Mechanisms of action and resistance in histone methylation-targeted therapy.

Epigenomes enable the rectification of disordered cancer gene expression, thereby providing new targets for pharmacological interventions. The clinical utility of targeting histone H3 lysine trimethylation (H3K27me3) as an epigenetic hallmark has been demonstrated1-7. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumour cells remain unclear. Here we show the potency and mechanisms of action and resistance of the EZH1-EZH2 dual inhibitor valemetostat in clinical trials of patients with adult T cell leukaemia/lymphoma. Administration of valemetostat reduced tumour size and demonstrated durable clinical response in aggressive lymphomas with multiple genetic mutations. Integrative single-cell analyses showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, including tumour suppressor genes. Nevertheless, subsequent long-term treatment encounters the emergence of resistant clones with reconstructed aggregate chromatin that closely resemble the pre-dose state. Acquired mutations at the PRC2-compound interface result in the propagation of clones with increased H3K27me3 expression. In patients free of PRC2 mutations, TET2 mutation or elevated DNMT3A expression causes similar chromatin recondensation through de novo DNA methylation in the H3K27me3-associated regions. We identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Targeting epigenetic drivers and chromatin homeostasis may provide opportunities for further sustained epigenetic cancer therapies.

Transcriptional profile of ethylene glycol monomethyl ether-induced testicular toxicity in rats.

To clarify the molecular mechanism of ethylene glycol monomethyl ether (EGME)-induced testicular toxicity, the potential for EGME-related changes in transcript levels of genes including spermatocyte-specific genes was evaluated in the testis of rats given single dosing of EGME at 200, 600, or 2000 mg/kg. Furthermore, the contribution of decreased testicular testosterone on EGME-induced spermatocyte toxicity was investigated by comparing to transcriptional profile due to a testosterone synthesis inhibitor, ketoconazole (KET), at 30 or 300 mg/kg. EGME at 600 mg/kg or more dose-dependently caused testicular toxicity characterized by degeneration and necrosis of spermatocytes at stage VII-XIV seminiferous tubules. The spermatocyte injury was well correlated with decreased spermatocyte-specific gene expression. Analysis of upstream regulators by the Ingenuity Pathways Analysis system suggested that up-regulation of oxidative stress, protein kinase activation, and histone acetylation was involved in EGME-induced spermatocyte toxicity. Interestingly, KET decreased testicular testosterone to a similar extent compared to the EGME treatment, but KET at up to 300 mg/kg did not show any histopathological abnormality or change in the expression of spermatocyte-specific genes. These results suggested that the decreased testicular testosterone have little impact on EGME-induced spermatocyte injury. In contrast, KET showed trends toward increases in Hsd3b2 and Hsd17b2 mRNAs, presumably resulting from inhibition of androgen synthesis. Transcriptome analysis clearly demonstrated the differential effects of EGME and KET on androgen synthesis. In conclusion, EGME caused spermatocyte toxicity correlated with decreased expression of spermatocyte-specific genes. Furthermore, oxidative stress, protein kinase activation, and histone acetylation were suggested to be involved in EGME-induced testicular toxicity.

MicroRNA profiles in a monkey testicular injury model induced by testicular hyperthermia.

