Identification of kynurenine and quinolinic acid as promising serum biomarkers for drug-induced interstitial lung diseases.

BACKGROUND: Drug-induced interstitial lung disease (DILD) is a lung injury caused by various types of drugs and is a serious problem in both clinical practice and drug development. Clinical management of the condition would be improved if there were DILD-specific biomarkers available; this study aimed to meet that need. METHODS: Biomarker candidates were identified by non-targeted metabolomics focusing on hydrophilic molecules, and further validated by targeted approaches using the serum of acute DILD patients, DILD recovery patients, DILD-tolerant patients, patients with other related lung diseases, and healthy controls. RESULTS: Serum levels of kynurenine and quinolinic acid (and kynurenine/tryptophan ratio) were elevated significantly and specifically in acute DILD patients. The diagnostic potentials of these biomarkers were superior to those of conventional lung injury biomarkers, Krebs von den Lungen-6 and surfactant protein-D, in discriminating between acute DILD patients and patients with other lung diseases, including idiopathic interstitial pneumonia and lung diseases associated with connective tissue diseases. In addition to identifying and evaluating the biomarkers, our data showed that kynurenine/tryptophan ratios (an indicator of kynurenine pathway activation) were positively correlated with serum C-reactive protein concentrations in patients with DILD, suggesting the potential association between the generation of these biomarkers and inflammation. Our in vitro experiments demonstrated that macrophage differentiation and inflammatory stimulations typified by interferon gamma could activate the kynurenine pathway, resulting in enhanced kynurenine levels in the extracellular space in macrophage-like cell lines or lung endothelial cells. Extracellular quinolinic acid levels were elevated only in macrophage-like cells but not endothelial cells owing to the lower expression levels of metabolic enzymes converting kynurenine to quinolinic acid. These findings provide clues about the molecular mechanisms behind their specific elevation in the serum of acute DILD patients. CONCLUSIONS: The serum concentrations of kynurenine and quinolinic acid as well as kynurenine/tryptophan ratios are promising and specific biomarkers for detecting and monitoring DILD and its recovery, which could facilitate accurate decisions for appropriate clinical management of patients with DILD.

Stratifin as a novel diagnostic biomarker in serum for diffuse alveolar damage.

Among the various histopathological patterns of drug-induced interstitial lung disease (DILD), diffuse alveolar damage (DAD) is associated with poor prognosis. However, there is no reliable biomarker for its accurate diagnosis. Here, we show stratifin/14-3-3σ (SFN) as a biomarker candidate found in a proteomic analysis. The study includes two independent cohorts (including totally 26 patients with DAD) and controls (total 432 samples). SFN is specifically elevated in DILD patients with DAD, and is superior to the known biomarkers, KL-6 and SP-D, in discrimination of DILD patients with DAD from patients with other DILD patterns or other lung diseases. SFN is also increased in serum from patients with idiopathic DAD, and in lung tissues and bronchoalveolar lavage fluid of patients with DAD. In vitro analysis using cultured lung epithelial cells suggests that extracellular release of SFN occurs via p53-dependent apoptosis. We conclude that serum SFN is a promising biomarker for DAD diagnosis.

Hepatic Adaptation to Therapeutic Doses of Acetaminophen: An Exploratory Study in Healthy Individuals.

