Volumetric muscle composition analysis in sporadic inclusion body myositis using fat-referenced magnetic resonance imaging: Disease pattern, repeatability, and natural progression.

INTRODUCTION/AIMS: Fat-referenced magnetic resonance imaging (MRI) has emerged as a promising volumetric technique for measuring muscular volume and fat in neuromuscular disorders, but the experience in inflammatory myopathies remains limited. Therefore, this work aimed at describing how sporadic inclusion body myositis (sIBM) manifests on standardized volumetric fat-referenced MRI muscle measurements, including within-scanner repeatability, natural progression rate, and relationship to clinical parameters. METHODS: Ten sIBM patients underwent whole-leg Dixon MRI at baseline (test-retest) and after 12 months. The lean muscle volume (LMV), muscle fat fraction (MFF), and muscle fat infiltration (MFI) of the quadriceps, hamstrings, adductors, medial gastrocnemius, and tibialis anterior were computed. Clinical assessments of IBM Functional Rating Scale (IBMFRS) and knee extension strength were also performed. The baseline test-retest MRI measurements were used to estimate the within-subject standard deviation (sw). 12-month changes were derived for all parameters. RESULTS: The MRI measurements showed high repeatability in all muscles; sw ranged from 2.7 to 18.0 mL for LMV, 0.7-1.3 percentage points (pp) for MFF, and 0.2-0.7 pp for MFI. Over 12 months, average LMV decreased by 7.4% while MFF and MFI increased by 3.8 pp and 1.8 pp, respectively. Mean IBMFRS decreased by 2.4 and mean knee extension strength decreased by 32.8 N. DISCUSSION: The MRI measurements showed high repeatability and 12-month changes consistent with muscle atrophy and fat replacement as well as a decrease in both muscle strength and IBMFRS. Our findings suggest that fat-referenced MRI measurements are suitable for assessing disease progression and treatment response in inflammatory myopathies.

Circulating microRNA Profiles Identify a Patient Subgroup with High Inflammation and Severe Symptoms in Schizophrenia Experiencing Acute Psychosis.

Schizophrenia is a complex and heterogenous psychiatric disorder. This study aimed to demonstrate the potential of circulating microRNAs (miRNAs) as a clinical biomarker to stratify schizophrenia patients and to enhance understandings of their heterogenous pathophysiology. We measured levels of 179 miRNA and 378 proteins in plasma samples of schizophrenia patients experiencing acute psychosis and obtained their Positive and Negative Syndrome Scale (PANSS) scores. The plasma miRNA profile revealed three subgroups of schizophrenia patients, where one subgroup tended to have higher scores of all the PANSS subscales compared to the other subgroups. The subgroup with high PANSS scores had four distinctively downregulated miRNAs, which enriched 'Immune Response' according to miRNA set enrichment analysis and were reported to negatively regulate IL-1ß, IL-6, and TNFα. The same subgroup had 22 distinctively upregulated proteins, which enriched 'Cytokine-cytokine receptor interaction' according to protein set enrichment analysis, and all the mapped proteins were pro-inflammatory cytokines. Hence, the subgroup is inferred to have comparatively high inflammation within schizophrenia. In conclusion, miRNAs are a potential biomarker that reflects both disease symptoms and molecular pathophysiology, and identify a patient subgroup with high inflammation. These findings provide insights for the precision medicinal strategies for anti-inflammatory treatments in the high-inflammation subgroup of schizophrenia.

Activation of Naturally Occurring Lecithin:Cholesterol Acyltransferase Mutants by a Novel Activator Compound.

Lecithin:cholesterol acyltransferase (LCAT) is a unique plasma enzyme able to esterify cholesterol, and it plays an important role in HDL maturation and promotion of reverse cholesterol transport. Familial LCAT deficiency (FLD; OMIM number 245900) is a rare recessive disease that results from loss-of-function mutations in the LCAT gene and has no cure. In this study, we assessed the in vitro efficacy of a novel small-molecule LCAT activator. Cholesterol esterification rate (CER) and LCAT activity were tested in plasma from six controls and five FLD homozygous carriers of various LCAT mutations at different doses of the compound (0.1, 1, and 10 µg/ml). In control plasma, the compound significantly increased both CER (P < 0.001) and LCAT activity (P = 0.007) in a dose-dependent manner. Both CER and LCAT activity increased by 4- to 5-fold, reaching maximum activation at the dose of 1 µg/ml. Interestingly, Daiichi Sankyo compound produced an increase in CER in two of the five tested LCAT mutants (Leu372--Arg and Val309--Met), while LCAT activity increased in three LCAT mutants (Arg147--Trp, Thr274--Ile and Leu372--Arg); mutant Pro254--Ser was not activated at any of the tested doses. The present findings form the basis for personalized therapeutic interventions in FLD carriers and support the potential LCAT activation in secondary LCAT defects. SIGNIFICANCE STATEMENT: We characterized the pharmacology of a novel small-molecule LCAT activator in vitro on a subset of naturally occurring LCAT mutants. Our findings form the basis for personalized therapeutic interventions for familial LCAT deficiency carriers, who can face severe complications and for whom no cure exists.