To characterize microRNAs (miRNAs) involved in testicular toxicity in cynomolgus monkeys, miRNA profiles were investigated using next-generation sequencing (NGS), microarray and reverse transcription-quantitative real-time-PCR (RT-qPCR) methods. First, to identify organ-specific miRNAs, we compared the expression levels of miRNAs in the testes to those in representative organs (liver, heart, kidney, lung, spleen and small intestine) obtained from naïve mature male and female monkeys (n = 2/sex) using NGS analysis. Consequently, miR-34c-5p, miR-202-5p, miR-449a and miR-508-3p were identified to be testicular-specific miRNAs in cynomolgus monkeys. Next, we investigated miRNA profiles after testicular-hyperthermia (TH) treatment to determine which miRNAs are involved in testicular injury. In this experiment, mature male monkeys were divided into groups with or without TH-treatment (n = 3/group) by immersion of the testes in a water bath at 43 °C for 30 min for 5 consecutive days. As a result, TH treatment induced testicular injury in all animals, which was characterized by decreased numbers of spermatocytes and spermatids. In a microarray analysis of the testis, 11 up-regulated (>2.0 fold) and 13 down-regulated (<0.5 fold) miRNAs were detected compared with those in the control animals. Interestingly, down-regulated miRNAs included two testicular-specific miRNAs, miR-34c-5p and miR-449a, indicating their potential use as biomarkers for testicular toxicity. Furthermore, RT-qPCR analysis revealed decreased expression levels of testicular miR-34b-5p and miR-34c-5p, which are enriched in meiotic cells, reflecting the decrease in pachytene spermatocytes and spermatids after TH treatment. These results provide valuable insights into the mechanism of testicular toxicity and potential translational biomarkers for testicular toxicity. Copyright © 2016 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.

Blood kidney injury molecule-1 is a biomarker of acute and chronic kidney injury and predicts progression to ESRD in type I diabetes.

Currently, no blood biomarker that specifically indicates injury to the proximal tubule of the kidney has been identified. Kidney injury molecule-1 (KIM-1) is highly upregulated in proximal tubular cells following kidney injury. The ectodomain of KIM-1 is shed into the lumen, and serves as a urinary biomarker of kidney injury. We report that shed KIM-1 also serves as a blood biomarker of kidney injury. Sensitive assays to measure plasma and serum KIM-1 in mice, rats, and humans were developed and validated in the current study. Plasma KIM-1 levels increased with increasing periods of ischemia (10, 20, or 30 minutes) in mice, as early as 3 hours after reperfusion; after unilateral ureteral obstruction (day 7) in mice; and after gentamicin treatment (50 or 200 mg/kg for 10 days) in rats. In humans, plasma KIM-1 levels were higher in patients with AKI than in healthy controls or post-cardiac surgery patients without AKI (area under the curve, 0.96). In patients undergoing cardiopulmonary bypass, plasma KIM-1 levels increased within 2 days after surgery only in patients who developed AKI (P<0.01). Blood KIM-1 levels were also elevated in patients with CKD of varous etiologies. In a cohort of patients with type 1 diabetes and proteinuria, serum KIM-1 level at baseline strongly predicted rate of eGFR loss and risk of ESRD during 5-15 years of follow-up, after adjustment for baseline urinary albumin-to-creatinine ratio, eGFR, and Hb1Ac. These results identify KIM-1 as a blood biomarker that specifically reflects acute and chronic kidney injury.

The first-in-human phase I study of a brain-penetrant mutant IDH1 inhibitor DS-1001 in patients with recurrent or progressive IDH1-mutant gliomas.

BACKGROUND: Approximately 70% of lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) mutations, resulting in the accumulation of oncometabolite D-2-hydroxyglutarate (D-2-HG); this leads to epigenetic dysregulation, oncogenesis, and subsequent clonal expansion. DS-1001 is an oral brain-penetrant mutant IDH1 selective inhibitor. This first-in-human study investigated the safety, pharmacokinetics, pharmacodynamics, and efficacy of DS-1001. METHODS: This was a multicenter, open-label, dose-escalation, phase I study of DS-1001 for recurrent/progressive IDH1-mutant (R132) glioma (N = 47) (NCT03030066). DS-1001 was administered orally at 125-1400 mg twice daily. Dose-escalation used a modified continual reassessment method. RESULTS: The maximum tolerated dose was not reached. Eight patients were continuing treatment at the data cutoff. Most adverse events (AEs) were grade 1-2. Twenty patients (42.6%) experienced at least 1 grade 3 AE. No grade 4 or 5 AEs or serious drug-related AEs were reported. Common AEs (>20%) were skin hyperpigmentation, diarrhea, pruritus, alopecia, arthralgia, nausea, headache, rash, and dry skin. The objective response rates were 17.1% for enhancing tumors and 33.3% for non-enhancing tumors. Median progression-free survival was 10.4 months (95% confidence interval [CI], 6.1 to 17.7 months) and not reached (95% CI, 24.1 to not reached) for the enhancing and non-enhancing glioma cohorts, respectively. Seven on-treatment brain tumor samples showed a significantly lower amount of D-2-HG compared with pre-study archived samples. CONCLUSIONS: DS-1001 was well tolerated with a favorable brain distribution. Recurrent/progressive IDH1-mutant glioma patients responded to treatment. A study of DS-1001 in patients with chemotherapy- and radiotherapy-naïve IDH1-mutated WHO grade 2 glioma is ongoing (NCT04458272).