PURPOSE: Acetaminophen (APAP) has hepatotoxic potential when overdosed. Recent studies have reported serum alanine aminotransferase (ALT) elevations that resolve spontaneously with continued use of the drug, referred to as adaptation, in several individuals receiving therapeutic doses of APAP. However, the clinical significance of these ALT elevations remains unclear. This study was performed to investigate the incidence and characteristics of hepatic adaptation to therapeutic doses of APAP in healthy individuals. METHODS: In a randomized, single-blind, placebo-controlled study, 242 healthy Japanese individuals were enrolled. Each person received 3 g/d of APAP (n = 202) or placebo (n = 40) for 28 days. All study participants underwent analysis of genetic polymorphisms of CYP2E1 and UGT1A1; measurements of plasma APAP concentration and urine metabolites (glucuronide, sulfate, cysteine, and mercapturate); liver function monitoring, including ALT, microRNA-122, and high-mobility group box 1. Individuals with ALT levels remaining below the upper limit of normal (ULN; 40 U/L) during the study period were defined as tolerant and those with ALT elevations above the ULN as susceptible. Susceptible individuals who developed ALT elevations exceeding 2 × ULN discontinued use of the study drug for tolerability consideration. Susceptible individuals who had ALT elevations that decreased toward the ULN spontaneously with continued use of the study drug were classified as adaptation. FINDINGS: In the APAP group, 129 individuals (66%) were classified as tolerant and 65 (34%) as susceptible. Among 65 susceptible individuals, 12 (18%) discontinued use of APAP because of ALT elevations (>2 × ULN), whereas 53 (82%) completed 28-day APAP dosing. Thirty of 65 susceptible individuals (46%) had adaptation within 28 days. In the placebo group, no individuals was withdrawn from the study because of elevated ALT levels, 33 individuals (89%) were classified as tolerant, and 4 (11%) were classified as susceptible. None had clinical signs of liver injury. ALT level correlated significantly with microRNA-122 but not with high-mobility group box 1. No association was found between plasma APAP concentrations and ALT levels. Urinary excretion of APAP mercapturate was higher in susceptible than in tolerant individuals (P = 0.018, Wilcoxon or Kruskal-Wallis test). The frequency of homozygotes and compound heterozygotes for UGT1A1∗28 and UGT1A1∗6 (∗28/∗28, ∗6/∗6, and ∗6/∗28) was higher in susceptible than in tolerant individuals (13.9% vs 3.9%; P = 0.011, χ2 test). IMPLICATIONS: These findings indicate that in healthy individuals, APAP at a therapeutic dose can cause transient and self-limiting ALT elevation, reflecting subclinical hepatocellular damage, and these ALT elevations may be associated with the disposition of APAP metabolites and genetic factors. UMIN-CTR identifier: UMIN000019607.

Peptide vaccinations elicited strong immune responses that were reboosted by anti-PD1 therapy in a patient with myxofibrosarcoma.

Peptide-based immunotherapy does not usually elicit strong immunological and clinical responses in patients with end-stage cancer, including sarcoma. Here we report a myxofibrosarcoma patient who showed a strong clinical response to peptide vaccinations and whose immune responses were reboosted by anti-PD1 therapy combined with peptide vaccinations. The 46-year-old man showed a strong response to the peptide vaccinations (papillomavirus binding factor peptide, survivin-2B peptide, incomplete Freund's adjuvant, and polyethylene glycol-conjugated interferon-alpha 2a) and subsequent wide necrosis and massive infiltration of CD8+ T cells in a recurrent tumor. The patient's immune responses weakened after surgical resection; however, they were reboosted following the administration of nivolumab combined with peptide vaccinations. Thus, anti-PD1 therapy combined with peptide vaccinations might be beneficial, as suggested by the observations in this sarcoma patient.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

Toxicogenomics discrimination of potential hepatocarcinogenicity of non-genotoxic compounds in rat liver.

Long-term carcinogenicity testing of a compound is exceedingly time-consuming and costly, and requires many test animals, whereas the Ames test, which is based on the assumption that any substance that is mutagenic may also exert carcinogenic potential, is useful as a short-term screening assay but has major drawbacks. Although, in fact, 90% of compounds that give a positive Ames test cause cancer in laboratory animals, a good proportion of compounds that give a negative Ames test are also carcinogens; that is, there is no good correlation between carcinogenicity and negative Ames test results. As an alternative to these two approaches, we have tried applying toxicogenomics to predict the carcinogenicity of a compound from the gene expression profile induced in vivo. To establish our model, male Sprague-Dawley rats were orally administered test compounds (12 hepatocarcinogens and 26 non-hepatocarcinogens) for 28 days. Analysis of liver gene expression data by Support Vector Machines (SVM) dividing compounds into 'for training' and 'for test' (20 cases assigned randomly) allowed a set of marker genes to be tested for prediction of hepatocarcinogenicity. The developed prediction model was then validated with reference to the concordance rate with training data and test data, and a good performance was obtained. We will have new gene expression data and continue the validation of our model.

