A novel support vector machine-based 1-day, single-dose prediction model of genotoxic hepatocarcinogenicity in rats.

The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment. This study aims to develop a 1-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. In the silico validation using the expression data of the Open TG-GATEs database indicates that this classifier distinguishes GHCs from other compounds with high accuracy. To further assess the model's effectiveness and reliability, we conducted multi-institutional 1-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR 24 h after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present 1-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.

Effect of ASP8062 on morphine self-administration and morphine-induced respiratory suppression in monkeys.

ASP8062 is an orally available GABAB receptor positive allosteric modulator (PAM). This study assessed the potential of ASP8062 for treating opioid use disorder (OUD). Three rhesus monkeys were pretreated with ASP8062 (0.3, 1 or 3 mg/kg) by oral administration 1 h prior to a 2-h morphine self-administration session (0.03 mg/kg, iv, per injection) under a fixed-ratio 5 schedule. We further examined the potential worsening of morphine-induced respiratory suppression by ASP8062 after coadministration of morphine (10 mg/kg, sc) and ASP8062 (10 mg/kg, po) in cynomolgus monkeys using a custom-made whole-body plethysmograph. Plasma concentrations of ASP8062 (3 or 10 mg/kg, po) were assessed in cynomolgus monkeys using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). ASP8062 at 3 mg/kg, po decreased the morphine self-administrations with significant differences from the vehicle-treated group (IC50 = 0.97 ± 0.36 mg/kg). Exposure levels at 3 mg/kg observed in monkeys were comparable to the clinical exposure levels which positive pharmacodynamic effects were previously shown. Further, ASP8062 did not potentiate morphine-induced respiratory suppression up to exposure levels higher than the clinically relevant dose. ASP8062 may reduce opioid use in OUD patients without affecting respiratory system, providing justification for further ASP8062 development as a potential treatment option for OUD.

Non-clinical assessment of cell therapy products: the perspective from five Asian countries/regions based on regulatory guidelines and the underpinning rationales.

BACKGROUND AIMS: Cell-based regenerative medicine is an innovative field that can potentially alter the overall survival and quality of life of patients with devastating diseases. Several cell therapy products (CTPs) have been approved within the last two decades, and more are under development. The establishment of an effective developmental strategy in accordance with the regulatory bodies of each country/region is crucial for fast delivery of each respective CTP. In particular, facilitating investigational new drug (IND) approval is important for accelerating the transition from non-clinical to clinical research/trial phases. METHODS: Here the authors compared the non-clinical prerequisites for initiating clinical studies in five Asian countries/regions (India, China, Korea, Taiwan and Japan) from an industry viewpoint. The authors first identified the differences and tried to clarify the perspectives/considerations underpinning the different requirements. RESULTS: The authors' findings revealed that differences in regulations and development experiences, especially with CTPs, have led to clear differences in the non-clinical study package and its corresponding study design. CONCLUSIONS: By sharing experiences of the research and development of CTPs among Asian countries/regions and including not only industry but also regulatory authorities, we will be able to expedite cross-border IND approval and eventually contribute to the early delivery of innovative CTPs to many Asian patients.

Serum miR-206 as a biomarker for drug-induced skeletal muscle injury in rats.

Creatine kinase (CK) and lactate dehydrogenase (LDH) serve as biomarkers for skeletal muscle injury in preclinical toxicity studies, but have a limitation regarding tissue specificity. Circulating miR-206 was recently reported to be a useful biomarker for skeletal muscle disorders in humans. Here, we sought to determine whether serum miR-206 can be used as a biomarker in preclinical toxicity studies to detect drug-induced skeletal muscle injury with higher sensitivity and specificity than the biomarkers CK, LDH, skeletal troponin I (sTnI), and myosin light chain 3 (Myl3). We established rat models of skeletal muscle injury through treatment with the muscle toxicant 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) as well as four in-house compounds. We found that serum miR-206 levels significantly increased after treatment with TMPD, and tended to be higher in rats treated with in-house compounds than in control rats. ROC analysis revealed that the specificity of serum miR-206 for detection of skeletal muscle injury was higher compared with those of other markers. Further, serum miR-206 levels were unchanged in rats with isoproterenol-induced cardiotoxicity. These findings demonstrate that serum miR-206 may serve as a highly specific biomarker for preclinical analysis of rats with drug-induced skeletal muscle injuries.

Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.

Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs.

MicroRNA-21 is associated with fibrosis in a rat model of nonalcoholic steatohepatitis and serves as a plasma biomarker for fibrotic liver disease.

Evidence indicates that hepatic fibrosis is the initial lesion of cirrhosis or hepatocellular carcinoma in diseases such as nonalcoholic steatohepatitis (NASH). To induce NASH, we fed rats a choline-deficient and iron-supplemented L-amino acid-defined (CDAA) diet. Histopathological examination revealed that fibrosis appeared from week 4 and progressed to bridging fibrosis from week 12. Using qRT-PCR assays, we detected increased expression of miR-21, Mmp-9, and Timp-1 in liver that peaked during week 4, when fibrosis was first detected. The expression pattern of miR-21 in plasma paralleled that in liver. Fibrosis tended to be resolved within 12 weeks of a recovery period after 12 weeks of feeding, and the expression of miR-21, Timp-1, and Mmp-9 decreased in liver. Comprehensive analyses of miRNA and mRNA expression in the liver using samples acquired at week 4 detected 16 miRNAs and 11 mRNAs that are mutually-interacting fibrosis-related factors. We therefore conclude that miR-21 was closely associated with fibrosis in a rat model of NASH and has potential as a plasma biomarker for hepatic fibrosis.

Detection of non-genotoxic hepatocarcinogens and prediction of their mechanism of action in rats using gene marker sets.

Several studies have successfully detected hepatocarcinogenicity in rats based on gene expression data. However, prediction of hepatocarcinogens with certain mechanisms of action (MOAs), such as enzyme inducers and peroxisome proliferator-activated receptor α (PPARα) agonists, can prove difficult using a single model and requires a highly toxic dose. Here, we constructed a model for detecting non-genotoxic (NGTX) hepatocarcinogens and predicted their MOAs in rats. Gene expression data deposited in the Open Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System (TG-GATEs) was used to investigate gene marker sets. Principal component analysis (PCA) was applied to discriminate different MOAs, and a support vector machine algorithm was applied to construct the prediction model. This approach identified 106 probe sets as gene marker sets for PCA and enabled the prediction model to be constructed. In PCA, NGTX hepatocarcinogens were classified as follows based on their MOAs: cytotoxicants, PPARα agonists, or enzyme inducers. The prediction model detected hepatocarcinogenicity with an accuracy of more than 90% in 14- and 28-day repeated-dose studies. In addition, the doses capable of predicting NGTX hepatocarcinogenicity were close to those required in rat carcinogenicity assays. In conclusion, our PCA and prediction model using gene marker sets will help assess the risk of hepatocarcinogenicity in humans based on MOAs and reduce the number of two-year rodent bioassays.

Modifying effects of 1,2-dichloropropane on N-nitrosobis(2-oxopropyl)amine-induced cholangiocarcinogenesis in male Syrian hamsters.

Based on the findings of epidemiological studies in Japan that occupational exposure to 1,2-dichloropropane (1,2-DCP) was associated with increased cholangiocarcinomas, 1,2-DCP has recently been classified as being carcinogenic to humans (Group 1). However, the cholangiocarcinogenicity of 1,2-DCP has not been demonstrated experimentally, and it was negative for cholangiocarcinogenicity in rats and mice. The present study determined the effects of 1,2-DCP on N-nitrosobis(2-oxopropyl)amine (BOP)-induced cholangiocarcinogenesis in male hamsters. We found that 1,2-DCP did not enhance the development of BOP-induced atypical biliary hyperplasia and did not induce any lesions in liver bile duct when administered alone. Notably, 1,2-DCP had no effect on the proliferative activity of bile duct epithelial cells regardless of BOP-initiation. These results demonstrate that 1,2-DCP lacks promoting effects on BOP-induced cholangiocarcinogenesis and suggest the possibility that 1,2-DCP is not cholangiocarcinogenic to the hamster in the present model. In addition, 1,2-DCP also lacks promoting effects on pancreatic, lung, and renal carcinogenesis. As the occurrence of occupational cholangiocarcinomas in Japan might be attributed to exposure to multiple chemicals, the results of the present study indicate that it will be necessary to determine the cholangiocarcinogenic effects of concurrent exposure of 1,2-DCP and the other halogen solvents to which workers with cholangiocarcinomas were exposed.