Exploratory Analysis of Circulating miRNA Signatures in Atrial Fibrillation Patients Determining Potential Biomarkers to Support Decision-Making in Anticoagulation and Catheter Ablation.

Novel biomarkers are desired to improve risk management for patients with atrial fibrillation (AF). We measured 179 plasma miRNAs in 83 AF patients using multiplex qRT-PCR. Plasma levels of eight (i.e., hsa-miR-22-3p, hsa-miR-128-3p, hsa-miR-130a-3p, hsa-miR-140-5p, hsa-miR-143-3p, hsa-miR-148b-3p, hsa-miR-497-5p, hsa-miR-652-3p) and three (i.e., hsa-miR-144-5p, hsa-miR-192-5p, hsa-miR-194-5p) miRNAs showed positive and negative correlations with CHA2DS2-VASc scores, respectively, which also showed negative and positive correlations with catheter ablation (CA) procedure, respectively, within the follow-up observation period up to 6-month after enrollment. These 11 miRNAs were functionally associated with TGF-ß signaling and androgen signaling based on pathway enrichment analysis. Seven of possible target genes of these miRNAs, namely TGFBR1, PDGFRA, ZEB1, IGFR1, BCL2, MAPK1 and DICER1 were found to be modulated by more than four miRNAs of the eleven. Of them, TGFBR1, PDGFRA, ZEB1 and BCL2 are reported to exert pro-fibrotic functions, suggesting that dysregulations of these eleven miRNAs may reflect pro-fibrotic condition in the high-risk patients. Although highly speculative, these miRNAs may potentially serve as potential biomarkers, providing mechanistic and quantitative information for pathophysiology in daily clinical practice with AF such as possible pro-fibrotic state in left atrium, which would enhance the risk of stroke and reduce the preference for performing CA.

Circulating miRNA Signature as a Potential Biomarker for the Prediction of Analgesic Efficacy of Hydromorphone.

No practical biomarkers currently exist for the prediction of the analgesic efficacy of opioids. Previously, we reported circulating miRNA signatures differentially regulated by µ-opioid receptor (MOR) agonists in healthy subjects. We hypothesized that these miRNAs could be potential pharmacodynamic biomarkers to estimate MOR stimulation, and predict the efficacy of opioids; i.e., patients with low MOR stimulation may be more vulnerable to strengthening of the MOR signal upon hydromorphone treatment. To test this hypothesis, plasma samples were obtained from 25 patients with cancer pain prior to the initiation of hydromorphone treatment and the circulating miRNA levels were evaluated, focusing on four miRNAs (i.e., hsa-miR-423-3p, hsa-let-7a-5p, hsa-miR-26a-5p, and hsa-let-7f-5p) and four miRNAs (i.e., hsa-miR-144-3p, hsa-miR-451a, hsa-miR-215, and hsa-miR-363-3p) that were most clearly up and downregulated by hydromorphone and oxycodone. The patients were classified into two classes with putative high and low MOR signal, estimated based on the plasma miRNA signature. A significant correlation was observed between the analgesic efficacy and the putative MOR signal level, and patients with low MOR signal achieved better pain control (i.e., ΔVAS < 0) through hydromorphone. These results suggested that plasma miRNA signatures could serve as clinical biomarkers for the prediction of the analgesic efficacy of hydromorphone.

Identification of Circulating miRNAs Differentially Regulated by Opioid Treatment.

Emerging evidence demonstrates functional contributions of microRNAs (miRNAs) to µ-opioid receptor (MOR) signaling, but the information so far has been mostly limited to their intracellular regulatory mechanisms. The present study aimed to investigate changes in plasma miRNA profiles elicited by opioid treatment in blood samples collected from clinical studies. Healthy male subjects were orally administered with hydromorphone or oxycodone and blood samples were collected at a specified time after the drug treatment. A total of 179 plasma miRNAs were measured using multiplex qRT-PCR. Nine and seventeen miRNAs were commonly upregulated (let-7a-5p, miR-423-3p, miR-199a-3p, miR-146a-5p, miR-23b-3p, miR-24-3p, miR-221-3p, miR-223-3p, and miR-146b-5p) and downregulated (miR-144-3p, miR-215, miR-363-3p, etc.), respectively, following opioid treatment. The MOR signaling-associated miRNAs, namely let-7 family miRNAs (i.e., let-7d-5p, let-7f-5p, let-7c, let-7e-5p), miR-103a-3p, miR-339-3p, miR-146a-5p, miR-23b-3p, miR-23a-3p, and miR-181a-5p, were differentially expressed following drug treatment. These differentially expressed miRNAs are circulating biomarker candidates that can be used to evaluate MOR stimulation and serve as novel clinical diagnostic tools for improving clinical outcomes.