A State-of-the-Art Roadmap for Biomarker-Driven Drug Development in the Era of Personalized Therapies.

Advances in biotechnology have enabled us to assay human tissue and cells to a depth and resolution that was never possible before, redefining what we know as the "biomarker", and how we define a "disease". This comes along with the shift of focus from a "one-drug-fits-all" to a "personalized approach", placing the drug development industry in a highly dynamic landscape, having to navigate such disruptive trends. In response to this, innovative clinical trial designs have been key in realizing biomarker-driven drug development. Regulatory approvals of cancer genome sequencing panels and associated targeted therapies has brought personalized medicines to the clinic. Increasing availability of sophisticated biotechnologies such as next-generation sequencing (NGS) has also led to a massive outflux of real-world genomic data. This review summarizes the current state of biomarker-driven drug development and highlights examples showing the utility and importance of the application of real-world data in the process. We also propose that all stakeholders in drug development should (1) be conscious of and efficiently utilize real-world evidence and (2) re-vamp the way the industry approaches drug development in this era of personalized medicines.

A new chronology for tsunami deposits prior to the 1700 CE Cascadia earthquake from Vancouver Island, Canada.

Coastal deposits at Tofino, Ucluelet, and Port Alberni in Vancouver Island along the Cascadia subduction zone were re-examined to improve the earthquake history of the southwest coast of Canada. We found sand sheets interbedded within peat and mud, suggesting deposition by strong flows in a low-energy environment. Based on limiting maximum and minimum ages derived from plant macrofossils, the age of one of the sand sheets below the tsunami deposits of the great Cascadia earthquake in 1700 CE was estimated to be 1330-1430 CE. Onshore paleoseismic evidence has been documented in Vancouver Island, northern Washington, and northern Oregon during this period. However, the newly constrained age is between those of coseismic subsidence Y and W events in southern Washington, which have been recognized as the 1700 CE and the penultimate Cascadia earthquakes, respectively. Moreover, the new age partly overlaps with the age of offshore paleoseismic evidence for T2, interpreted to have originated from the penultimate Cascadia earthquake, based on offshore turbidite records. The new chronology prior to the 1700 CE Cascadia tsunami deposit from Vancouver Island contributes to a better understand of the timing of the penultimate Cascadia earthquake.

Construction of a CYP2E1-template system for prediction of the metabolism on both site and preference order.

We have constructed an in silico system for the prediction of CYP2E1-mediated reaction using a two-dimensional template derived from substrate structures. Although CYP2E1 prefers small-size molecules for the substrates, the enzyme mediates oxidations of large-size molecules, such as benzo[a]pyrene. Overlays of these substrates, to assemble their sites of oxidation into a specific area, suggested a range of regions frequently occupied. The region, having a benzo[a]pyrene-like shape, was thus used as a CYP2E1 template. In this system, atoms in substrates, except for hydrogen atoms, were placed on corners of honeycomb structures of the template after having expanded the structures. Using published data for the metabolism on more than 80 substrates of CYP2E1, the core template was further refined to verify the adjacent area and to define the relative contribution of template positions for the catalysis. The positions on the template were classified into four different point (0-3) groups, depending on relative usage. In addition, we set independent points (-5 to 3) for specific positions to incorporate three-dimensional or functional information. Total scores from both position-occupancy and -function points were calculated for all the orientations of possible conformers of test substrates, and the scores were found to predict the relative abundance (i.e., order) as well as the regioselectivity of human CYP2E1 reactions with high fidelities.