[Complete disappearance of pulmonary metastases in a case of rectal cancer treated with oral tegafur-uracil (UFT) and leucovorin (LV)].

We report a case of complete remission after treatment with tegafur-uracil (UFT)/Leucovorin (LV) therapy for pulumonary metastasis of rectal cancer. A 56-year-old male was admitted to our hospital with a diagnosis of rectal cancer (Ra, type2). Chest CT on admission demonstrated bilateral lung metastases (rt S2 and lt S4). After anterior resection of the primary tumor, oral UFT/LV was administered (UFT 400 mg/LV 75 mg, 4-week administration and 1-week no-administration period) on an outpatient basis. After 2 courses, chest CT revealed reduction of both metastases, and complete resection of the metastases by video assisted thoracic surgery (VATS) was planned. Pathological findings of a specimen revealed no residual cancer cells, indicating a complete response to UFT/LV therapy. After these treatments, combined therapy of UFT/LV was continued for 3 months, and the single administration of UFT was continued for 1 year. The patient experienced no adverse reactions, and has had no recurrent disease in 4 years. Oral UFT/LV therapy is considered to be a promising regimen for patients with resectable metastatic lesion from a standpoint of clinical efficacy and safety.

Effect of nuclear receptor downregulation on hepatic expression of cytochrome P450 and transporters in chronic hepatitis C in association with fibrosis development.

Analysis of mRNAs from liver biopsy samples of patients with chronic hepatitis C revealed that the levels of nuclear receptor expression were correlated with those of drug-metabolizing enzymes and transporters in relation to the development of fibrosis. Overall, the median mRNA level was largely dependent on fibrosis stage (F), and that for stage 3 patients (F3) was about 50% less than that for F1 patients. Levels of expression of AhR, together with CAR and PXR, were lowest in livers of F3 patients. Multivariate linear regression analysis revealed that AhR expression appeared to be involved in the regulation of CYP1A2, 2E1, 2D6, UGT1A, MDR1/3, MRP2/3, NTCP and OCT1 in the livers of patients with chronic hepatitis C. These results suggest that downregulation of AhR during the progression of liver fibrosis is associated with decreased expression levels of these phase I and II enzymes and drug transporters during inflammation-related signal transduction between AhR and other nuclear receptors.

Prediction model of potential hepatocarcinogenicity of rat hepatocarcinogens using a large-scale toxicogenomics database.

The present study was performed to develop a robust gene-based prediction model for early assessment of potential hepatocarcinogenicity of chemicals in rats by using our toxicogenomics database, TG-GATEs (Genomics-Assisted Toxicity Evaluation System developed by the Toxicogenomics Project in Japan). The positive training set consisted of high- or middle-dose groups that received 6 different non-genotoxic hepatocarcinogens during a 28-day period. The negative training set consisted of high- or middle-dose groups of 54 non-carcinogens. Support vector machine combined with wrapper-type gene selection algorithms was used for modeling. Consequently, our best classifier yielded prediction accuracies for hepatocarcinogenicity of 99% sensitivity and 97% specificity in the training data set, and false positive prediction was almost completely eliminated. Pathway analysis of feature genes revealed that the mitogen-activated protein kinase p38- and phosphatidylinositol-3-kinase-centered interactome and the v-myc myelocytomatosis viral oncogene homolog-centered interactome were the 2 most significant networks. The usefulness and robustness of our predictor were further confirmed in an independent validation data set obtained from the public database. Interestingly, similar positive predictions were obtained in several genotoxic hepatocarcinogens as well as non-genotoxic hepatocarcinogens. These results indicate that the expression profiles of our newly selected candidate biomarker genes might be common characteristics in the early stage of carcinogenesis for both genotoxic and non-genotoxic carcinogens in the rat liver. Our toxicogenomic model might be useful for the prospective screening of hepatocarcinogenicity of compounds and prioritization of compounds for carcinogenicity testing.

Mechanism-based biomarker gene sets for glutathione depletion-related hepatotoxicity in rats.

