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.

Muscle relaxant and neurotoxic activities of intrathecal baclofen in rats.

Intrathecal baclofen therapy by the continuous intrathecal infusion of baclofen has been shown to be an effective treatment for spasticity in patients with spinal cord injury, cerebral palsy, traumatic brain injury, multiple sclerosis and other disorders. To demonstrate the efficacy and safety of intrathecal baclofen therapy, we investigated the muscle relaxant and neurotoxic activities of intrathecal baclofen in rats, compared with intravenous baclofen. Intrathecal and intravenous administration of baclofen dose-dependently inhibited the anemic decerebrate rigidity with ED(50) values of 0.31microg/animal (=1.1-1.3microg/kg) and 0.43mg/kg, respectively. Intrathecal administration of baclofen induced no noticeable changes in a spontaneous electroencephalogram at 30microg/animal. Intravenous administration of baclofen induced an abnormal electroencephalogram with flat waves in all the animals and the no-observed-effect level was estimated to be 5mg/kg. In some animals, intravenous administration of baclofen induced sporadic spikes or sharp waves with background flat waves, indicating inhibitory and excitatory effects on the central nervous system. In conclusion, intrathecal administration of baclofen dose-dependently inhibited anemic decerebrate rigidity in rats and the effective dose was more than 300 times lower than that of intravenous baclofen. The safety margin of intrathecal baclofen was greater than that of intravenous baclofen (> or =97 versus 12). These results suggest that intrathecal baclofen therapy is superior to systemic baclofen therapy in both efficacy and safety.

Effects of estradiol treatment and/or ovariectomy on spontaneous hemorrhagic lesions in the pancreatic islets of Sprague-Dawley rats.

The present study was conducted to investigate the effect of estradiol treatment and/or ovariectomy (OVX) on non-neoplastic lesions in the pancreatic islets of Sprague-Dawley rats. Males were divided into non-treatment (naïve) and beta-estradiol 3-benzoate (EB) treatment groups and females into naïve, sham-operation, OVX, and OVX plus EB treatment groups. EB was subcutaneously administered once a week from seven to twenty-six weeks of age. The animals were euthanized at twelve, eighteen, and twenty-six weeks of age, and the serum estradiol concentrations were measured in conjunction with the pancreatic islet histopathology. The histological stages of pancreatic findings were classified into three groups, hemorrhagic, fibrotic, and inflammatory lesions, and the incidence of each type of lesion was enumerated. In males, both the total and individual incidence of pancreatic lesions increased age dependently in the naïve group. EB treatment significantly decreased the total incidence at twenty-six weeks. This alteration consisted of fibrotic and inflammatory lesions, but not hemorrhagic lesions. Additionally, the incidence of hemorrhagic lesions was at the same level between male naïve and male EB groups at twelve weeks, despite a markedly higher concentration of serum estradiol in the EB group. In females, a similar tendency was seen, and the total incidence was generally low in the naïve group, whereas it was increased by OVX. OVX plus EB treatment tended to decrease the incidence accompanied by a marked increase in estradiol concentrations. In conclusion, estrogen was shown to inhibit the development of pancreatic islet lesions toward inflammation and fibrosis but did not inhibit the occurrence of hemorrhagic lesions.

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.

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.

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats.

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

Gene expression profiles in the articular cartilage of juvenile rats receiving the quinolone antibacterial agent ofloxacin.