(+)-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.

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.

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.

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.

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.

Species-specific regulation of PXR/CAR/ER-target genes in the mouse and rat liver elicited by o, p'-DDT.

BACKGROUND: Dichlorodiphenyltrichloroethane (DDT) is a persistent estrogenic organochlorine pesticide that is a rodent hepatic tumor promoter, with inconclusive carcinogenicity in humans. We have previously reported that o, p'-DDT elicits primarily PXR/CAR-mediated activity, rather than ER-mediated hepatic responses, and suggested that CAR-mediated effects, as opposed to ER-mediated effects, may be more important in tumor promotion in the rat liver. To further characterize species-specific hepatic responses, gene expression analysis, with complementary histopathology and tissue level analyses were investigated in immature, ovariectomized C57BL/6 mice treated with 300 mg/kg o, p'-DDT, and compared to Sprague-Dawley rat data. RESULTS: Rats and mice exhibited negligible histopathology with rapid o, p'-DDT metabolism. Gene expression profiles were also similar, exhibiting PXR/CAR regulation with the characteristic induction of Cyp2b10 and Cyp3a11. However, PXR-specific target genes such as Apoa4 or Insig2 exhibited more pronounced induction compared to CAR-specific genes in the mouse. In addition, mouse Car mRNA levels decreased, possibly contributing to the preferential activation of mouse PXR. ER-regulated genes Cyp17a1 and Cyp7b1 were also induced, suggesting o, p'-DDT also elicits ER-mediated gene expression in the mouse, while ER-mediated effects were negligible in the rat, possibly due to the inhibitory effects of CAR on ER activities. In addition, o, p'-DDT induced Gadd45a, Gadd45b and Cdkn1, suggesting DNA damage may be an additional risk factor. Furthermore, elevated blood DHEA-S levels at 12 h after treatment in the mouse may also contribute to the endocrine-related effects of o, p'-DDT. CONCLUSION: Although DDT is known to cause rodent hepatic tumors, the marked species differences in PXR/CAR structure, expression patterns and ligand preference as well as significant species-specific differences in steroidogenesis, especially CYP17A1 expression and activity, confound the extrapolation of these results to humans. Nevertheless, the identification of potential modes of action as well as species-specific responses may assist in the selection and further development of more appropriate models for assessing the toxicity of DDT to humans and wildlife.

o,p'-DDT elicits PXR/CAR-, not ER-, mediated responses in the immature ovariectomized rat liver.

Technical-grade dichlorodiphenyltrichloroethane (DDT) is an agricultural pesticide and malarial vector control agent that has been designated a potential human hepatocarcinogen. The o,p'-enantiomer exhibits estrogenic activity that has been associated with the carcinogenicity of DDT. The temporal and dose-dependent hepatic estrogenicity of o,p'-DDT was investigated using complementary DNA microarrays in immature ovariectomized Sprague-Dawley rats with complementary histopathology and tissue-level analysis. Animals were gavaged with 300 mg/kg o,p'-DDT either once or once daily for 3 consecutive days. Liver samples were examined 2, 4, 8, 12, 18, or 24 h after a single dose or following three daily doses. For dose-response studies, a single dose of 3, 10, 30, 100, or 300 mg/kg body weight o,p'-DTT was administered for 3 consecutive days. Genes associated with drug metabolism (Cyp2b2 and Cyp3a2), the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), cell proliferation (Ccnd1, Ccnb1, Ccnb2, and Stmn1), and oxidative stress (Gclm and Hmox1) were significantly induced. Cyp2b2 exhibited dose-dependent regulation and was significantly induced across all time points, while cell proliferation- and oxidative stress-related genes exhibited transient induction. The induction of Cyp2b2 and Cyp3a2 mRNA levels suggest PXR/CAR activation, consistent with expression of genes associated with oxidative stress. Few genes known to be estrogen receptor (ER) regulated were differentially expressed when compared to the hepatic gene expression profile elicited by ethynyl estradiol in immature ovariectomized C57BL/6 mice using the same study design and analysis methods. These data indicate that o,p'-DDT elicits PXR/CAR-, not ER-, mediated gene expression in the rat liver. Based on the species-specific differences in CAR regulation, the extrapolation of rodent DDT hepatocarcinogenicity to humans warrants further investigation.