Hepatic transcriptome and proteome responses against diethyl maleate-induced glutathione depletion in the rat.

Hepatic transcriptome and proteome responses against glutathione depletion were investigated by Affymetrix GeneChip Microarray and 2-dimensional gel electrophoresis (2D-DIGE), followed by MALDI-TOF-MS analysis and utilizing a glutathione-depleted rat model treated with diethyl maleate (DEM). Hepatic glutathione content decreased to 1.29 µmol/g liver (25.5% compared to control) after DEM treatment, and there were no apparent hepatotoxic signs estimated by blood chemistry examinations. A total of 247 and 213 annotated gene probe sets exhibited greater than twofold up- and down-regulation compared with controls, respectively. The up-regulated gene list contained a number of glutathione depletion-responsive genes reported previously, such as Trib3, Srxn1, Myc, Asns, Igfbp1, Txnrd1, or Hmox1, suggesting that these genes are robust mRNA biomarkers for evaluating hepatic glutathione depletion. In the 2D-DIGE analysis, proteins for a total of 361 spots were identified by MALDI-TOF-MS analysis. Of the identified proteins, 5 and 14 proteins showed up- and down-regulation, respectively. Some proteins exhibited differential expression in the protein level but not in the mRNA level, including L-FABP, MAWDBP, aldo-keto reductase family 1 member A1, catalase and ATP synthase subunit beta, suggesting that these proteins would be potential protein biomarkers for evaluating glutathione depletion. Moreover, up-regulation of FABP1 protein along with up-regulation of PPARα-regulated gene transcripts (i.e., Acot2 and Acot4) is indicative of PPARα activation, which may contribute to hepatocellular protection against glutathione depletion-induced oxidative stress. The up-regulation of L-FABP1 was detected by proteome data but not by transcriptome data, demonstrating the advantage of utilizing transcriptomics and proteomics combination to investigate glutathione depletion-induced molecular dynamics.

Intersubject and Intrasubject Variability of Potential Plasma and Urine Metabolite and Protein Biomarkers in Healthy Human Volunteers.

A limited understanding of intersubject and intrasubject variability hampers effective biomarker translation from in vitro/in vivo studies to clinical trials and clinical decision support. Specifically, variability of biomolecule concentration can play an important role in interpretation, power analysis, and sampling time designation. In the present study, a wide range of 749 plasma metabolites, 62 urine biogenic amines, and 1,263 plasma proteins were analyzed in 10 healthy male volunteers measured repeatedly during 12 hours under tightly controlled conditions. Three variability components in relative concentration data are determined using linear mixed models: between (intersubject), time (intrasubject), and noise (intrasubject). Biomolecules such as 3-carboxy-4-methyl-5-propyl-2-furanpropanoate, platelet-derived growth factor C, and cathepsin D with low noise potentially detect changing conditions within a person. If also the between component is low, biomolecules can easier differentiate conditions between persons, for example cathepsin D, CD27 antigen, and prolylglycine. Variability over time does not necessarily inhibit translatability, but requires choosing sampling times carefully.

Neutralization of IL-10 exacerbates cycloheximide-induced hepatocellular apoptosis and necrosis.

