RESUMEN
The metabolomic profiles of rat primary hepatocytes following treatment with rotenone, FCCP, or (+)-usnic acid were determined using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Significant and similar changes in the levels of 283 biochemical metabolites were associated with the three treatments compared with solvent control samples. Overall, the three treatments generated similar global biochemical profiles, with some minor differences associated with rotenone treatment. All three treatments resulted in a shift in energy metabolism as demonstrated by decreased glycogen stores and glycolysis. A reduced antioxidant response was detected in cells following all treatments. In addition, bile acid biosynthesis decreased as a potential consequence of increased oxidative stress by all three treatments. Conversely, rotenone treatment induced a number of changes after 1 hr, which were not detected in FCCP- or (+)-usnic acid-treated samples; these changes were not sustained over time and included increased NAD+ salvage and lysine degradation. In conclusion, these biochemical profiles could provide new insights into the mechanism(s) of mitochondrial toxicity.
Asunto(s)
Benzofuranos/efectos adversos , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/efectos adversos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Rotenona/efectos adversos , Animales , Ácidos y Sales Biliares/metabolismo , Células Cultivadas , Cromatografía Liquida , Metabolismo Energético/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Glucógeno/metabolismo , Glucólisis/efectos de los fármacos , Metabolómica , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas Endogámicas F344RESUMEN
Genotoxicity and carcinogenicity profiles of drugs occasionally vary across species due to species difference in drug metabolic profile. To clarify the effect of species differences in the metabolic profile on micronucleus induction, we conducted an in vitro micronucleus test for seven clastogens (benzo[a]pyrene: BaP, cyclophosphamide monohydrate: CPA coumarin, diclofenac, piroxicam, lansoprazole, and chlorpheniramine) with rat, mouse, monkey, dog, or human liver S9. BaP, CPA, coumarin, diclofenac, piroxicam, and lansoprazole induced micronucleus formation with all species of S9s, whereas chlorpheniramine did not induce micronucleus formation in any of the S9s. BaP and CPA revealed remarkable species differences in micronucleus induction, whereas coumarin, diclofenac, piroxicam, and lansoprazole did not present any differences. Interestingly, the amounts of hydroxy-BaP-epoxides and phosphamide mustard, which might be associated with micronucleus induction by BaP and CPA, respectively, were correlated with the degree of micronucleus induction among the five species. In conclusion, the species difference in micronucleus induction by BaP and CPA was attributable to the differences in the metabolic profiles of these drugs among species. Our results indicate that it is crucial to understand the effect of species differences in the metabolic profile of drug candidates on genotoxicity and carcinogenicity potential and to predict their risk in human.
Asunto(s)
Hígado/metabolismo , Metaboloma , Mutágenos/toxicidad , Animales , Perros , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones Endogámicos C3H , Pruebas de Micronúcleos , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
A high incidence of positive results is obtained with in vitro genotoxicity tests, which do not correlate with the in vivo negative results in many cases. To address this issue, the metabolic profile of rat liver 9000 × g supernatant fraction (S9) pretreated with phenobarbital (PB) and 5,6-benzoflavone (BNF) was characterized. Furthermore, the in vitro micronucleus tests of 10 compounds were performed with PB-BNF-induced rat S9. PB-BNF increased cytochrome P450 (CYP) activity and CYP1A1, CYP1A2, CYP2B1/2, CYP2C6, CYP3A1, and CYP3A2 expression in rat S9, whereas it decreased CYP2C11 and CYP2E1 expression. PB-BNF-induced S9 enhanced the micronucleus induction (MI) of benzo[a]pyrene (BaP), cyclophosphamide (CPA), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), which are metabolized by CYP1A1, CYP2C6, and CYP1A2, respectively. In contrast, coumarin and chlorpheniramine showed MI with PB-BNF-induced S9 despite the fact that they show negative results in the in vivo studies. Furthermore, diclofenac, piroxicam, lansoprazole, and caffeine showed MI regardless of the enzyme induction by PB-BNF, whereas phenacetin did not show MI. These results indicate that PB-BNF-induced rat S9 is effective in detecting the genotoxic potential of promutagens, such as BaP, CPA, and PhIP, but not of coumarin and chlorpheniramine, probably due to the differences in the in vitro and in vivo metabolic profile and its exposure levels of the drugs.