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.

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.

Characterization of resistance to bromobenzene-induced hepatotoxicity by microarray.

In our previous study, we demonstrated that the initial hepatic injury caused by bromobenzene (BB) was no longer detected in rats despite subsequent dosing, indicating that the liver acquired resistance to BB-induced hepatotoxicity. In this experiment, microarray analysis was conducted to characterize this resistance. The liver samples for the analysis utilized were obtained from previous experiments where F344 rats were treated intraperitoneally with BB (150 mg/kg). At 24 hr post-dose, hepatic injury was confirmed by monitoring the AST values and then the rats were maintained at the same dosing regimen for an additional 8 days. The gene expression profiles of the BB-treated rat livers were compared with a vehicle-treated group by Affymetrix RG_U34A arrays. As results, a decreased expression level of CYP3A9 and an increased expression level of GST Yc2 and glutathione peroxidase (GPX) were detected. These changes indicated suppression of the phase I reaction and induction of the phase II reaction (glutathione conjugation). Increased expression levels of epoxide hydrolase (EH) and NAD(P)H:quinone oxidoreductase (NQO1) also suggested the involvement of EH- and NQO1-mediated hydrolysis other than glutathione conjugation with resistance in the phase II reaction. Moreover, an increased expression level of abcc3 (multidrug resistance protein 3; Mrp3) was significantly noted. Based on the present findings, it was suggested that Mrp3 in the phase III reaction (drug elimination) contributed to the resistance to BB hepatotoxicity in addition to the suppression of the phase I reaction (metabolic activation) and the induction of the phase II reaction (detoxification). Among them, the factors which contributed most were considered to be the increased GST Yc2 and Mrp3, based on the degree of the gene expression changes.

Gene expression profiling of rat liver treated with serum triglyceride-decreasing compounds.

We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for interpretation of plasma triglyceride (TG) decrease, we extracted 218 probe sets of rat hepatic genes from data of 15 drugs that decreased the plasma TG level but differentially affected food consumption. Pathway and gene ontology analysis revealed that the genes belong to amino acid metabolism, lipid metabolism and xenobiotics metabolism. Principal component analysis (PCA) showed that 12 out of 15 compounds were separated in the direction of PC1, and these 12 were separated in the direction of PC2, according to their hepatic gene expression profiles. It was found that genes with either large or small eigenvector values in principal component PC 2 were those reported to be regulated by peroxisome proliferator-activated receptor (PPAR)alpha or constitutive androstane receptor (CAR), respectively. In fact, WY-14,643, clofibrate, gemfibrozil and benzbromarone, reported to be PPARalpha activators, distributed to the former, whereas propylthiouracil, omeprazole, phenobarbital, thioacetamide, methapyrilene, sulfasalazine and coumarin did to the latter. We conclude that these identified 218 probe sets could be a useful source of biomarkers for classification of plasma TG decrease, based on the mechanisms involving PPARalpha and CAR.

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.

Identification of glutathione depletion-responsive genes using phorone-treated rat liver.

To identify candidate biomarker gene sets to evaluate the potential risk of chemical-induced glutathione depletion in livers, we conducted microarray analysis on rat livers administered with phorone (40, 120 and 400 mg/kg), a prototypical glutathione depletor. Hepatic glutathione content was measured and glutathione depletion-responsive gene probe sets (GSH probe sets) were identified using Affymetrix Rat Genome 230 2.0 GeneChip by the following procedure. First, probe sets, whose signal values were inversely correlated with hepatic glutathione content throughout the experimental period, were statistically identified. Next, probe sets, whose average signal values were greater than 1.5-fold compared to those of controls 3 hr after phorone treatment, were selected. Finally, probe sets without unique Entrez Gene ID were removed, ending up with 161 probe sets in total. The usefulness of the identified GSH probe sets was verified by a toxicogenomics database. It was shown that signal profiles of the GSH probe sets in rats treated with bromobenzene were strongly altered compared with other chemicals. Focusing on bromobenzene, time-course profiles of hepatic glutathione content and gene expression revealed that the change in gene expression profile was marked after the bromobenzene treatment, whereas hepatic glutathione content had recovered after initial acute depletion, suggesting that the gene expression profile did not reflect the hepatic glutathione content itself, but rather reflects a perturbation of glutathione homeostasis. The identified GSH probe sets would be useful for detecting glutathione-depleting risk of chemicals from microarray data.