Cycloheximide (CHX)-induced liver injury in rats has been characterized by hepatocellular apoptosis and necrosis. We previously reported that Kupffer cell inactivation causes a reduction of IL-10 production, resulting in the exacerbation of CHX-induced liver injury. In this study, we directly evaluate the role of IL-10 in liver injury by a pretreatment with anti-IL-10 neutralizing antibody (IL-10Ab). Rats were given goat IgG or IL-10Ab before being treated with CHX (CHX group or IL-10Ab/CHX group). In the CHX group, the CHX treatment markedly induced hepatic mRNA and serum protein levels of IL-10. The up-regulation of IL-10 was significantly suppressed in the IL-10Ab/CHX group. Blocking IL-10 in the IL-10Ab/CHX group led to greater increases in hepatic mRNA and serum levels of proinflammatory cytokines, such as TNF-alpha and IL-6. The IL-10Ab/CHX group developed more severe hepatocellular apoptosis, neutrophil transmigration, and necrotic change of hepatocytes compared with the CHX group. The caspase activities and mRNA levels of Cc120, LOX-1, and E-selectin in the livers were significantly higher in the IL-10Ab/CHX group than the CHX group. These results demonstrate that IL-10 plays an important role in counteracting the effect of proinflammatory cytokines, such as a TNF signaling cascade, and in attenuating the CHX-induced liver injury.

Sensitivity of liver injury in heterozygous Sod2 knockout mice treated with troglitazone or acetaminophen.

Recently, it was reported that the intraperitoneal administration of 30 mg/kg/day troglitazone to heterozygous superoxide dismutase 2 gene knockout (Sod2+/-) mice for twenty-eight days caused liver injury, manifested by increased serum ALT activity and hepatic necrosis. Therefore, we evaluated the reproducibility of troglitazone-induced liver injury in Sod2+/- mice, as well as their validity as an animal model with higher sensitivity to mitochondrial toxicity by single-dose treatment with acetaminophen in Sod2+/- mice. Although we conducted a repeated dose toxicity study in Sod2+/- mice treated orally with 300 mg/kg/day troglitazone for twenty-eight days, no hepatocellular necrosis was observed in our study. On the other hand, six hours and twenty-four hours after an administration of 300 mg/kg acetaminophen, plasma ALT activity was significantly increased in Sod2+/- mice, compared to wild-type mice. In particular, six hours after administration, hepatic centrilobular necrosis was observed only in Sod2+/- mice. These results suggest that Sod2+/- mice are valuable as an animal model with higher sensitivity to mitochondrial toxicity. On the other hand, it was suggested that the mitochondrial damage alone might not be the major cause of the troglitazone-induced idiosyncratic liver injury observed in humans.

Influence of Kupffer cell inactivation on cycloheximide-induced hepatic injury.

In our previous study, we found that cycloheximide (CHX) induces hepatocellular necrosis as well as hepatocellular apoptosis. This article evaluates the role of Kupffer cells on cycloheximide-induced hepatic injury using gadolinium chloride (GdCl(3)) for the inhibition of Kupffer cells. One group of rats was treated with CHX (CHX group), and another was treated with GdCl(3) before being treated with the same dose of CHX (GdCl(3)/CHX group). The necrotic change in the GdCl(3)/CHX group was exacerbated under the induction of hepatocellular apoptosis by the CHX treatment. A substantial diminution of the number of ED1- or ED2-positive cells was demonstrated in the GdCl(3)/CHX group compared to the CHX group. In addition, the degree of decrease in ED2-positive cells was more apparent than that in ED1-positive cells. Increases in the mRNA levels of IL-10 and Stat3 were observed in the CHX group, but not in the GdCl(3)/CHX group. On the other hand, the hepatic mRNA levels of chemokines and adhesion molecules such as Ccl20, LOX-1, and E-selectin were significantly increased only in the GdCl(3)/CHX group. Thus, Kupffer cell inactivation by the GdCl(3) treatment leads to a loss of the capacity to produce IL-10, supposedly resulting in the enhancement of pro-inflammatory cytokine activities such as tumor necrosis factor (TNF) signaling. These events are suggested to be a factor of the inflammatory exacerbation in the livers of the GdCl(3)/CHX group. In conclusion, Kupffer cells may play a role in protecting hepatic necroinflammatory changes by releasing anti-inflammatory cytokines following the hepatocellular apoptosis resulting from CHX treatment.