Chemical-induced glutathione depletion is thought to be caused by two types of toxicological mechanisms: PHO-type glutathione depletion [glutathione conjugated with chemicals such as phorone (PHO) or diethyl maleate (DEM)], and BSO-type glutathione depletion [i.e., glutathione synthesis inhibited by chemicals such as l-buthionine-sulfoximine (BSO)]. In order to identify mechanism-based biomarker gene sets for glutathione depletion in rat liver, male SD rats were treated with various chemicals including PHO (40, 120 and 400 mg/kg), DEM (80, 240 and 800 mg/kg), BSO (150, 450 and 1500 mg/kg), and bromobenzene (BBZ, 10, 100 and 300 mg/kg). Liver samples were taken 3, 6, 9 and 24 h after administration and examined for hepatic glutathione content, physiological and pathological changes, and gene expression changes using Affymetrix GeneChip Arrays. To identify differentially expressed probe sets in response to glutathione depletion, we focused on the following two courses of events for the two types of mechanisms of glutathione depletion: a) gene expression changes occurring simultaneously in response to glutathione depletion, and b) gene expression changes after glutathione was depleted. The gene expression profiles of the identified probe sets for the two types of glutathione depletion differed markedly at times during and after glutathione depletion, whereas Srxn1 was markedly increased for both types as glutathione was depleted, suggesting that Srxn1 is a key molecule in oxidative stress related to glutathione. The extracted probe sets were refined and verified using various compounds including 13 additional positive or negative compounds, and they established two useful marker sets. One contained three probe sets (Akr7a3, Trib3 and Gstp1) that could detect conjugation-type glutathione depletors any time within 24h after dosing, and the other contained 14 probe sets that could detect glutathione depletors by any mechanism. These two sets, with appropriate scoring systems, could be promising biomarkers for preclinical examination of hepatotoxicity.

The RXR agonists PA024 and HX630 have different abilities to activate LXR/RXR and to induce ABCA1 expression in macrophage cell lines.

Release of cellular cholesterol by ATP-binding cassette transporter (ABC)A1 and apolipoproteins is a major source of plasma high-density lipoprotein (HDL). Expression of ABC transporter A1 (ABCA1) is directly stimulated by liver X receptor (LXR)/retinoid X receptor (RXR) activation. We evaluated the abilities of two RXR agonists, PA024 and HX630, to increase ABCA1 expression. In differentiated THP-1 cells, the two agonists efficiently enhanced ABCA1 mRNA expression and apoA-I-dependent cellular cholesterol release. However, in RAW264 cells and undifferentiated THP-1 cells, PA024 was highly effective while HX630 was inactive in increasing ABCA1 mRNA. In parallel, the two agonists had different abilities to activate ABCA1 promoter in an LXR-responsive-element (LXRE)-dependent manner and to directly stimulate LXRalpha/RXR transactivation. The ability of HX630 to enhance ABCA1 expression was correlated closely with the cellular PPARgamma mRNA level. Moreover, HX630 was able to activate PPARgamma/RXR. Transfection of PPARgamma in RAW264 cells induced HX630-mediated activation of LXRE-dependent transcription and ABCA1 promoter, suggesting the ability of HX630 to activate PPARgamma-LXR-ABCA1 pathway. We conclude that RXR agonist PA024 and HX630 have different abilities to activate LXR/RXR, and that the cell-type-dependent effect of HX630 on ABCA1 expression and HDL generation is closely associated with this defect.

Safety assessment of biopharmaceuticals: Japanese perspective on ICH S6 guideline maintenance.

Safety assessment of biopharmaceuticals in preclinical studies is guided by the ICH S6 guideline issued in 1997. Along with enormous experiences and knowledge on safety assessment of some classes of biopharmaceuticals over the last decade, the necessity and feasibility of updating the guideline has been discussed. According to a recommendation by safety experts at the ICH meeting in Chicago in 2006, regional discussions of ICH S6 were held in the USA, EU and Japan. The meeting to clarify the values, challenges and recommendations for ICH S6 from Japanese perspective was held as a part of the first Drug Evaluation Forum in Tokyo on August 10, 2007. Of utmost importance, the "case-by-case" approach must be preserved as the basic principle of the ICH S6 guideline. It is our opinion that oligonucleotides, siRNA, aptamers and related molecules should be excluded from ICH S6 and may be more appropriate for separate guidance. However, based on experiences and accumulated knowledge, there are a number of issues that can be updated including new types of biopharmaceuticals such as bioconjugates, use of homologous proteins and transgenic animals, reproductive/developmental toxicity studies in non-human primates, in vitro cardiac ion channel assay and alternative approaches for carcinogenicity assessment. Preliminary recommendations for some of these topics were outlined at the meeting. The overall Japanese recommendation is that the ICH S6 guideline should be updated to address these topics.

Decreased expression of cytochromes P450 1A2, 2E1, and 3A4 and drug transporters Na+-taurocholate-cotransporting polypeptide, organic cation transporter 1, and organic anion-transporting peptide-C correlates with the progression of liver fibrosis in chronic hepatitis C patients.

Patients with chronic hepatitis C viral infection underwent liver biopsies and laboratory studies for evaluation and to determine subsequent treatment. Changes in status of drug metabolism and disposition may vary with chronic hepatitis C stage and should be assessed. Total RNA was extracted from liver biopsy specimens (n = 63) and reverse transcribed to yield cDNA. Relative mRNA levels of drug-metabolizing enzymes, transporters, nuclear receptors, and proinflammatory cytokines were analyzed with normalization to glyceraldehyde 3-phosphate dehydrogenase expression. mRNAs encoding cytochromes P450 1A2, 2E1, and 3A4, and drug transporters, Na(+)-taurocholate-cotransporting polypeptide, organic anion-transporting peptide-C, and organic cation transporter 1 showed remarkable decreases, and tumor necrosis factor-alpha showed an increase according to fibrosis stage progression. HepG2 cells and primary hepatocytes of two human individuals were treated with interleukin 1beta, interleukin 6, or tumor necrosis factor-alpha. CYP1A2 and Na(+)-taurocholate-cotransporting polypeptide mRNA levels significantly decreased in HepG2 cells with interleukin 1beta and interleukin 6 treatments. CYP2E1 and organic cation transporter 1 mRNA levels significantly decreased with tumor necrosis factor-alpha treatment only in HepG2. These results suggested that down-regulation of CYP1A2, 2E1, and 3A4, and drug transporters, Na(+)-taurocholate-cotransporting polypeptide, organic anion-transporting peptide-C, and organic cation transporter 1, manifested in livers of patients with chronic hepatitis C viral infection, was associated, at least in part, with the elevated production of proinflammatory cytokines, including tumor necrosis factor-alpha.

beta-Estradiol induces synaptogenesis in the hippocampus by enhancing brain-derived neurotrophic factor release from dentate gyrus granule cells.

We investigated the effect of beta-estradiol (E2) on synaptogenesis in the hippocampus using organotypic hippocampal slice cultures and subregional hippocampal neuron cultures. E2 increased the expression of PSD95, a postsynaptic marker, specifically in stratum lucidum of Cornu Ammonis 3 (CA3SL) in cultured hippocampal slices. E2 also increased the spine density at the proximal site of CA3 apical dendrites in CA3SL and PSD95 was clustered on these spine heads. The effects of E2 on the expression of PSD95 and the spine density disappeared when the dentate gyrus (DG) had been excised at 1 day in vitro (DIV). FM1-43 analysis of subregional hippocampal neuron cultures which were comprised of Ammon's horn neurons, DG neurons, or a mixture of these neurons, revealed that E2 increased the number of presynaptic sites in the cultures that contained DG neurons. K252a, a potent inhibitor of the high affinity receptor of brain-derived neurotrophic factor (BDNF), and function-blocking antibody to BDNF (BDNFAB) completely inhibited the effects of E2 in hippocampal slice cultures and subregional neuron cultures, whereas ICI182,780 (ICI), a strong antagonist of nuclear estrogen receptors (nERs), did not. Expression of BDNF in DG neurons was markedly higher than that in Ammon's horn neurons and E2 did not affect these expression levels. E2 significantly increased the BDNF release from DG neurons. KT5720, a specific inhibitor of 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), and Rp-adenosine 3', 5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMP), a non-hydrolyzable diastereoisomer and a potent inhibitor of PKA, completely suppressed the E2-induced increase in BDNF release, whereas ICI and U0126, a potent inhibitor of MAP kinase kinase (MEK), did not. These results suggest that E2 induces synaptogenesis between mossy fibers and CA3 neurons by enhancing BDNF release from DG granule cells in a nER-independent and PKA-dependent manner.

Extracellular ATP counteracts the ERK1/2-mediated death-promoting signaling cascades in astrocytes.

Oxidative stress is the main cause of neuronal death in pathological conditions. Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species, activates many intracellular signaling cascades including src family and mitogen-activated protein kinases (MAPKs), some of which are critically involved in the induction of cellular damage. We previously showed that H(2)O(2)-induced cell death in astrocytes and adenosine 5(')-triphosphate (ATP), acting on P2Y(1) receptors, had a protective effect. Here, we examined the H(2)O(2)-induced changes in intracellular signaling cascades that promote cell death in astrocytes, showing the molecular mechanisms by which the activation of P2Y(1) receptors counteracts such signals. Although H(2)O(2) activated three MAPKs including ERK1/2, p38, and JNK, only the activation of ERK1/2 participated in the H(2)O(2)-evoked cell death. H(2)O(2) induced a sustained activation of ERK1/2 mainly in the nucleus region, which was well in accordance with the H(2)O(2)-induced cell death. H(2)O(2) also activated the src tyrosine kinase family, which was an upstream signal for ERK1/2. Activation of P2Y(1) receptors by 2methylthio-ADP (2MeSADP) inhibited the H(2)O(2)-evoked activation of src tyrosine kinase, resulting in the inhibition of the phosphorylated-ERK1/2 accumulation in the nucleus. 2MeSADP enhanced the gene expression and activity of protein tyrosine phosphatase (PTP), which was responsible for the inhibition of src tyrosine kinase. Thioredoxin reductase, another cytoprotective gene we previously showed to be upregulated by 2MeSADP, also controlled the activity of PTP. Taken together, ATP, acting on P2Y(1) receptors, upregulates the PTP expression and its activity, which counteracts the H(2)O(2)-promoted death signaling cascades including ERK1/2 and its upstream signal src tyrosine kinase in astrocytes.

Gene expression property of high-density three-dimensional tissue of HepG2 cells formed in radial-flow bioreactor.

In our previous study, we examined three-dimensional culture using 5-ml radial-flow bioreactor (RFB) and showed that genes encoding cell cycle related proteins were suppressed in a stable phase. In this study, we analyzed the gene expression profiles of RFB-cultivated HepG2 cells and found that vascular endothelial growth factor (VEGF) production was strongly induced in the stable phase compared with the growth phase or static two-dimensional culture. When human umbilical vein endothelial cells (HUVECs) were grown under the conditioned medium of the stable phase, it was found that the formation of new blood vessels was induced in the angiogenesis model. DNA microarray analysis showed that the expression levels of both genes related to cell cycle arrest and which are known as tumor markers have increased in the stable phase. This result suggests that HepG2 cells in the stable phase maintain an active tumor phenotype. In addition, the expression of genes induced in the hypoxic condition was also induced in the stable phase. When the culture was carried out under a higher dissolved oxygen (DO) concentration, VEGF production did not decrease significantly and the new blood-vessel-forming ability of the conditioned medium was not suppressed. This suggests that the induction of VEGF production in a stable phase is not affected by DO during the tested level. These results suggest that the RFB cell culture system may be used to assess tumor progression mechanism under three-dimensional condition in vitro.

Metabolism of acrylamide to glycidamide and their cytotoxicity in isolated rat hepatocytes: protective effects of GSH precursors.

