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1.
Drug Metab Dispos ; 42(6): 1039-43, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24711249

RESUMO

Chimeric mice with humanized liver (PXB mice) have been generated by transplantation of urokinase-type plasminogen activator/severe combined immunodeficiency mice with human hepatocytes. The purpose of the present study was to clarify the protein expression levels of metabolizing enzymes and transporters in humanized liver of PXB mice transplanted with hepatocytes from three different donors, and to compare their protein expressions with those of human livers to validate this human liver model. The protein expression levels of metabolizing enzymes and transporters were quantified in microsomal fraction and plasma membrane fraction, respectively, by means of liquid chromatography-tandem mass spectrometry. Protein expression levels of 12 human P450 enzymes, two human UDP-glucuronosyltransferases, eight human ATP binding cassette (ABC) transporters, and eight human solute carrier transporters were determined. The variances of protein expression levels among samples from mice humanized with hepatocytes from all donors were significantly greater than those from samples obtained from mice derived from each individual donor. Compared with the protein expression levels in human livers, all of the quantified metabolizing enzymes and transporters were within a range of 4-fold difference, except for CYP2A6, CYP4A11, bile salt export pump (BSEP), and multidrug resistance protein 3 (MDR3), which showed 4- to 5-fold differences between PXB mouse and human livers. The present study indicates that humanized liver of PXB mice is a useful model of human liver from the viewpoint of protein expression of metabolizing enzymes and transporters, but the results are influenced by the characteristics of the human hepatocyte donor.


Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Glucuronosiltransferase/biossíntese , Fígado/metabolismo , Espectrometria de Massas em Tandem , Transportadores de Cassetes de Ligação de ATP/análise , Animais , Criança , Pré-Escolar , Quimera , Cromatografia Líquida/métodos , Sistema Enzimático do Citocromo P-450/análise , Feminino , Glucuronosiltransferase/análise , Hepatócitos/química , Hepatócitos/metabolismo , Humanos , Fígado/química , Masculino , Camundongos , Camundongos SCID , Espectrometria de Massas em Tandem/métodos
2.
Drug Metab Dispos ; 40(1): 93-103, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21979928

RESUMO

The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 µM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/química , Hepatócitos/química , Proteínas de Membrana Transportadoras/química , Animais , Técnicas de Cultura de Células , Células Cultivadas , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Dexametasona/química , Dexametasona/metabolismo , Glucuronosiltransferase/genética , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/biossíntese , Ratos , Espectrometria de Massas em Tandem/métodos
3.
Drug Metab Dispos ; 40(1): 83-92, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21994437

RESUMO

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Microssomos Hepáticos/metabolismo , RNA Mensageiro/biossíntese , Adulto , Fatores Etários , Idoso , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Ativação Enzimática/genética , Feminino , Glucuronosiltransferase/química , Glucuronosiltransferase/genética , Humanos , Masculino , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Adulto Jovem
4.
Chem Res Toxicol ; 23(1): 152-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19928966

RESUMO

Drug development of a potential analgesic agent 5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine was withdrawn because of its limited hepatotoxic effects in humans that could not be predicted from regulatory animal or in vitro studies. In vivo formation of glutathione conjugates and covalent binding of a model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine were investigated in the present study after intravenous administration to chimeric mice with a human or rat liver because of an interesting capability of human cytochrome P450 1A2 in forming a covalently bound metabolite in vitro. Rapid distribution and elimination of radiolabeled 5-n-butyl-pyrazolo[1,5-a]pyrimidine in plasma or liver fractions were seen in chimeric mice after intravenous administration. However, similar covalent binding in liver was detected over 0.17-24 h after intravenous administration. Radio-LC analyses revealed that the chimeric mice with humanized liver preferentially gave the 3-hydroxylated metabolite and its glutathione conjugate in the plasma and liver. On the contrary, chimeric mice with a rat liver had some rat-specific metabolites in vivo. Analyses by electrophoresis with accelerator mass spectrometry of in vivo radiolabeled liver proteins in chimeric mice revealed that bioactivated 5-n-butyl-pyrazolo[1,5-a]pyrimidine bound nonspecifically to a variety of microsomal proteins including human P450 1A2 as well as cytosolic proteins in the livers from chimeric mice with humanized liver. These results suggest that the hepatotoxic model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine was activated by human liver microsomal P450 1A2 to reactive intermediate(s) in vivo in humanized chimeric mice and could relatively nonspecifically bind to biomolecules such as P450 1A2 and other proteins.


Assuntos
Analgésicos/metabolismo , Fígado/metabolismo , Pirazóis/metabolismo , Pirimidinas/metabolismo , Analgésicos/química , Animais , Quimera , Citocromo P-450 CYP1A2/metabolismo , Eletroforese em Gel Bidimensional/métodos , Glutationa/metabolismo , Humanos , Masculino , Espectrometria de Massas/métodos , Camundongos , Microssomos Hepáticos/metabolismo , Ligação Proteica , Pirazóis/química , Pirimidinas/química
5.
Drug Metab Dispos ; 36(12): 2429-33, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18784266

RESUMO

Chimeric mice having humanized livers were constructed by transplantation of human hepatocytes. In this study, we investigated whether these mice have a capacity for drug metabolism similar to that of humans by examining hydroxylation of S-warfarin, which is predominantly metabolized to S-7-hydroxywarfarin, catalyzed by CYP2C9, in humans but not mice. The 7-hydroxylating activity of chimeric mouse liver microsomes toward S-warfarin was approximately 10-fold higher than that of control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice. The 7-hydroxylase activity of chimeric mouse liver microsomes was markedly inhibited by sulfaphenazole, as was that of human liver microsomes, whereas the activity of control mice was unaffected. The CYP2C isoform in chimeric mouse liver was also confirmed to be the human isoform, CYP2C9, by immunoblot analysis. In the present in vivo study, the level of S-7-hydroxywarfarin in plasma of chimeric mice was approximately 7-fold higher than that in control mice, in agreement with the in vitro data. Thus, the CYP2C isoform in chimeric mice functions in vivo and in vitro as a human isoform, CYP2C9. These results suggest that chimeric mice with humanized liver could be useful for predicting drug metabolism in humans, at least regarding CYP2C9-dependent metabolism.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Fígado/metabolismo , Quimeras de Transplante/metabolismo , Varfarina/análogos & derivados , Varfarina/metabolismo , Adolescente , Animais , Área Sob a Curva , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Criança , Pré-Escolar , Citocromo P-450 CYP2C9 , Hepatócitos/transplante , Humanos , Masculino , Camundongos , Camundongos SCID , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Sulfafenazol/farmacologia , Quimeras de Transplante/sangue , Varfarina/sangue , Varfarina/farmacocinética
6.
Drug Metab Dispos ; 36(7): 1202-5, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18332084

RESUMO

Aldehyde oxidase-mediated oxidation of N(1)-methylnicotinamide to N(1)-methyl-2-pyridine-5-carboxamide (2-PY) and N(1)-methyl-4-pyridone-5-carboxamide (4-PY) in chimeric mice constructed by transplanting human hepatocytes into urokinase-type plasminogen activator-transgenic severe combined immunodeficient mice was examined in vivo and in vitro. The activity in liver cytosol of chimeric mice with a high replacement index was approximately 4-fold higher than that in control mice. Furthermore, the oxidation products in control mice were 2-PY and 4-PY, whereas, in chimeric mice, the major product was 2-PY, as in humans. The aldehyde oxidase in chimeric mouse liver was confirmed to be of human type by immunoblotting analysis. The ratio of pyridones (2-PY/4-PY) excreted in the urine of chimeric mice was closer to that of humans than to that of control mice. Thus, the aldehyde oxidase in chimeric mice has human-type functional characteristics.


Assuntos
Aldeído Oxidase/metabolismo , Quimera , Fígado/metabolismo , Niacinamida/análogos & derivados , Animais , Catálise , Cromatografia Líquida de Alta Pressão , Humanos , Fígado/enzimologia , Camundongos , Camundongos SCID , Niacinamida/metabolismo
7.
J Biomater Appl ; 32(2): 150-161, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28610487

RESUMO

The purpose of this study was to evaluate various types of meniscus scaffolds that mimic the meniscus structure, and to establish a novel cell-free meniscus scaffold with polyglycolic acid or poly-l-lactic acid. Four types of scaffolds were implanted into Japanese white rabbits: poly-l-lactic acid sponge poly-l-lactic acid, PGA-coated PLLA sponge, PGA lamination, and film-coated PGA lamination. Samples were harvested at 8 and 12 weeks after implantation, and a compression stress test was performed. The meniscus size and Ishida scores were evaluated for regenerated tissue. Immunohistochemistry was analyzed by anti-type I, II and X collagen antibodies to investigate the structure of the regenerated tissue, and by anti-iNOS antibody to investigate the inflammatory tissue of the meniscus. The cell nuclei of lymphocytes and foreign body multinucleated giant cells were counted in hematoxylin and eosin staining. Modified Mankin scores for cartilage degeneration were used for assessment after Safranin-O/Fast Green staining. The biomechanical test showed that l- and film-coated PGA lamination exhibited greater strength than s- and PGA-coated PLLA sponge. At 12 weeks, the size of meniscus and the Ishida score in implanted film-coated PGA lamination were improved significantly compared with the defect groups. The type II collagen staining intensity in the PGA lamination lamination is significantly higher than the defect at eight weeks. The staining intensity of iNOS and number of lymphocytes significantly increased in sponge poly-l-lactic acid at eight weeks, and increased in p-PLLA at 12 weeks. Foreign body multinucleated giant cells in implantation groups appeared, especially at eight weeks. The Mankin score for film-coated PGA lamination was significantly lower than for the defect at 12 weeks. Novel meniscal scaffolds especially PGA should possess not only biological but also biomechanical functions. In conclusions, film-coated PGA lamination was the beneficial property for meniscus scaffold from the points of better biomechanical function, good regeneration, and less inflammation with chondroprotective effects.


Assuntos
Materiais Biocompatíveis/química , Menisco/lesões , Menisco/cirurgia , Poliésteres/química , Ácido Poliglicólico/química , Alicerces Teciduais/química , Animais , Fenômenos Biomecânicos , Força Compressiva , Menisco/patologia , Menisco/fisiologia , Coelhos , Regeneração
8.
Cancer Res ; 63(3): 674-81, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12566313

RESUMO

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate a keratinocyte-specific null mutation of Pten in mice (k5Pten(flox/flox) mice). k5Pten(flox/flox) mice exhibit wrinkled skin because of epidermal hyperplasia and hyperkeratosis and ruffled, shaggy, and curly hair. Histological examination revealed that skin morphogenesis is accelerated in k5Pten(flox/flox) mice. Within 3 weeks of birth, 90% of k5Pten(flox/flox) mice die of malnutrition possibly caused by hyperkeratosis of the esophagus. All k5Pten(flox/flox) mice develop spontaneous tumors within 8.5 months of birth, and chemical treatment accelerates the onset of tumors. k5Pten(flox/flox) keratinocytes are hyperproliferative and resistant to apoptosis and show increased activation of the Pten downstream signaling mediators Akt/protein kinase B (PKB) and extracellular signal-regulated kinase. Pten is thus an important regulator of normal development and oncogenesis in the skin.


Assuntos
Transformação Celular Neoplásica/genética , Folículo Piloso/citologia , Queratinócitos/enzimologia , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Serina-Treonina Quinases , Neoplasias Cutâneas/genética , Pele/patologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Apoptose/fisiologia , Divisão Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Ativação Enzimática , Feminino , Hiperplasia/enzimologia , Hiperplasia/genética , Queratinócitos/patologia , Queratinócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Pele/enzimologia , Anormalidades da Pele/enzimologia , Anormalidades da Pele/genética , Anormalidades da Pele/patologia , Neoplasias Cutâneas/enzimologia , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética
10.
Biochim Biophys Acta ; 1623(2-3): 82-7, 2003 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-14572905

RESUMO

Concentrations of 1,5-anhydroglucitol (1,5-AG), which is a major circulating polyol, decrease in patients with diabetes mellitus. In both insulinoma-derived RINr and MIN6 cells, 1,5-AG stimulated insulin release within the range of 0.03-0.61 mM in a dose-dependent manner. Insulin release was maximally stimulated by 1,5-AG to levels that reached 25% and 100% greater than that of control (1,5-AG-free group) in RINr and MIN6 cells, respectively. A physiological concentration of 1,5-AG stimulated insulin release after a 5-min incubation and this action was maintained for 60 min. In addition, at approximately 1/200 the concentration of glucose, 1,5-AG had additive action with 20 mM glucose. The action of 1,5-AG on insulin secretion with other types of saccharides and polyol was similarly additive. Mannnoheptulose and diazoxide suppressed the stimulative action of 1,5-AG on insulin release. The secretagogue action of 1,5-AG seemed to be independent on an increase in the intracellular content of cAMP and ATP. These results suggest that 1,5-AG can stimulate insulin secretion through a mechanism that completely differs from that of glucose.


Assuntos
Desoxiglucose/farmacologia , Insulina/metabolismo , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Carboidratos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxiglucose/fisiologia , Humanos , Secreção de Insulina , Xilitol/farmacologia
11.
J Invest Dermatol ; 119(5): 1183-8, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12445210

RESUMO

Stem cell factor can induce mast cell proliferation and melanocyte activation. Vasoactive intestinal peptide has been suggested to play a part in inflammatory dermatoses, such as atopic dermatitis. The aim of this study was to investigate the possible role of stem cell factor in atopic dermatitis by analyzing epidermal stem cell factor production induced by vasoactive intestinal peptide and cytokines. Full-length type stem cell factor transcript was detected in normal human epidermal keratinocytes, and a human epidermal keratinocyte cell line DJM-1, as well as normal human dermal fibroblasts, using reverse transcription-polymerase chain reaction. Spliced-type stem cell factor transcript was detected in both DJM-1 cells and normal human epidermal keratinocytes. Western blot analysis with stem cell factor antibody revealed a protein of the known molecular size of membrane-bound stem cell factor in the lysates of all three cell types. Stem cell factor immunoreactivity was found in the cytoplasm and the membrane of both DJM-1 cells and normal human epidermal keratinocytes using confocal laser scanning microscope. We examined the effects of vasoactive intestinal peptide and cytokines on stem cell factor production of DJM-1 cells using enzyme-linked immunosorbent assays. Stem cell factor contents significantly increased in culture supernatants of DJM-1 cells treated with 1000 nm vasoactive intestinal peptide and/or cytokines, including interleukins 4 and 13, tumor necrosis factor-alpha, and interferon-gamma. Overall, these results suggest that several inflammatory cytokines (T helper 1 and 2) and vasoactive intestinal peptide from mast cells and nerve endings are capable of inducing stem cell factor production from epidermal keratinocytes in atopic dermatitis.