Quinolone antibacterial agents are extensively utilized in antimicrobial chemotherapy. However, they have been reported to induce arthropathy in juvenile animals, and the mechanism has not been clarified. In the present study, to investigate the molecular details of the chondrotoxicity of the quinolone ofloxacin (OFLX), it was orally administered by gavage at a dose level of 900 mg/kg once to male juvenile Sprague-Dawley rats, 3 weeks of age. Then gene expression profiles in the articular cartilage of the distal femur were analyzed at 2, 4, 8 and 24h post-dose. In the GeneChip analysis, the expression of 134 gene probes in the OFLX-treated group showed statistically significant differences with at least 1.5-fold difference from the control. Among them, intracellular signaling cascade- and stress response-related genes changed at 2h post-dose; cell death- and inflammatory response-related genes at 4 and 8h post-dose; basic-leucine zipper transcription factor and stress response-related genes at 8 and 24h post-dose; stress response-, proteolysis- and glycoprotein-related genes at 24h post-dose. In a quantitative real-time reverse transcription-polymerase chain reaction analysis, up-regulated Dusp1 (intracellular signaling cascade-related gene), Tnfrsf12a (cell death-related gene), Ptgs2, Fos (inflammatory response-related genes), Mt1a, Plaur (stress response-related genes) and Mmp3 (proteolysis-related gene) and down-regulated Sstr1 and Has2 (glycoprotein-related genes) were observed with dose dependency in the articular cartilage of juvenile rats treated with OFLX at 100, 300 and 900 mg/kg. The expression of Tnfrsf12a, Ptgs2, Plaur and Mmp3 was also noted in chondrocytes around the cartilage lesions by in situ hybridization. In conclusion, our results suggest that cytokines, chemokines and/or proteases produced by up-regulation of cell death-, inflammatory response-, stress response- and proteolysis-related genes play a important role in the onset of OFLX-induced chondrotoxicity in juvenile rats.

Relationship between GST Yp induction and hepatocyte proliferation in rats treated with phase II drug metabolizing enzyme inducers.

Butylated hydroxyanisole (BHA) and 1,2-bis(2-pyridyl)ethylene (2PY-e) are phase II drug metabolizing enzyme inducers which cause hepatomegaly without hepatocyte hypertrophy and induce glutathione S-transferase Yp (GST Yp, pi-class GST), which is known as a tumor marker. To evaluate the relationship between GST Yp induction and hepatocyte proliferation, male F344/DuCrj rats were treated with BHA, 2PY-e, or phenobarbital (PB) for three or seven days. All three chemicals caused increases in liver weight after three and seven days. Immunohistochemical examinations revealed that BHA and 2PY-e induced GST Yp in the hepatocytes of the periportal and centrilobular areas at three and seven days, respectively, whereas PB did not. Significant increases in the BrdU labeling indices were found in the livers of rats in each of the three-day treatment groups, but the labeling index of rat livers treated with BHA was decreased to the control level at seven days, although the high labeling indices of 2PY-e and PB persisted at seven days. Double immunostaining confirmed that BrdU-positive nuclei corresponded to GST Yp-positive hepatocytes in both BHA and 2PY-e treated rats. These results suggest that the GST Yp induction caused by BHA or 2PY-e has some kind of relationship with hepatocyte proliferation.

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats.

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.

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.

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.

Data-intensive drug development in the information age: applications of Systems Biology/Pharmacology/Toxicology.

Pharmaceutical companies continuously face challenges to deliver new drugs with true medical value. R&D productivity of drug development projects depends on 1) the value of the drug concept and 2) data and in-depth knowledge that are used rationally to evaluate the drug concept's validity. A model-based data-intensive drug development approach is a key competitive factor used by innovative pharmaceutical companies to reduce information bias and rationally demonstrate the value of drug concepts. Owing to the accumulation of publicly available biomedical information, our understanding of the pathophysiological mechanisms of diseases has developed considerably; it is the basis for identifying the right drug target and creating a drug concept with true medical value. Our understanding of the pathophysiological mechanisms of disease animal models can also be improved; it can thus support rational extrapolation of animal experiment results to clinical settings. The Systems Biology approach, which leverages publicly available transcriptome data, is useful for these purposes. Furthermore, applying Systems Pharmacology enables dynamic simulation of drug responses, from which key research questions to be addressed in the subsequent studies can be adequately informed. Application of Systems Biology/Pharmacology to toxicology research, namely Systems Toxicology, should considerably improve the predictability of drug-induced toxicities in clinical situations that are difficult to predict from conventional preclinical toxicology studies. Systems Biology/Pharmacology/Toxicology models can be continuously improved using iterative learn-confirm processes throughout preclinical and clinical drug discovery and development processes. Successful implementation of data-intensive drug development approaches requires cultivation of an adequate R&D culture to appreciate this approach.