Naoki Kiyosawa

A toxicogenomics approach for early assessment of potential non-genotoxic hepatocarcinogenicity of chemicals in rats

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 24 h 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.

Ethylene Glycol Monomethyl Ether–Induced Toxicity Is Mediated through the Inhibition of Flavoprotein Dehydrogenase Enzyme Family

Ethylene glycol monomethyl ether (EGME) is a widely used industrial solvent known to cause adverse effects to human and other mammals. Organs with high metabolism and rapid cell division, such as testes, are especially sensitive to its actions. In order to gain mechanistic understanding of EGME-induced toxicity, an untargeted metabolomic analysis was performed in rats. Male rats were administrated with EGME at 30 and 100 mg/kg/day. At days 1, 4, and 14, serum, urine, liver, and testes were collected for analysis. Testicular injury was observed at day 14 of the 100 mg/kg/day group only. Nearly 1900 metabolites across the four matrices were profiled using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Statistical analysis indicated that the most significant metabolic perturbations initiated from the early time points by EGME were the inhibition of choline oxidation, branched-chain amino acid catabolism, and fatty acid β-oxidation pathways, leading to the accumulation of sarcosine, dimethylglycine, and various carnitine- and glycine-conjugated metabolites. Pathway mapping of these altered metabolites revealed that all the disrupted steps were catalyzed by enzymes in the primary flavoprotein dehydrogenase family, suggesting that inhibition of flavoprotein dehydrogenase–catalyzed reactions may represent the mode of action for EGME-induced toxicity. Similar urinary and serum metabolite signatures are known to be the hallmarks of multiple acyl-coenzyme A dehydrogenase deficiency in humans, a genetic disorder because of defects in primary flavoprotein dehydrogenase reactions. We postulate that disruption of key biochemical pathways utilizing flavoprotein dehydrogenases in conjugation with downstream metabolic perturbations collectively result in the EGME-induced tissue damage.

Gene expression profiles in pregnant rats treated with T-2 toxin

Pregnant rats on day 13 of gestation were treated orally with T-2 toxin at a single dose of 2 mg/kg and sacrificed at 24 hours after treatment. Histopathologically, apoptosis was increased in the liver, placenta and fetal liver. Microarray analysis was performed to examine the gene expression in the liver, placenta, and fetal liver. The results of microarray analysis showed that the changes in the expression of apoptosis genes, metabolic enzymes and oxidative stress-related genes were detected in these tissues. Suppression of phase I and II enzymes-related genes expression in the liver, and suppression of phase II enzymes-related genes expression in the placenta and fetal liver were observed. Semiquantitive RT-PCR analysis also showed the same results as those of microarray analysis. From the results of microarray analysis and histopathological examination, T-2 toxin seems to induce oxidative stress in these tissues, following the changes in metabolism-related genes expression. These changes may alter the intracellular environments resulting in the induction of apoptosis. Further studies on the gene expression profiles at the earlier time point are necessary to clarify the detailed mechanisms of T-2 toxin-induced toxicity in pregnant rats.

Practical Application of Toxicogenomics for Profiling Toxicant-Induced Biological Perturbations

A systems-level understanding of molecular perturbations is crucial for evaluating chemical-induced toxicity risks appropriately, and for this purpose comprehensive gene expression analysis or toxicogenomics investigation is highly advantageous. The recent accumulation of toxicity-associated gene sets (toxicogenomic biomarkers), enrichment in public or commercial large-scale microarray database and availability of open-source software resources facilitate our utilization of the toxicogenomic data. However, toxicologists, who are usually not experts in computational sciences, tend to be overwhelmed by the gigantic amount of data. In this paper we present practical applications of toxicogenomics by utilizing biomarker gene sets and a simple scoring method by which overall gene set-level expression changes can be evaluated efficiently. Results from the gene set-level analysis are not only an easy interpretation of toxicological significance compared with individual gene-level profiling, but also are thought to be suitable for cross-platform or cross-institutional toxicogenomics data analysis. Enrichment in toxicogenomics databases, refinements of biomarker gene sets and scoring algorithms and the development of user-friendly integrative software will lead to better evaluation of toxicant-elicited biological perturbations.

Toxicogenomic Biomarkers for Liver Toxicity

Toxicogenomics (TGx) is a widely used technique in the preclinical stage of drug development to investigate the molecular mechanisms of toxicity. A number of candidate TGx biomarkers have now been identified and are utilized for both assessing and predicting toxicities. Further accumulation of novel TGx biomarkers will lead to more efficient, appropriate and cost effective drug risk assessment, reinforcing the paradigm of the conventional toxicology system with a more profound understanding of the molecular mechanisms of drug-induced toxicity. In this paper, we overview some practical strategies as well as obstacles for identifying and utilizing TGx biomarkers based on microarray analysis. Since clinical hepatotoxicity is one of the major causes of drug development attrition, the liver has been the best documented target organ for TGx studies to date, and we therefore focused on information from liver TGx studies. In this review, we summarize the current resources in the literature in regard to TGx studies of the liver, from which toxicologists could extract potential TGx biomarker gene sets for better hepatotoxicity risk assessment.