Resistance to the skeletal muscle injury expressed by repeated treatment with compound A that has HMG-CoA reductase inhibitory activity.

It has been noted that chemical-induced initial insult is sometimes no longer detected in examinations after additional consecutive treatments, suggesting that the target organs acquire resistance to the chemical toxicity. In this study, whether acquired resistance to the skeletal muscle toxicity is observed during repeated treatment of a toxic dose of Compound A that has a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitory activity was examined. F344 male rats (7-weeks old) were given a mixed diet with 0.12% Compound A (corresponding to approximately 100 mg/kg/day) for up to 56 days. Blood samples were obtained from the tail vein periodically during the dosing period, and utilized for the measurement of creatine kinase (CK) as a marker of skeletal muscle injury. In the necropsies on Days 4, 8, 11, 28, 42 and 56, the skeletal muscles from the rectus femoris were removed for histopathology or gene expression analysis. A satellite group was provided to measure the plasma concentrations of Compound A and M1, the active metabolite of Compound A. CK levels increased from Day 9 and reached approximately 30 times those of the controls on Day 12. Histopathology of the skeletal muscle on Day 11 revealed severe necrosis of the muscle fibers. However, in spite of continuous treatments to the damaged rats, the CK levels decreased after that and returned to normal levels on Day 18. No skeletal muscle injury was observed on Days 42 and 56. There were no marked differences in the exposure levels of Compound A and M1 between Days 8 (prior to CK elevation) and 28 (post CK elevation). As for the most significant changes in the gene expression analysis for the skeletal muscle on Days 42 and 56, the probe for IkappaBa, which is known as an inhibitor for nuclear factor-kappaB (NF-kappaB), increased 2-fold compared to the control. Furthermore, an increased probe for CCAAT/enhancer-binding protein (C/EBP) delta, a transcriptional factor, and a decreased probe for cAMP-response element-binding protein (CBP)/p300, a transcriptional coactivator, were also noted significantly on Day 56. These changes in the gene expression analysis suggested suppressed NF-kappaB-mediated transactivation, which was responsible for the protective effects on the muscle injury. Based on the present findings, the resistance to skeletal muscle injury observed in this study may be attributable to the suppressed NF-kappaB-mediated transactivation, but not to the decreased exposure to toxicants.

Molecular mechanism investigation of cycloheximide-induced hepatocyte apoptosis in rat livers by morphological and microarray analysis.

Male F344 rats were intravenously treated with 6 mg/kg cycloheximide (CHX), and microarray analysis was conducted on their livers 1, 2 and 6h after the CHX treatment. The histopathological examination and serum chemistry results indicated a mild hepatic cell death 2 and 6h after the CHX treatment, respectively. Multi-focal hepatocellular necrosis with slight neutrophil infiltration was observed 6h after the CHX treatment. The TUNEL staining results showed that the number of apoptotic hepatocytes was the highest 2h after the CHX treatment. Dramatic increases in the mRNA levels of ATF3 and CHOP genes, both of which were reported to play roles in the ER stress-mediated apoptosis pathway, were observed from 1h after the CHX treatment. In addition, increase of GADD45, p21 and p53 mRNA levels also suggested a time course-related stimulation of hepatocellular apoptotic signals. These results suggest that the hepatocyte apoptosis induced by the CHX treatment is triggered by ER stress. The hepatic mRNA levels of proinflammatory genes, such as TNFalpha, IL-1alpha and beta, were also increased 1 and 2h after the CHX treatment, supposedly mediated by the activated Kupffer cells engulfing the apoptotic hepatocytes.