Altered gene expression profile in ovarian follicle in rats treated with indomethacin and RU486.

It is well-known that indomethacin (the cyclooxygenase 1 & 2 inhibitor) and RU486 (or mifepristone, the progesterone receptor antagonist) block follicular rupture in rats. To characterize genetic alterations in unruptured follicles, gene expression profiles in ovarian follicle were analyzed in indomethacin- and RU486-treated female Sprague-Dawley rats. Ovaries are collected at 22:00 on the proestrus day and 10:00 on the following estrus day after a single dose of indomethacin and RU486. Histopathologically, changes depicting responses to LH surge were observed in ovaries, uteri and vagina. Total RNA was extracted from pre-ovulatory follicles or unruptured follicles collected by laser microdissection and analyzed by Genechip(®). Among genes showing statistically significant changes compared to control groups, following changes were considered relevant to induction of unruptured follicles. In indomethacin-treated rats, Wnt4 was down-regulated, suggesting effect on tissue integrity and steroid genesis. In RU486-treated rats, Adamts1, Adamts9, Edn2, Ednra, Lyve1, Plat, and Pparg were down-regulated. These changes suggest effects on proteolysis for extra cellular matrix or surrounding tissue (Adamts1 & 9, and Plat), constriction of smooth muscle surrounding follicles (Edn2, Ednra, and Pparg), follicular fluid (Lyve1), and angiogenesis (Pparg). Down-regulation of angiogenesis related genes (Angpt2, Hmox1, and Vegfa) was observed in both treatment groups. Here, we clarify genetic alterations induced by the inhibition of cyclooxygenase or progesterone receptor.

Comprehensive analysis of DNA methylation and gene expression of rat liver in a 2-stage hepatocarcinogenesis model.

Recent studies have shown that epigenetic alterations correlate with carcinogenesis in various tissues. Identification of these alterations might help characterize the early stages of carcinogenesis. We comprehensively analyzed DNA methylation and gene expression in livers obtained from rats exposed to nitrosodiethylamine (DEN) followed by a promoter of hepatic carcinogenesis, phenobarbital (PB). The combination of DEN and PB induced marked increases in number and area of glutathione S-transferase-placental form (GST-P)-positive foci in the liver. In the liver of rats that received 30 mg/kg of DEN, pathway analysis revealed alterations of common genes in terms of gene expression and DNA methylation, and that these alterations were related to immune responses. Hierarchical clustering analysis of the expression of common genes from public data obtained through the Toxicogenomics Project-Genomics Assisted Toxicity Evaluation system (TG-GATEs) showed that carcinogenic compounds clustered together. MBD-seq and GeneChip analysis indicated that major histocompatibility complex class Ib gene RT1-CE5, which has an important role in antigen presentation, was hypomethylated around the promoter region and specifically induced in the livers of DEN-treated rats. Further, immunohistochemical analysis indicated that the co-localization of GST-P and protein homologous to RT1-CE5 was present at the foci of some regions. These results suggest that common genes were altered in terms of both DNA methylation and expression in livers, with preneoplastic foci indicating carcinogenic potential, and that immune responses are involved in early carcinogenesis. In conclusion, the present study identified a specific profile of DNA methylation and gene expression in livers with preneoplastic foci. Early epigenetic perturbations of immune responses might correlate with the early stages of hepatocarcinogenesis.