Scoring multiple toxicological endpoints using a toxicogenomic database

As information regarding microarray data sets and toxicogenomic biomarkers grows rapidly, the process of analyzing data and interpreting the results is increasingly complicated. To facilitate data analysis, a simple expression ratio-based scoring method called the TGP1 score was previously proposed [Kiyosawa, N., Shiwaku, K., Hirode, M., Omura, K., Uehara, T., Shimizu, T., Mizukawa, Y., Miyagishima, T., Ono, A., Nagao, T., Urushidani, T., 2006. Utilization of a one-dimensional score for surveying chemical-induced changes in expression levels of multiple biomarker gene sets using a large-scale toxicogenomics database. J. Toxicol. Sci. 31, 433-448]. Although the TGP1 score has demonstrated its efficacy for rapid comprehension of large-scale toxicogenomic data sets, inclusion of low quality gene expression data in the biomarker gene set produced flaws in the calculated score. To overcome this shortcoming, we tested a new scoring method called the differentially expressed gene score (D-score), where Detection Call as well as signal log ratios generated by MAS5 algorithm on Affymetrix GeneChip data were considered for the calculation. Four prototypical toxicants, namely acetaminophen, phenobarbital, clofibrate and acetamidofluorene, were used for detailed analysis. A toxicogenomics database (TG-GATEs) was utilized as a reference data set. The D-score successfully alleviated the effects of low quality data on the score calculation, and captured the overall direction of expression changes as well as the magnitude of expression change level of a set of genes, highlighting the affected toxicological endpoints elicited by chemical treatment. The D-score will be useful for high-throughput toxicity screening using a toxicogenomic database and biomarkers.

Methemoglobinemia Induced by 1,2-Dichloro-4-nitrobenzene in Mice with a Disrupted Glutathione S-Transferase Mu 1 Gene

A specific substrate to Mu class glutathione S-transferase (GST), 1,2-dichloro-4-nitrobenzene (DCNB), was administered to mice with a disrupted GST Mu 1 gene (Gstm1-null mice) to investigate the in vivo role of murine Gstm1 in toxicological responses to DCNB. A single oral administration of DCNB at doses of 500 and 1000 mg/kg demonstrated a marked increase in blood methemoglobin (MetHB) in Gstm1-null mice but not in wild-type mice. Therefore, Gstm1-null mice were considered to be more predisposed to methemoglobinemia induced by a single dosing of DCNB. In contrast, 14-day repeated-dose studies of DCNB at doses up to 600 mg/kg demonstrated a marked increase in blood MetHB in both wild-type and Gstm1-null mice. However, marked increases in the blood reticulocyte count, relative spleen weight, and extramedullary hematopoiesis in the spleen were observed in Gstm1-null mice compared with wild-type mice. In addition, microarray and quantitative reverse transcription-polymerase chain reaction analyses in the spleen showed exclusive up-regulation of hematopoiesis-related genes in Gstm1-null mice. These changes were considered to be adaptive responses to methemoglobinemia and attenuated the higher predisposition to methemoglobinemia observed in Gstm1-null mice in the single-dose study. In toxicokinetics monitoring, DCNB concentrations in plasma and blood cells were higher in Gstm1-null mice than those in wild-type mice, resulting from the Gstm1 disruption. In conclusion, it is suggested that the higher exposure to DCNB due to Gstm1 disruption was reflected in methemoglobinemia in the single-dose study and in adaptive responses in the 14-day repeated-dose study.

(+)-Usnic Acid-induced Myocardial Toxicity in Rats

(+)-Usnic acid (UA) has been known to be a strong uncoupler, and mitochondrial and endoplasmic reticulum (ER)–related stresses are suggested to be involved in the mechanism of hepatotoxicity. However, it has not been clarified whether UA causes toxicity in other mitochondria-rich organs such as the heart. We elucidated whether UA induces cardiotoxicity and its mechanism. UA was orally administered to rats for 14 days, and laboratory and histopathological examinations were performed in conjunction with toxicogenomic analysis. As a result, there was no alteration in blood chemistry, whereas cytoplasmic rarefaction of myocardium was observed microscopically. This finding corresponded to the swollen mitochondria observed ultrastructurally. Immunohistochemically, expression of prohibitin, indicating mitochondrial imbalance, increased in the sarcoplasmic area. Toxicogenomic analysis highlighted the upregulation of gene groups consisting of oxidative stress, ER stress, and amino acid limitation. Interestingly, the number of upregulated genes was larger in the amino acid limitation-related gene group than that in other groups, implying that amino acid limitation might be one of the sources of oxidative stress, not only mitochondria and ER-originated stresses. In conclusion, the heart was manifested to be one of the target organs of UA. Mitochondrial imbalance with complex stresses may be involved in the toxic mechanism.

Gene set-level network analysis using a toxicogenomics database

Toxicogenomics data sets on rat livers covering 118 compounds were subjected to inference of a gene set-level, not individual gene-level, network structure. Expression changing levels for 58 gene sets was used for network inference with a Gaussian graphical model algorithm. The established network contained reasonable relationships, such as ones between the blood glucose level and glycolysis-related genes or the blood transaminase level and cellular injury-related genes, indicating that the gene set-level network inference successfully highlighted biological pathway-level interactions. In addition, the robustness of the inferred network structure was investigated using microarray data on bromobenzene-treated rat livers, where the gene set-level activation exhibited time-dependent propagation through neighbored nodes (i.e. gene sets) on the network, indicating that the network structure was robust and comparable with an external microarray data set. Accumulating such robust gene sets with toxicity-associated subnetwork structures would lead to a better understanding of the molecular mechanisms of drug-elicited toxicities.

Toxicogenomic investigation on rat testicular toxicity elicited by 1,3-dinitrobenzene

Rats were treated with a single oral dose of 10, 25 and 50mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli-germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity.