary, our toxicogenomic model is particularly useful for predicting the future onset of proximal tubular injury.

Identification of metabolomic biomarkers for drug-induced acute kidney injury in rats.

Nephrotoxicity is a common side effect observed during both nonclinical and clinical drug development investigations. The present study aimed to identify metabolomic biomarkers that could provide early and sensitive indication of nephrotoxicity in rats. Metabolomic analyses were performed using capillary electrophoresis–time‐of‐flight mass spectrometry on rat plasma collected at 9 and 24 h after a single dose of 2‐bromoethylamine or n‐phenylanthranilic acid and at 24 h after 7 days of repeated doses of gentamicin, cyclosporine A or cisplatin. Among a total of 169 metabolites identified, 3‐methylhistidine (3‐MH), 3‐indoxyl sulfate (3‐IS) and guanidoacetate (GAA) were selected as candidate biomarkers. The biological significance and reproducibility of the observed changes were monitored over time in acute nephrotoxicity model rats treated with a single dose of cisplatin, with the glomerular filtration rate monitored by determination of creatinine clearance. Increased plasma levels of 3‐MH and 3‐IS were related to a decline in glomerular filtration due to a renal failure. In contrast, the decrease in plasma GAA, which is synthesized from arginine and glycine in the kidneys, was considered to reflect decreased production due to renal malfunction. Although definitive validation studies are required to confirm their usefulness and reliability, 3‐MH, 3‐IS and GAA may prove to be valuable plasma biomarkers for monitoring nephrotoxicity in rats. Copyright

MiRNA expression atlas in male rat

MicroRNAs (miRNAs) are small (~22 nucleotide) noncoding RNAs that play pivotal roles in regulation of gene expression. The value of miRNAs as circulating biomarkers is now broadly recognized; such tissue-specific biomarkers can be used to monitor tissue injury and several pathophysiological conditions in organs. In addition, miRNA profiles of normal organs and tissues are important for obtaining a better understanding of the source of modulated miRNAs in blood and how those modulations reflect various physiological and toxicological conditions. This work was aimed at creating an miRNA atlas in rats, as part of a collaborative effort with the Toxicogenomics Informatics Project in Japan (TGP2). We analyzed genome-wide miRNA profiles of 55 different organs and tissues obtained from normal male rats using miRNA arrays. The work presented herein represents a comprehensive dataset derived from normal samples profiled in a single study. Here we present the whole dataset with miRNA profiles of multiple organs, as well as precise information on experimental procedures and organ-specific miRNAs identified in this dataset.

Toxicogenomics discrimination of potential hepatocarcinogenicity of non‐genotoxic compounds in rat liver

Long‐term carcinogenicity testing of a compound is exceedingly time‐consuming and costly, and requires many test animals, whereas the Ames test, which is based on the assumption that any substance that is mutagenic may also exert carcinogenic potential, is useful as a short‐term screening assay but has major drawbacks. Although, in fact, 90% of compounds that give a positive Ames test cause cancer in laboratory animals, a good proportion of compounds that give a negative Ames test are also carcinogens; that is, there is no good correlation between carcinogenicity and negative Ames test results. As an alternative to these two approaches, we have tried applying toxicogenomics to predict the carcinogenicity of a compound from the gene expression profile induced in vivo. To establish our model, male Sprague–Dawley rats were orally administered test compounds (12 hepatocarcinogens and 26 non‐hepatocarcinogens) for 28 days. Analysis of liver gene expression data by Support Vector Machines (SVM) dividing compounds into ‘for training’ and ‘for test’ (20 cases assigned randomly) allowed a set of marker genes to be tested for prediction of hepatocarcinogenicity. The developed prediction model was then validated with reference to the concordance rate with training data and test data, and a good performance was obtained. We will have new gene expression data and continue the validation of our model. Copyright

Genomic biomarkers for cardiotoxicity in rats as a sensitive tool in preclinical studies

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver‐operating characteristic (ROC) analysis using more cardiotoxic and non‐cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi‐gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi‐gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi‐gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs. Copyright

Toxicogenomic biomarkers for renal papillary injury in rats.