Detection of initiating potential of non-genotoxic carcinogens in a two-stage hepatocarcinogenesis study in rats.

We previously reported a toxicogenomics-based prediction model for hepatocarcinogens in which the expression patterns of signature genes following repeated doses of either genotoxic or non genotoxic compounds were similar. Based on the results of our prediction model, we hypothesized that repeated doses of non-genotoxic carcinogens might have initiating potential. Here, we conducted a two stage hepatocarcinogenesis study in rats exposed to the initiating agent nitrosodiethylamine (DEN), and hepatotoxic compounds thioacetamide (TAA), methapyrilene (MP) and acetaminophen (APAP) for 1-2 weeks followed by the liver tumor promoter phenobarbital (PB). The duration of initial treatment was determined based on positive results from our prediction model. Combined treatment of 3 or 30 mg/kg of genotoxic DEN and PB induced marked increases in altered hepatocellular foci and a DEN dose-dependent increase in the number and area of glutathione S-transferase-placental form (GST-P)-positive foci. A low number of altered hepatocellular foci were also observed in rats treated with TAA at a dose of 45 mg/kg.MP at a dose of 100 mg/kg induced a very low number of foci, but APAP did not. Hierarchical clustering analysis using gene expression data revealed that 2-week treatment with TAA at a dose of 30 mg/kg and MP at 45 mg/kg induced specific expression of DNA damage-related genes, similar to 1-week treatment with DEN at a dose of 30 mg/kg. These results suggest that TAA and MP induce DNA damage, which partially supports our hypothesis. Although this study does not indicate whether tumor growth in response to these compounds can be assessed in this model, our results suggest that cumulative treatment with non genotoxic TAA might have initiating potential in the liver.

Identification of urinary miRNA biomarkers for detecting cisplatin-induced proximal tubular injury in rats.

Despite increased focus in recent years on urinary microRNA (miRNA) biomarkers in patients with diabetes and chronic kidney diseases, few studies have explored urinary miRNA markers in drug-induced nephrotoxicity. Here, we attempted to identify urinary miRNA markers suitable for use in detecting cisplatin-induced nephrotoxicity in rats. Cisplatin (6mg/kg) was given as a single intraperitoneal injection to male Sprague-Dawley (SD) rats, and urine collected from Days 4 to 5 for 17h under fed or fasted conditions. MiRNAs were identified using TaqMan(®) Rodent microRNA PCR cards, and rats were euthanized 5 days after administration. Levels of 25 miRNAs were significantly increased in the urine of cisplatin-treated rats under both fed and fasted conditions, while the levels of these miRNAs were decreased in either or both the cortex or outer medulla of the kidney. Analysis of time and dose dependency in the urine from rats treated with cisplatin (1, 3, and 6mg/kg) on Days 1, 3, and 7, showed levels of 25 miRNAs were increased in urine and their appearance correlated with the severity of necrosis in the proximal tubules. Four miRNAs (let-7g-5p, miR-93-5p, miR-191a-5p and miR-192-5p) in urine were measured by absolute quantification, and a strong correlation was found between relative and absolute quantification methods. In summary, we identified 25 miRNAs in urine that were able to be used as non-invasive biomarkers for the detection of cisplatin-induced proximal tubular injury in rats. This study is the first step in demonstrating the potential utility of urinary miRNAs in assessing nephrotoxicity. Further study, such as collaborative programs currently underway in the HESI consortium, will clarify the usability of identified miRNA markers in measurement of other nephrotoxicants and injury-site specificity.

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.

Identification of novel liver-specific mRNAs in plasma for biomarkers of drug-induced liver injury and quantitative evaluation in rats treated with various hepatotoxic compounds.