Asunto(s)
Hígado/metabolismo , Pruebas de Micronúcleos , Mutágenos/toxicidad , Animales , Línea Celular , Cricetulus , Sistema Enzimático del Citocromo P-450/metabolismo , Masculino , Fenobarbital/farmacología , Ratas Sprague-Dawley , beta-naftoflavona/farmacologíaRESUMEN
We investigated the relationship between metabolic activities of cytochrome P450 (CYP) isozymes present in microsomal fractions derived from the livers of 78 donors and micronucleus induction by cyclophosphamide (CPA). Consequently, a wide inter-individual variation in CYP activities was observed among the 78 donors. The CYP activities were partially correlated with the metabolic phenotypes predicted for the donors based on their single nucleotide polymorphisms. In addition, CPA induced micronucleus formation was seen for 47 out of 52 donors whose samples were tested with CPA doses ranging from 18.8 to 100 µg/mL. The CPA dose at which micronucleated cells were observed varied among the donors. Furthermore, a close correlation was identified between the catalytic activities of the CYP2B6, CYP2C9, CYP2C19, and CYP3A4 isozymes and micronucleus induction by CPA. To elucidate the mechanism underlying CPA-induced micronucleus formation in vitro tests were conducted on expression systems of CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Additionally, the metabolites of CPA generated by the expression systems were quantified by a liquid chromatography tandem mass spectrometer. Interestingly, several metabolites including the 4-hydroxyl form of CPA (4-OH-CPA) and phosphamide mustard were detected in the CYP2B6, CYP2C19, and CYP3A4 expression systems, but not in the CYP2C9 and CYP2D6 system. The presence of these metabolites was correlated with micronucleus induction by CPA. The absence of CPA metabolites in the CYP2C9 expression system might be associated with the lower 4-hydroxylase activity of this system. The present results suggest that inter-individual variability in the metabolic capacity of each donor was associated with potential micronucleus induction due to CPA. Additionally, CPA metabolites like 4-OH-CPA and phosphamide mustard produced by human CYP2B6, CYP2C9, CYP2C19, and CYP3A4 are suggested to be major determinants of micronucleus induction by CPA.
Asunto(s)
Ciclofosfamida/farmacología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Mutágenos/farmacología , Cromatografía Liquida , Humanos , Hígado/metabolismo , Pruebas de Micronúcleos , Polimorfismo de Nucleótido Simple/genética , Espectrometría de Masas en TándemRESUMEN
Previously, we have demonstrated the potential of plasma 2-hydroxyglutarate (2HG) as an easily detectable biomarker for skeletal muscle injury in rats. Here, we examined whether plasma 2HG was superior to conventional skeletal muscle damage biomarkers, including aspartate aminotransferase (AST), creatine kinase (CK), and skeletal muscle-type CK isoenzyme (CK-MM) levels, in rats. Skeletal muscle injury was induced in 4- or 9-week-old male Fischer 344 rats by cerivastatin (CER) or tetramethyl-p-phenylenediamine (TMPD) administration. Plasma 2HG levels were measured on days 4, 8, and 11 (CER group) and at 6 and 24 hr post-administration (TMPD group). Plasma AST, CK, and CK-MM activities and histopathological changes in the rectus femoris muscle were evaluated at the study endpoints. In the CER group, AST, CK, and CK-MM increased in 4- and 9-week-old rats, whereas increases in CK (4- and 9-week-old rats) and CK-MM (4-week-old rats) were not obvious in the TMPD group. In both 4- and 9-week-old rats, plasma 2HG increased on day 8 and at 24 hr post-administration in the CER and TMPD groups, respectively. Histopathological analysis revealed myofiber vacuolation and necrosis in both groups. The histopathological damage to the rectus femoris muscle was more severe in the CER than in the TMPD group. Increased plasma 2HG was associated with CER- and TMPD-induced skeletal muscle injuries in rats and was not affected by age differences or repeated blood collection. The results suggest that plasma 2HG is superior to CK and CK-MM as a biomarker for mild skeletal muscle injury.