Toxicoproteomic investigation of the molecular mechanisms of cycloheximide-induced hepatocellular apoptosis in rat liver.

C/EBP homologous protein (CHOP) is a transcriptional factor and is induced under conditions such as the unfolded protein response or amino acid starvation. A previous study showed that the transcriptional level of CHOP was highly increased in rat liver in which hepatocellular apoptosis was induced by cycloheximide (CHX) treatment. Here, we investigated the relationship between hepatocellular apoptosis and CHOP-mediated apoptotic pathway, and studied the mechanisms of induction of CHOP gene in the liver of rats treated with CHX. Male F344 rats were treated intravenously with 6mg/kg CHX, and sacrificed at 1, 2 and 6h after the treatment. In the gene expression assay using quantitative RT-PCR, the genes related to CHOP-mediated apoptosis such as the C/EBPbeta, ATF3 and ATF4 genes were significantly increased corresponding to the induction of hepatocellular apoptosis in rats treated with CHX. However the GRP78/Bip gene, which serves as a representative marker for the unfolded protein response, did not change after the treatment. Toxicoproteomics using two-dimensional difference gel electrophoresis and mass spectrometry indicated that GRP78/Bip was inactivated by the CHX treatment. Furthermore, the CHX-treated animals exhibited a significant decrease of phosphorylated Akt/PKB (protein kinase B). These results indicate that the protein synthesis inhibition by CHX induces the CHOP gene through a pathway similar to that of amino acid starvation, and that Akt/PKB inactivation enhances the CHOP-mediated hepatocellular apoptosis.

Utilization of a toxicogenomic biomarker for evaluation of chemical-induced glutathione deficiency in rat livers across the GeneChip data of different generations.

Previously, we reported 69 probe sets (GSH probe sets) of RG U34A GeneChip that were useful for the evaluation of chemical-induced glutathione depletion in rat livers. The aim of the present study was to investigate whether these probe sets could be applied to the analysis of RAE 230A GeneChip data. Since a straightforward data comparison of RG U34A and RAE 230A GeneChips could not overcome the generation-dependent discrepancy in signal profiles, we tried two methods to improve the data compatibility between the two GeneChips. First, we re-calculated the signal values by excluding the probes with poor-overlapping sequences between the two GeneChips, but the data compatibility did not improve from the view point of Spearman's and Pearson's correlation coefficients. On the other hand, the PCA result demonstrated that an adjustment of the baseline signal level between the RG U34A and RAE 230A GeneChip data on vehicle-treated rats dramatically improved the data compatibility, suggesting that the GSH probe sets identified from RG U34A GeneChip data can be utilized in RAE 230A GeneChip data as well. Such a baseline adjustment of signal data is an easy and practical way to utilize biomarkers across GeneChip data of different generations.

Prediction of regioselectivity and preferred order of metabolisms on CYP1A2-mediated reactions. Part 1. Focusing on polycyclic arenes and the related chemicals.

This prediction system is based on placements of CYP1A2-substrates on a hexagonal-grid template in a way following the rule generated from the relationship of the substrate-structure and selective-area uses on the template, and also the rule for a triggering-event to initiate the catalytic reactions. Clear relationship found between the placements and preferred-order of regioselective reactions from the comparison of experimental data of polyaromatic hydrocarbon (PAH)-substrates was implemented in this system. The template generated is consisted of two flat-shape regions (Thin- and Thick-Areas) and Site of Oxidation. The latter is located at an overlapping region of Thin- and Thick-Areas and has a shape of cubic-like block. Thin-Area and Thick-Area are not on the same plane, and bend slightly at their overlapping parts. Two Entrances and two Gatekeepers are situated each near the top and middle bottleneck-parts of Thin-Area and Thick-Area to restrict the substrate entry. A procedure of stepwise movement of substrates resided on Thick-Area is introduced to define the regioselectivity. A specific part (termed Trigger-Region) showing high placement rates was found at a region away from Site of Oxidation on the template, assuming that substrate-sittings at Trigger-Region are essential for the initiation of catalytic reactions. These observations lead us an idea of simultaneous bi-molecule placement to fulfill both the essential sitting at Trigger-Region and regioselectivity of metabolisms, although a uni-molecule sitting co-covering Trigger-Region is also possible to contribute for the metabolite formation. Substrates of CYP1A2 are verified from distinct points including selective use of Thin-Area, Trigger-molecule Harboring Area, dual access routs, Facial-side Movement as well as their sizes. In addition, inhibitory actions as well region/stereo selective oxid/reductions of both PAH and non-PAH molecules are verified with their supposable interaction mechanisms in this prediction system.

