Noriyo Niino

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.

(+)-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.

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.

Comprehensive Evaluation of (+)-Usnic Acid–induced Cardiotoxicity in Rats by Sequential Cross-omics Analysis

Two-week administration of (+)-usnic acid (UA) induces mitochondrial swelling of cardiomyocytes, and toxicogenomic analysis of the heart revealed upregulation of oxidative stress, amino acid limitation, and endoplasmic reticulum stress–related genes in rats. To analyze the pathogenesis, UA was orally administrated to rats for 1, 4, 7, and 14 days, and sequential histopathological, genomic, and metabolomic analyses were performed on the heart, liver, and plasma. As a result, mitochondrial swelling of cardiomyocytes was observed on day 15 preceded by genomic upregulation on days 5 and 8. Of the focused gene groups, amino acid limitation–related genes represented by Mthfd2 showed numerically higher values or upregulation from day 5, which was sustained through the experimental period. On the contrary, oxidative stress–related genes were upregulated temporally on day 5. In metabolomic analysis, amino acids such as taurocholate and their metabolites fluctuated in concert with the upregulation of amino acid limitation–related genes in the heart, liver, and plasma. Moreover, accumulations of bile acids were manifested in all the tested tissues, while no histopathological change was seen in the liver. Increased bile acids might have an indirect effect on the myocardium; however, more detailed analysis is required. In conclusion, amino acid limitation was suggested as the pivotal toxic trigger of UA-induced cardiotoxicity.

A monkey model of acetaminophen-induced hepatotoxicity; phenotypic similarity to human

Species-specific differences in the hepatotoxicity of acetaminophen (APAP) have been shown. To establish a monkey model of APAP-induced hepatotoxicity, which has not been previously reported, APAP at doses up to 2,000 mg/kg was administered orally to fasting male and female cynomolgus monkeys (n = 3-5/group) pretreated intravenously with or without 300 mg/kg of the glutathione biosynthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO). In all the animals, APAP at 2,000 mg/kg with BSO but not without BSO induced hepatotoxicity, which was characterized histopathologically by centrilobular necrosis and vacuolation of hepatocytes. Plasma levels of APAP and its reactive metabolite N-acethyl-p-benzoquinone imine (NAPQI) increased 4 to 7 hr after the APAP treatment. The mean Cmax level of APAP at 2,000 mg/kg with BSO was approximately 200 µg/mL, which was comparable to high-risk cutoff value of the Rumack-Matthew nomogram. Interestingly, plasma alanine aminotransferase (ALT) did not change until 7 hr and increased 24 hr or later after the APAP treatment, indicating that this phenotypic outcome was similar to that in humans. In addition, circulating liver-specific miR-122 and miR-192 levels also increased 24 hr or later compared with ALT, suggesting that circulating miR-122 and miR-192 may serve as potential biomarkers to detect hepatotoxicity in cynomolgus monkeys. These results suggest that the hepatotoxicity induced by APAP in the monkey model shown here was translatable to humans in terms of toxicokinetics and its toxic nature, and this model would be useful to investigate mechanisms of drug-induced liver injury and also potential translational biomarkers in humans.

Establishment of an in vitro cytotoxicity assay platform using primary monkey cardiomyocytes

To establish an in vitro cytotoxicity assay platform using monkey cardiomyocytes, we isolated primary cardiomyocytes from fetal cynomolgus monkeys at different gestation days (from day 39 to 90) using the trypsin and collagenase digestion method, which was identical to the standard procedure for rat cardiomyocytes. Under these conditions, the primary cells obtained from monkeys at gestation day 63 or earlier showed spontaneous beating, with >80% cells being viable from all fetuses. Transcriptome analysis of the monkey cardiomyocytes indicated that the cells have essential components of cardiac functions, such as myosins, α-actin, cardiac troponins, and calcium-related molecules. The susceptibility to doxorubicin-induced cytotoxicity in monkey cardiomyocytes was comparable to that in rat cardiomyocytes, as evaluated based on intracellular ATP levels. Microarray analysis with Ingenuity Pathway Analysis revealed that doxorubicin predominantly increased the expression of several key genes involved in the endoplasmic reticulum stress pathway in monkey cardiomyocytes than in rat cardiomyocytes. In conclusion, we isolated primary monkey cardiomyocytes that showed similar sensitivity to doxorubicin as compared with rat cardiomyocytes. This in vitro monkey cardiomyocyte assay platform would serve as a powerful tool for the investigation of the interspecies differences in drug-induced cardiotoxicity and its underlying mechanism.

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification. A fixed volume (200 µL) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step. Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/µL, for most of the examined miRNAs. This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

Comprehensive Analysis of Circulating microRNA Specific to the Liver, Heart, and Skeletal Muscle of Cynomolgus Monkeys

Circulating microRNAs (miRNAs) could represent sensitive and specific biomarkers for tissue injury. However, their utility as biomarkers in nonclinical toxicological studies using nonhuman primates is limited by a lack of information on their organ specificity and circulating levels under resting condition of the animals. Herein, liver, heart, and skeletal muscle-specific expression patterns of miRNAs were determined in 27 tissues/organs from male and female monkeys (n =2/sex) by next-generation sequencing (NGS) analysis. This analysis revealed organ-specific miRNAs in the liver (miR-122), heart (miR-208a and miR-499a), and skeletal muscle (miR-206). Next, plasma was collected from conscious-naive male and female cynomolgus monkeys (n = 25/sex) to better understand the expressions of organ-specific circulating miRNAs. The absolute values of circulating miRNAs were quantified using a Taqman microRNA assay. MiR-1, miR-133a, and miR-208b showed preferential expression in the heart and skeletal muscles, whereas miR-192 was abundant in the liver, stomach, small intestine, and kidney. These miRNAs had identical sequences to their human counterparts. Six organ-specific miRNAs (miR-1, miR-122, miR-133a, miR-192, miR-206, and miR-499a) could be evaluated quantitatively by quantitative real-time reverse transcription polymerase chain reaction with or without preamplification. No significant sex differences were noted for these circulating miRNAs. For their circulation levels, miR-133a showed more than 900-fold interindividual variation, whereas miR-122 showed only a 20-fold variation. In conclusion, we profiled circulating organ-specific miRNAs for the liver, heart, and skeletal muscle of cynomolgus monkeys.