Circulating liver-specific mRNAs such as albumin (Alb) and α-1-microglobulin/bikunin precursor (Ambp) have been reported to be potential biomarkers for drug-induced liver injury (DILI). We identified novel circulating liver-specific mRNAs and quantified them, together with the two previously reported mRNAs, in plasma from rats treated with various hepatotoxicants to validate circulating liver-specific mRNAs as biomarkers for DILI. Among six genes selected from the database, high liver specificity of apolipoprotein h (Apoh) and group-specific component (Gc) mRNAs were confirmed by reverse transcription (RT)-PCR and the copy numbers of these mRNAs elevated in plasma from rats treated with thioacetamide. Liver-specific mRNAs (Alb, Ambp, Apoh, and Gc) were quantified by real-time RT-PCR in plasma from rats with single dosing of seven hepatotoxicants. There were noticeable interindividual and intercompound variabilities in the severity of liver injury. The levels of four mRNAs increased almost in parallel and correlated with changes in the alanine aminotransferase (ALT) values and the hepatocellular necrosis scores at 24h after dosing. It was noteworthy that the magnitude of the increases in mRNA levels was greater than that in the ALT value. Time course analysis within 24h after dosing revealed that the timing of the increase was different among mRNA species, and the plasma levels of Alb and Gc mRNAs increased substantially earlier than the ALT values, suggesting that patterns of changes in circulating liver-specific mRNAs indicate the progression of liver injury. These results strongly support the reliability and usefulness of the four circulating liver-specific mRNAs as biomarkers for DILI.

Transitional gene expression profiling in ovarian follicle during ovulation in normal-cycle rats.

Evaluation of ovarian toxicity requires an understanding of the physiological changes related to the estrous cycle in the ovary. The authors investigated the transitional gene expression profile of ovulatory follicles in rats that show normal estrous cyclicity. Ovaries were collected at 10:00 and 22:00 on the proestrus day and at 10:00 on the estrus day. Ovarian follicles or early corpora lutea were isolated using laser microdissection, and extracted total RNA was analyzed using microarray technology. Clustering analysis revealed four different expression patterns: transient up- or down-regulation only at 22:00 on the proestrus day (pattern 1), up- or down-regulation only at 10:00 on the estrus day (pattern 2), continuous increase at 22:00 on the proestrus day and at 10:00 on the estrus day (pattern 3), and up- or down-regulation at 22:00 on the proestrus day and level maintenance at 10:00 on the estrus day (pattern 4). In addition, these probe sets were functionally categorized in each pattern using the Ingenuity Pathways Analysis database. These data will aid in understanding the physiology of ovulation and may be useful in assessing ovarian toxicity and its mechanism, such as in investigations of chemical-induced ovulatory impairment.

Possible involvement of oxidative stress in piperonyl butoxide induced hepatocarcinogenesis in rats.

To clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by piperonyl butoxide (PBO), male F344 rats were administered an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Two weeks later, the rats were administered a PBO-containing (0, 1, or 2%) diet for 6 weeks and subjected to a two-third partial hepatectomy 1 week later. After sacrificing them on week 8, their livers were histopathologically examined and analyzed for gene expression using a microarray and real-time RT-PCR. Reactive oxygen species (ROS) products were also measured using liver microsomes. Hepatocytes exhibited centrilobular hypertrophy and increased glutathione S-transferase placental form (GST-P) positive foci formation. ROS products increased significantly in liver microsomes. In the microarray analysis, the expressions of genes related to metabolism and oxidative stress - NAD(P)H dehydrogenase, quinone 1 (Nqo1), UDP-glucuronosyltransferase (UDPGTR-2), glutathione peroxidase 2 (Gpx2), glutathione reductase (GRx) - multidrug resistance associated protein 3 (Abcc3), and solute carrier family 7 (cationic amino acid transporter, y+ system) member 5 (Slc7a5) were up-regulated in the PBO group in comparison to the 0% PBO group; this was confirmed by real-time RT-PCR. Additionally, a significant up-regulation of stress response related genes such as CYP1A1 was observed in PBO-treated groups in real-time RT-PCR. HPLC analysis revealed that the level of 8-OHdG in the 2% PBO group was significantly higher than that in the 0% PBO group. This suggests that PBO has the potential to generate ROS via metabolic pathways and induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in rats.