Microarray analysis of T-2 toxin-induced liver, placenta and fetal liver lesions in pregnant rats.

Pregnant rats on day 13 of gestation were treated orally with 2 mg/kg of T-2 toxin and sacrificed at 1, 3, 6, 9 and 12 h after the treatment (HAT). Histopathologically, the number of apoptotic cells was increased in the liver, placenta and fetal liver (peaked at 6, 12 and 9-12 HAT, respectively). To examine the gene expression profiles, we performed microarray analysis of these tissues at two selected time points based on the results of the TdT-mediated dUTP nick end labeling (TUNEL) staining. Increased expression of oxidative stress- and apoptosis-related genes was detected in the liver of dams, placenta and fetal liver of pregnant rats treated with T-2 toxin at the peak time point of apoptosis. Decreased expression of lipid metabolism- and drug-metabolizing enzyme-related genes was also detected in these tissues. The results suggested that the mitogen-activated protein kinase (MAPK) pathway might be involved in the mechanism of T-2 toxin-induced apoptosis. In addition, increased expression of the c-jun gene was consistently observed in these tissues. Our results suggest that the mechanism of T-2 toxin-induced toxicity in pregnant rats is due to oxidative stress followed by the activation of the MAPK pathway, finally inducing apoptosis. The c-jun gene may play an important role in T-2 toxin-induced apoptosis.

MicroRNA profiling in ethylene glycol monomethyl ether-induced monkey testicular toxicity model.

To establish and characterize ethylene glycol monomethyl ether (EGME)-induced testicular toxicity model in cynomolgus monkeys, EGME at 0 or 300 mg/kg was administered orally to sexually mature male cynomolgus monkeys (n = 3/group) for 4 consecutive days. Circulating and testicular microRNA (miRNA) profiles in this model were investigated using miRNA microarray or real-time quantitative reverse transcription-PCR methods. EGME at 300 mg/kg induced testicular toxicity in all the monkeys, which was characterized histopathologically by decreases in pachytene spermatocytes and round spermatids, without any severe changes in general conditions or clinical pathology. In microarray analysis, 16 down-regulated and 347 up-regulated miRNAs were detected in the testis, and 326 down-regulated but no up-regulated miRNAs were detected in plasma. Interestingly, miR-1228 and miR-2861 were identified as abundant miRNAs in plasma and the testis of control animals, associated presumably with apoptosis and cell differentiation, respectively, and were prominently increased in the testis of EGME-treated animals, reflecting the recovery from EGME-induced testicular damages via stimulating cell proliferation and differentiation of sperm. Furthermore, down-regulation of miR-34b-5p and miR-449a, which are enriched in meiotic cells like pachytene spermatocytes, was obvious in the testis, suggesting that these spermatogenic cells were damaged by the EGME treatment. In conclusion, EGME-induced testicular toxicity in cynomolgus monkeys was shown, and this model would be useful for investigating the mechanism of EGME-induced testicular toxicity and identifying testicular biomarkers. Additionally, testicular miR-34b-5p and miR-449a were suggested to be involved in damage of pachytene spermatocytes.