Renal papillary injury is a common side effect observed during nonclinical and clinical investigations in drug development. The present study aimed to identify genomic biomarkers for early and sensitive detection of renal papillary injury in rats. We hypothesized that previously identified genomic biomarkers for tubular injury might be applicable for the sensitive detection of papillary injury in rats. We selected 18 genes as candidate biomarkers for papillary injury based on previously published studies and analyzed their expression profiles by RT-PCR in each kidney region, namely the cortex, cortico-medullary junction, and papilla in various nephrotoxicity models. Comparative analysis of gene expression profiles revealed that some genes were commonly upregulated or downregulated in the renal papilla, reflecting papillary injuries induced by 2-bromoethylamine hydrobromide, phenylbutazone, or n-phenylanthranilic acid. By applying receiver operator characteristics analysis, six candidate biomarkers were identified and their usefulness was confirmed by using an independent data set. The three top-ranked genes, Timp1, Igf1, and Lamc2, exhibited the best prediction performance in an external data set with area under the curve (AUC) values of greater than 0.91. An optimized support vector machine model consisting of three genes achieved the highest AUC value of 0.99. In conclusion, even though definitive validation studies are required for the establishment of their usefulness and reliability, these identified genes may prove to be the most promising candidate genomic biomarkers of renal papillary injury in rats.

Comparative gene and protein expression analyses of a panel of cytokines in acute and chronic drug-induced liver injury in rats.

Drug-induced liver injury (DILI) is a significant safety issue associated with medication use, and is the major cause of failures in drug development and withdrawal in post marketing. Cytokines are signaling molecules produced and secreted by immune cells and play crucial roles in the progression of DILI. Although there are numerous reports of cytokine changes in several DILI models, a comprehensive analysis of cytokine expression changes in rat liver injury induced by various compounds has, to the best of our knowledge, not been performed. In the past several years, we have built a public, free, large-scale toxicogenomics database, called Open TG-GATEs, containing microarray data and toxicity data of the liver of rats treated with various hepatotoxic compounds. In this study, we measured the protein expression levels of a panel of 24 cytokines in frozen liver of rats treated with a total of 20 compounds, obtained in the original study that formed the basis of the Open TG-GATEs database and analyzed protein expression profiles combined with mRNA expression profiles to investigate the correlation between mRNA and protein expression levels. As a result, we demonstrated significant correlations between mRNA and protein expression changes for interleukin (IL)-1β, IL-1α, monocyte chemo-attractant protein (MCP)-1/CC-chemokine ligand (Ccl)2, vascular endothelial growth factor A (VEGF-A), and regulated upon activation normal T cell expressed and secreted (RANTES)/Ccl5 in several different types of DILI. We also demonstrated that IL-1β protein and MCP-1/Ccl2 mRNA were commonly up-regulated in the liver of rats treated with different classes of hepatotoxicants and exhibited the highest accuracy in the detection of hepatotoxicity. The results also demonstrate that hepatic mRNA changes do not always correlate with protein changes of cytokines in the liver. This is the first study to provide a comprehensive analysis of mRNA-protein correlations of factors involved in various types of DILI, as well as additional insights into the importance of understanding complex cytokine expression changes in assessing DILI.

Underestimation of urinary biomarker-to-creatinine ratio resulting from age-related gain in muscle mass in rats

Recent efforts have been made to identify useful urinary biomarkers of nephrotoxicity. Furthermore, the application of urine to the other toxicities as new biomarker source has been recently expanded. Meanwhile, correction of urinary biomarker concentrations according to fluctuations in urine flow rate is required for adequate interpretation of the alteration. The urinary biomarker-to-creatinine ratio (UBCR) is widely used because of the convenience, while the urinary biomarker-excretion rate is regarded as the gold standard corrective method. Because creatinine is a catabolite in energy production in muscles, we hypothesized that altered muscle mass could affect creatinine kinetics, ultimately affecting UBCR. However, no study has examined this hypothesis. In this study, we examined the influence of muscle mass gain on UBCR, using male Sprague-Dawley rats during the growth phase, 6-12-week old. Both plasma creatinine and excretion of urinary creatinine (Ucr excretion) showed increases with muscle mass gain in rats, in which the alterations of UBCR were lowered. The renal mRNA level of the organic cation transporter-2 (Oct2), a creatinine transporter, showed an age-related increase, whereas the mRNA level of multidrug and toxin extrusions-1 (Mate1) remained constant. Multiple regression analysis showed that the increase in creatinine clearance highly contributed to the age-related increase in Ucr excretion compared to the mRNA levels of Oct2 and Mate1. This suggested that the age-related increase in Ucr excretion may be attributable to the increased transglomerular passage of creatinine. In conclusion, the results suggest that muscle mass gain can affect creatinine kinetics, leading to underestimation of UBCR. Therefore, it is important to understand the characteristics of the corrective method when using urinary biomarker, the failure of which can result in an incorrect diagnosis.