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.

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.

Ethylene glycol monomethyl ether-induced toxicity is mediated through the inhibition of flavoprotein dehydrogenase enzyme family.

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

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.

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.

Gene expression profiling of methapyrilene-induced hepatotoxicity in rat.

The present study was conducted as a model case of the toxicogenomics approach for analyzing toxicological mechanisms and toxicity assessments in the early stage of drug development by comparing with classical toxicology data. Methapyrilene (MP) 100 mg/kg produced obvious histopathological changes in liver of rats by single or repeated dose up to 28 days with significant elevation of ALT and AST. In the middle dose groups (30 mg/kg MP), no apparent changes were noted in blood biochemical data by single dosing or repeated dosing up to one week, and no obvious histopathological changes were observed except a slight hypertrophy in the hepatocytes. Comprehensive gene expression changes were analyzed using Affymetrix GeneChip and differentially expressed probe sets were statistically extracted. These contained many genes related to "glutathione metabolism", "apoptosis", "MAPK signaling pathway" and "regulation of cell cycle", which were all thought to be involved in the development of presently observed phenotypes. In the high dose groups, TGP1 scores (developed in our system in order to overview the responsiveness of drugs to multiple marker gene lists) for these categories were markedly increased from the early time point after single dose and kept their high expression throughout the repeated dose period. In the middle dose groups, the increment of the scores were noted not only at the time points when apparent pathological changes emerged, but also at the earlier stage of repeated dosing and even after single dosing. We conclude that toxicogenomics would enable a more sensitive assessment at the earlier time point than classical toxicology evaluation.

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.

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.

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.

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.

Utilization of a one-dimensional score for surveying chemical-induced changes in expression levels of multiple biomarker gene sets using a large-scale toxicogenomics database.

A large-scale toxicogenomcis database has now been constructed in the Toxicogenomics Project in Japan (TGP). To facilitate the analytical procedures for such large-scale microarray data, we developed a simple one-dimensional score, named TGP1 which expresses the trend of the changes in expression of biomarker genes as a whole. To evaluate the usefulness of the TGP1 score, microarray data of rat liver and rat hepatocytes deposited in the TGP database were scored for three biomarker gene sets, i.e., carcinogenesis-related, PPARalpha-regulated and glutathione depletion-related gene sets. The TGP1 scoring system gave reasonable results, i.e., the scores for carcinogenesis-related genes were high in omeprazole-, chlorpromazine-, hexachlorobenzene-, sulfasalazine- and Wy-14,643-treated rat livers, that for PPARalpha-regulated genes were high in clofibrate-, Wy-14,643-, gemfibrozil-, benzbromarone- and aspirin-treated rat livers as well as rat hepatocytes, and for glutathione deficiency-related genes were high in omeprazole-, bromobenzene-, acetaminophen- and coumarin-treated rat liver. We concluded that the TGP1 score is useful for surveying the expression changes in multiple biomarker gene sets for a large-scale toxicogenomics database, which would reduce the time of doing conventional multivariate statistical analysis. In addition, the TGP1 score can be applied to screening of compatible biomarker gene sets between rat liver and rat hepatocytes, like PPARalpha-regulated gene sets, which will allow us to develop an appropriate in vitro system for drug safety assessment in vivo.

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.

Evaluation of glutathione deficiency in rat livers by microarray analysis.

Hepatic glutathione content was measured and gene expression data were obtained using an Affymetrix RG U34 array after treatment with tap water containing 20mM l-buthionine (S, R)-sulfoximine (BSO) to male F344 rats for four consecutive days. Both Spearman's and Pearson's correlation coefficients were calculated between the glutathione content and the mRNA content level obtained from the microarray analysis individually. Sixty-nine gene probes, which were statistically significant (Spearman's correlation, P < 0.05) and showed a Pearson's correlation coefficients (Pearson's r) less than -0.8 between mRNA content and hepatic glutathione content, were identified as glutathione deficiency-correlated probes. By comparing the hepatic gene expression profiles between BSO- and butylated hydroxyanisole (BHA)-treated rats, 14 probes of genes that showed an increase in the corresponding gene mRNA levels only after the BSO treatment were thought to be good indicators of glutathione deficiency. A principal component analysis successfully illustrated the time-course of hepatic gene expression after the treatment with acetaminophen, phenobarbital and clofibrate, and the expression profiles were thought to reflect the changes in hepatic glutathione levels. The identified gene probes in the present study would be useful as markers for assessing hepatocellular glutathione deficiency, or oxidative stress level, based on microarray data.