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxicity and prevention. Metabolism and cytotoxicity of AA and its epoxide glycidamide (GA) were studied in the hepatocytes freshly isolated from male Sprague-Dawley rats. The isolated hepatocytes metabolized AA to GA. The formation of GA followed Michaelis-Menten kinetic parameters yielded apparent Km = 0.477 +/- 0.100 and 0.263 +/- 0.016 mM, Vmax = 6.5 +/- 2.1 and 26.4 +/- 3.0 nmol/h/10(6) cells, and CLint = 14 +/- 5 and 100 +/- 12 microl/h/10(6) cells for the hepatocytes from untreated and acetone-treated rats, respectively. There were lower Km and marked increases in Vmax (four-fold) and in CLint (sevenfold) in acetone-treated rat hepatocytes. The data suggest that CYP2E1 played a major role in metabolizing AA to more toxic GA. Both AA and GA induced a concentration- and time-dependent glutathione (GSH) depletion of the hepatocytes. From decreasing rates of GSH contents in hepatocytes, the parameters of glutathione S-transferase (GST) in hepatocytes to AA and GA were calculated to be Km = 1.4 and 1.5 mM, Vmax = 21 and 33 nmol/h/10(6) cells, and CLint = 15 and 23 microl/h/10(6) cells, respectively. GA 1.5-times more readily depleted GSH content than AA. GA decreased the viability of hepatocytes at 3 mM, but AA did not. These data indicate that GA is more toxic than AA as assessed by intracellular GSH depletion and loss of viability of hepatocytes. GSH precursors such as N-acetylcysteine and methionine provided significant anti-cytotoxic effects on the decrease of GSH content and cell viability of hepatocytes induced by GA and AA.

Frequency distribution of phenol sulfotransferase 1A1 activity in platelet cells from healthy Japanese subjects.

AIMS: To determine the distribution of sulfotransferase 1A1 (SULT1A1) activities, we used trans-4-hydroxytamoxifen (OHT) as a substrate to test samples from a Japanese population to examine whether the SULT1A1*2 allele can account for the wide distribution of OHT sulfating activity. We also studied genetic mutations other than the SULT1A1*2 allele to determine the cause of differences in SULT1A1 protein expression and activity. METHODS: The subjects were 103 healthy Japanese adults. Identification of SULT1A1 genotypes was performed using a polymerase chain reaction-restriction fragment length polymorphism method. SULT1A1 activity in platelet cytosol was assayed using OHT as a substrate. SULT1A1 protein was detected using Western blotting analysis. Mutations other than SULT1A1*2 in the SULT1A1 gene were detected using sequencing analysis. RESULTS: SULT1A1*2 allele frequency was found to be 16.5%, while SULT1A1 activity ranged from 63 to 1860pmol sulfated/h/mg platelet protein (260+/-241pmol sulfated/h/mg platelet protein, median+/-S.D.) using OHT as a substrate. The median values in subjects with SULT*1/*2 (221+/-113pmol sulfated/h/mg platelet protein, range 63-442, n=26) and SULT*2/*2 (124+/-66pmol sulfated/h/mg platelet protein, range 74-231, n=4) were significantly lower than that in subjects with SULT*1/*1 (303+/-267pmol sulfated/h/mg platelet protein, range 97-1859, n=73). A novel G148C mutation was found in one subject, who showed the lowest OHT sulfating activity, for a frequency of 0.49%. CONCLUSION: There was wide variety of OHT sulfating activities found among the present healthy Japanese subjects. The SULT1A1*2 allele was found to be a common variant allele and was associated with decreased OHT sulfating activity. These observations may be related to inter-individual variations of OHT pharmacokinetics and the pharmacologic effects of tamoxifen seen in Japanese patients with breast cancer.

5Alpha-bile alcohols function as farnesoid X receptor antagonists.

The farnesoid X receptor (FXR) is a bile acid/alcohol-activated nuclear receptor that regulates lipid homeostasis. Unlike other steroid receptors, FXR binds bile acids in an orientation that allows the steroid nucleus A ring to face helix 12 in the receptor, a crucial domain for coactivator-recruitment. Because most naturally occurring bile acids and alcohols contain a cis-oriented A ring, which is distinct from that of other steroids and cholesterol metabolites, we investigated the role of this 5beta-configuration in FXR activation. The results showed that the 5beta-(A/B cis) bile alcohols 5beta-cyprinol and bufol are potent FXR agonists, whereas their 5alpha-(A/B trans) counterparts antagonize FXR transactivation and target gene expression. Both isomers bound to FXR, but their ability to induce coactivator-recruitment and thereby induce transactivation differed. These findings suggest a critical role for the A-ring orientation of bile salts in agonist/antagonist function.