Asunto(s)
Compuestos de Anilina/toxicidad , Glutaratos/sangre , Músculo Esquelético/lesiones , Piridinas/toxicidad , Músculo Cuádriceps/lesiones , Animales , Biomarcadores/sangre , Modelos Animales de Enfermedad , Masculino , Músculo Esquelético/patología , Miofibrillas/patología , Necrosis , Músculo Cuádriceps/patología , Ratas Endogámicas F344 , Factores de Tiempo , Vacuolas/patologíaRESUMEN
To identify new candidate biomarkers for skeletal muscle toxicity, an unbiased metabolomic analysis was performed in rats treated with two distinct myotoxicants, cerivastatin (CER) and tetramethyl-p-phenylenediamine (TMPD). Skeletal muscle toxicity was induced in male Fischer 344 rats by administering CER or TMPD and monitored using established endpoints, such as increased plasma creatine kinase (CK) activity and histopathology, and a metabolomic analysis of skeletal muscle and plasma samples. Plasma CK levels in CER-treated rats were markedly elevated at Day 11; however, those in TMPD-treated rats showed a statistically significant decrease at 24 hr after dosing. Light microscopy revealed that vacuolated or necrotic fibers were evident in all CER-treated rats on Day 11, and slightly vacuolated fibers were observed in TMPD-treated rats at 6 and 24 hr after dosing. Metabolomic analysis of the rectus femoris indicated increases in 2-hydroxyglutarate (2HG) in CER-treated rats and hexanoylcarnitine in CER- and TMPD-treated rats. There were also increases in plasma 2HG in CER-treated rats on Days 8 and 11 and in TMPD-treated rats at 24 hr after dosing and increases in plasma hexanoylcarnitine in CER-treated rats on Day 11 and in TMPD-treated rats at 6 and 24 hr after dosing. These experiments demonstrated the potential of plasma 2HG and hexanoylcarnitine as specific and easily detectable biomarkers for skeletal muscle toxicity in rats and demonstrated the value of metabolomics for biomarker detection and identification in toxicological studies.
Asunto(s)
Compuestos de Anilina/toxicidad , Carnitina/análogos & derivados , Glutaratos/sangre , Músculo Esquelético/metabolismo , Enfermedades Musculares/inducido químicamente , Enfermedades Musculares/diagnóstico , Piridinas/toxicidad , Compuestos de Anilina/administración & dosificación , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Carnitina/sangre , Carnitina/metabolismo , Creatina Quinasa/sangre , Modelos Animales de Enfermedad , Glutaratos/metabolismo , Masculino , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Enfermedades Musculares/patología , Piridinas/administración & dosificación , Ratas Endogámicas F344RESUMEN
The widely used analgesic-antipyretic drug acetaminophen (APAP) is known to cause serious liver necrosis at high doses in man and experimental animals. For studies of toxic processes, 1H NMR spectroscopy of biofluids allows monitoring of endogenous metabolite profiles that alter characteristically in response to changes in physiological status. Herein, a 1H NMR metabolomics approach was applied to the investigation of APAP toxicity in rats and the effect of phenobarbital (PB) on APAP-induced hepatotoxicity. Metabolite differences due to hepatotoxicity were observed in 1H NMR spectra of serum and urine, and enhanced APAP hepatotoxicity by pretreatment with PB was clearly shown by a principal components analysis of the spectral data. NMR spectra of APAP-dosed rat urine provided profiles of APAP-related compounds together with endogenous metabolites. By comparison of endogenous and APAP-related metabolite spectra with those from rats pretreated with PB, it was possible to show the importance of oxidative metabolism of APAP to N-acetyl-p-benzoquinone, an essential step in APAP hepatotoxicity.
Asunto(s)
Acetaminofén/metabolismo , Acetaminofén/toxicidad , Analgésicos no Narcóticos/metabolismo , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Acetaminofén/sangre , Acetaminofén/orina , Analgésicos no Narcóticos/sangre , Analgésicos no Narcóticos/orina , Animales , Benzoquinonas/sangre , Benzoquinonas/metabolismo , Benzoquinonas/toxicidad , Benzoquinonas/orina , Inducción Enzimática , Glutatión/sangre , Glutatión/metabolismo , Glutatión/toxicidad , Glutatión/orina , Iminas/sangre , Iminas/metabolismo , Iminas/toxicidad , Iminas/orina , Hígado/efectos de los fármacos , Hígado/metabolismo , Hepatopatías/metabolismo , Imagen por Resonancia Magnética , Masculino , Metabolómica , Oxidación-Reducción , Estrés Oxidativo , Fenobarbital/metabolismo , Fenobarbital/farmacología , Ratas , Ratas Endogámicas F344RESUMEN
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.
Asunto(s)
Perfilación de la Expresión Génica , Glutatión , Hígado/efectos de los fármacos , Maleatos/toxicidad , Proteoma/metabolismo , Transcriptoma/efectos de los fármacos , Animales , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Glutatión/genética , Glutatión/metabolismo , Hígado/metabolismo , Pruebas de Función Hepática , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteómica/métodos , Ratas , Ratas Endogámicas F344 , Toxicogenética/métodos , Regulación hacia ArribaRESUMEN
To determine the optimum timing of partial hepatectomy (PH) in a previously developed mouse liver micronucleus test (Igarashi and Shimada, 1997), the relation between DNA damage and micronucleus was examined using the in vivo alkaline comet assay and the micronucleus test on the liver of the same individual mouse. Five genotoxic carcinogens, 1-nitropyrene (1-NP) (125 mg/kg), cyclophosphamide (CP) (50 mg/kg), methylmethan sulfonate (MMS) (80 mg/kg), mitomycin C (MMC) (2 mg/kg) and diethylnitrosamine (DEN) (50 mg/kg) were intraperitoneally dosed to each group consisting of 4 male ddY mice. The mice were subjected to PH 3, 8 or 24 hr after dosing of each carcinogen, and comet assay was performed using the removed liver. The regenerated hepatocyte was sampled five days after PH, and the incidence of micronucleus was measured. CP, MMS, MMC and DEN induced DNA damage at 8 and 24 hr after dosing, while 1-NP induced DNA damage only 8 hr after dosing. All five carcinogens induced micronuclei whenever PH was performed. In the case of CP, the peak of DNA damage was 24 hr after dosing and the timing of PH did not remarkably affect the incidence of micronuclei. The other 4 carcinogens showed peak DNA damage at 8 hr and the highest incidence of micronuclei when PH was operated 24 hr after dosing. In conclusion, we are the first to show the relation of induction between DNA damage and micronucleus in the liver from the same mouse, and tentatively showed the optimal timing of PH as 24 hr after dosing.
Asunto(s)
Carcinógenos/toxicidad , Ensayo Cometa/métodos , Daño del ADN , Hígado , Micronúcleos con Defecto Cromosómico/inducido químicamente , Pruebas de Micronúcleos/métodos , Animales , Hepatectomía , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/cirugía , Masculino , Ratones , Ratones Endogámicos , Factores de TiempoRESUMEN
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.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Toxicogenética/métodos , Biomarcadores/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , TranscriptomaRESUMEN
Glutathione plays an important role as not only a scavenger of reactive oxygen species but also in the conjugation or detoxification of electrophilic reactive metabolites, which has been thought to be one of the causes for idiosyncratic drug toxicity (IDT). Therefore, toxic responses to the reactive metabolites have been expected to be expressed more strongly in a glutathione-depleted condition. In the present study, we attempted to establish an in vitro cytotoxicity assay method to evaluate the toxicity of the reactive metabolite using rat primary cultured hepatocytes with cellular glutathione depletion by l-buthionine-S,R-sulfoximine. Also, we investigated whether the IDT risk is predictable by comparing the cytotoxic sensitivity between glutathione-depleted hepatocytes and untreated hepatocytes. Consequently, 10 drugs of 42 approved drugs, which were classified into 4 IDT categories (Withdrawn, Black box warning, Warning, and Safe), demonstrated higher cytotoxic sensitivity in the glutathione-depleted hepatocytes. Furthermore, a correlation was observed between the incidence of drugs with higher cytotoxic sensitivity in the glutathione-depleted hepatocytes and the IDT risk. The incidence was 50% in the Withdrawn category, 38% in the Black box warning category, 22% in the Warning category, and 8% in the Safe category. These results suggest that the IDT risk of some drugs may be predicted by comparing the cytotoxic sensitivity between them. Additionally, this method may be useful as a screening in the early stage of drug development where leads/candidates are optimized.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Glutatión/deficiencia , Hepatocitos/efectos de los fármacos , Pruebas de Toxicidad/métodos , Acetaminofén/metabolismo , Acetaminofén/toxicidad , Animales , Butionina Sulfoximina/farmacología , Células Cultivadas , Aprobación de Drogas , Hepatocitos/metabolismo , Ratas , RiesgoRESUMEN
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.
Asunto(s)
Flavoproteínas Transportadoras de Electrones/metabolismo , Inhibidores Enzimáticos/toxicidad , Glicoles de Etileno/toxicidad , Testículo/efectos de los fármacos , Animales , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/metabolismo , Epidídimo/efectos de los fármacos , Epidídimo/patología , Glicoles de Etileno/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Masculino , Metabolómica , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Recuento de Espermatozoides , Espermatozoides/efectos de los fármacos , Espermatozoides/patología , Testículo/metabolismo , Testículo/patologíaRESUMEN
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.
Asunto(s)
Biomarcadores , Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Hígado/metabolismo , Toxicogenética/métodos , Algoritmos , Animales , Bromobencenos/toxicidad , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/fisiología , Hígado/efectos de los fármacos , Masculino , Redes y Vías Metabólicas/fisiología , Modelos Estadísticos , Fenotipo , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Biología de Sistemas/métodosRESUMEN
In order to characterize the hepatic effects of phenobarbital (PB) and clofibrate (CPIB) in dogs, PB and CPIB were administered to male beagle dogs for 14 days, and biochemical and histopathological examinations and comprehensive genomic and proteomic analyses, including GeneChip analysis and proteomics analysis using the 2-dimension difference gel electrophoresis (2D-DIGE) technique, were performed. Both compounds caused centrilobular hepatocellular hypertrophy, which were related to smooth endoplasmic reticulum (SER) proliferation in PB-treated dogs and to mitochondrial proliferation in CPIB-treated dogs. In the PB-treated dogs, drug-metabolizing enzyme induction was observed by Western blot and genomic analyses. CYP proteins could not be detected by the 2D-DIGE analysis, but increases in several endoplasmic reticulum (ER)-related proteins were observed. In the CPIB-treated dogs, drug-metabolizing enzyme induction was not clearly observed by any of Western blot, genomic and proteomic analyses. Genomic and proteomic analyses revealed that mitochondrial genes and proteins, including carnitine palmytoiltransferase II, acyl-CoA deheydrogenase and hydroxyacyl-CoA dehydrogenase, pyruvate carboxylase and ATP synthase beta chain were induced. There is a relatively good correlation among the morphology and the genomic and proteomic data, but some differences exist between the genomic and proteomic data. Comprehensive evaluation using these techniques in addition to morphological evaluation may provide a useful tool for safety assessment of the liver.
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Clofibrato , Hepatomegalia/inducido químicamente , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenobarbital , Proteómica , Acil-CoA Deshidrogenasa/metabolismo , Animales , Carnitina O-Palmitoiltransferasa/metabolismo , Perros , Electroforesis en Gel Bidimensional , Retículo Endoplásmico Liso , Hígado/citología , Hígado/ultraestructura , Masculino , Microscopía Electrónica de Transmisión , Mitocondrias Hepáticas/genéticaRESUMEN
As information regarding microarray data sets and toxicogenomic biomarkers grows rapidly, the process of analyzing data and interpreting the results is increasingly complicated. To facilitate data analysis, a simple expression ratio-based scoring method called the TGP1 score was previously proposed [Kiyosawa, N., Shiwaku, K., Hirode, M., Omura, K., Uehara, T., Shimizu, T., Mizukawa, Y., Miyagishima, T., Ono, A., Nagao, T., Urushidani, T., 2006. 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. J. Toxicol. Sci. 31, 433-448]. Although the TGP1 score has demonstrated its efficacy for rapid comprehension of large-scale toxicogenomic data sets, inclusion of low quality gene expression data in the biomarker gene set produced flaws in the calculated score. To overcome this shortcoming, we tested a new scoring method called the differentially expressed gene score (D-score), where Detection Call as well as signal log ratios generated by MAS5 algorithm on Affymetrix GeneChip data were considered for the calculation. Four prototypical toxicants, namely acetaminophen, phenobarbital, clofibrate and acetamidofluorene, were used for detailed analysis. A toxicogenomics database (TG-GATEs) was utilized as a reference data set. The D-score successfully alleviated the effects of low quality data on the score calculation, and captured the overall direction of expression changes as well as the magnitude of expression change level of a set of genes, highlighting the affected toxicological endpoints elicited by chemical treatment. The D-score will be useful for high-throughput toxicity screening using a toxicogenomic database and biomarkers.
Asunto(s)
Bases de Datos Genéticas , Determinación de Punto Final , Expresión Génica/efectos de los fármacos , Pruebas de Toxicidad/métodos , Toxicogenética/métodos , 2-Acetilaminofluoreno/toxicidad , Acetaminofén/toxicidad , Algoritmos , Animales , Biomarcadores/análisis , Clofibrato/toxicidad , Interpretación Estadística de Datos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenobarbital/toxicidad , Ratas , Ratas Sprague-Dawley , Pruebas de Toxicidad/estadística & datos numéricos , Toxicogenética/estadística & datos numéricosRESUMEN
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.
Asunto(s)
Acetaminofén/farmacología , Cromanos/farmacología , Hígado/efectos de los fármacos , Hígado/lesiones , Superóxido Dismutasa/genética , Tiazolidinedionas/farmacología , Adenosina Trifosfato/metabolismo , Alanina Transaminasa/sangre , Fosfatasa Alcalina/sangre , Animales , Aspartato Aminotransferasas/sangre , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Técnicas de Inactivación de Genes/métodos , Hepatocitos/citología , Hepatocitos/metabolismo , Heterocigoto , L-Lactato Deshidrogenasa/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/metabolismo , Modelos Animales , Necrosis/patología , Sensibilidad y Especificidad , Factores de Tiempo , TroglitazonaRESUMEN
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.
RESUMEN
DNA is damaged by reactive oxygen species (ROS) and such damage is age-dependent. Blood chemical parameters also change age-dependently. Glutathione (GSH) plays an important role as an antioxidant. However, the effects of GSH on DNA damage and blood chemistry are unclear. Therefore, this study was aimed to evaluate GSH contribution to DNA damage and changes of blood chemical parameters in aged and young rats. The GSH content in the livers and kidneys of aged rats (20 months) were lower than that in young rats (9 weeks of age) with higher DNA damage detected by a comet assay. There was a negative correlation between the GSH content and the DNA damage in the liver and kidney. L-buthionine (S,R)-sulfoximine (BSO; 0, 5, 20 mM), which inhibits GSH synthesis, was administered in drinking water for 28 days to young and aged rats (8 weeks and 19 months of age at the start of the administration). The treatment significantly decreased GSH levels in the heart, liver, lung and kidney of either the young or aged rats without causing DNA damage in those organs. When compared with young rats, aged rats showed higher levels in aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, globulin, creatinine, sodium and chloride and lower levels in alkaline phosphatase, triglyceride, albumin/globulin and inorganic phosphorus. However, BSO did not change these parameters in young or aged rats. These results showed that there was a negative correlation between GSH and DNA damage during aging, but the BSO-induced GSH depletion did not affect DNA damage or blood chemistry levels in young and aged rats under these study conditions.
Asunto(s)
Envejecimiento/metabolismo , Daño del ADN , Glutatión/metabolismo , Envejecimiento/sangre , Animales , Análisis Químico de la Sangre , Butionina Sulfoximina/toxicidad , Ensayo Cometa , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratas , Ratas Endogámicas F344RESUMEN
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.
Asunto(s)
Hidroxianisol Butilado/toxicidad , Proliferación Celular/efectos de los fármacos , Etilenos/toxicidad , Glutatión Transferasa/biosíntesis , Hepatocitos/citología , Hígado/efectos de los fármacos , Fenobarbital/farmacología , Piridinas/toxicidad , Animales , Bromodesoxiuridina/metabolismo , Inducción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Hepatocitos/enzimología , Isoenzimas , Hígado/enzimología , Hígado/patología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas F344RESUMEN
Glutathione S-transferase (GST) theta 1 (GSTT1) has been regarded as one of the key enzymes involved in phase II reactions because of its unique substrate specificity. In this study, we generated mice with the disrupted Gstt1 gene (Gstt1-null mice) by gene targeting and analyzed the metabolic properties in cytosolic and in vivo studies. The resulting Gstt1-null mice failed to express the Gstt1 mRNA and GSTT1 protein by reverse transcriptase-polymerase chain reaction analysis and two-dimensional fluorescence difference gel electrophoresis/mass spectrometry analysis, respectively. However, the Gstt1-null mice appeared to be normal and were fertile. In an enzymatic study using cytosolic samples from the liver and kidney, GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), dichloromethane (DCM), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was markedly lower in Gstt1-null mice than in the wild-type controls, despite there being no difference in GST activity toward 1-choloro-2,4-dinitrobenzene between Gstt1-null mice and the wild-type controls. Gstt1-null mice had GST activity of only 8.7 to 42.1% of the wild-type controls to EPNP, less than 2.2% of the wild-type controls to DCM, and 13.2 to 23.9% of the wild-type controls to BCNU. Plasma BCNU concentrations after a single i.p. administration of BCNU to Gstt1-null mice were significantly higher, and there was a larger area under the curve(5-60) min (male, 2.30 times; female, 2.28 times, versus the wild-type controls) based on the results. In conclusion, Gstt1-null mice would be useful as an animal model of humans with the GSTT1-null genotype.