Assuntos
Citocinas/farmacologia , Queratinócitos/efeitos dos fármacos , Fator de Células-Tronco/genética , Peptídeo Intestinal Vasoativo/farmacologia , Vasodilatadores/farmacologia , Processamento Alternativo , Linhagem Celular , Membrana Celular/química , Citoplasma/química , Dermatite Atópica/fisiopatologia , Ensaio de Imunoadsorção Enzimática , Células Epidérmicas , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Queratinócitos/citologia , Queratinócitos/fisiologia , Microscopia Confocal , RNA Mensageiro/análise , Fator de Células-Tronco/análise
12.
J Dermatol ; 29(5): 280-9, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12081159

RESUMO

The accumulation of mast cells around tumors is a well-recognized phenomenon in a number of malignancies, including basal cell carcinoma, melanoma, and breast cancer. However, little information exists regarding mast cells within tumor nests. To clarify the role of mast cells infiltrating in skin cancers, we examined the morphological features of mast cells within tumors of five cases of angiosarcoma, including two patients with Stewart-Treves syndrome, by electron microscopy. In light microscopy, mast cells were observed within tumor nests at various densities and exhibited weak staining intensity with toluidine blue. By electron microscopy, most of the tumor-infiltrating mast cells exhibited anaphylactic or piecemeal degranulation, indicating that the mast cells had been activated in situ. Some mast cells were noted in close apposition to tumor cells, suggesting the existence of direct cell-to-cell interactions. Tumor cells adjacent to mast cells showed no degenerative changes. In conclusion, these results suggest that careful histologic examination in combination with electron microscopy should enable us to identify more mast cells within cancer lesions with greater sensitivity than in a number of prior reports. Furthermore, the close proximity of mast cells and surrounding tumor cells suggests some biologically significant role of mast cells in the development of angiosarcoma, including tumor growth as well as host immunity and stromal reaction.


Assuntos
Hemangiossarcoma/patologia , Mastócitos/imunologia , Mastócitos/ultraestrutura , Neoplasias Cutâneas/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha , Evolução Fatal , Feminino , Hemangiossarcoma/imunologia , Hemangiossarcoma/terapia , Humanos , Masculino , Microscopia Eletrônica , Sensibilidade e Especificidade , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/terapia
13.
J Pharm Sci ; 100(9): 4037-43, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21544820

RESUMO

Mass spectrometry (MS)-based multiplexed multiple reaction monitoring quantification of proteins has recently evolved as a versatile tool for accurate, absolute quantification of proteins. The purpose of this study was to examine the validity of the present method with regard to standard bioanalytical criteria for drug transporters, cytochrome P450 (CYP) enzymes and uridine 5'-diphospho-glucuronosyltransferases (UGTs). Membrane preparations from human liver tissue were used for target protein quantification. As a result, the determination coefficients (r(2)) of all targets were greater than 0.986. In the absence of matrix, inaccuracy values (expressed as % deviation) were -8.1% to 20.3%, whereas imprecision values (expressed as % coefficient of variation) were within 15.9%. In the presence of matrix, which consisted of digested plasma membrane fraction for transporters and digested microsomal membrane fraction for CYP enzymes and UGTs, respectively, the inaccuracy was -15.3%-8.1%, and the imprecision were within 18.9%. Sufficient sample stability of membrane fraction was shown for three freeze-thaw cycles, 32 days at -20°C, and in processed samples for 7 days at 10°C. In conclusion, this study demonstrated, for the first time, that the MS-based assay with nano-liquid chromatography provides adequate reliability and robustness for the quantification of selected drug transporters, P450 enzymes and UGTs.


Assuntos
Proteínas de Transporte/metabolismo , Cromatografia Líquida/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem/métodos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Humanos , Microssomos Hepáticos/enzimologia , Nanotecnologia , Reprodutibilidade dos Testes
14.
Drug Metab Pharmacokinet ; 25(3): 262-73, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20610885

RESUMO

The formation of morphine-3-glucuronide (M-3-G, pharmacologically inactive) and morphine-6-glucuronide (M-6-G, active metabolite) by liver microsomes from humans and rodents, including chimeric mice carrying human liver, was evaluated in the presence of fatty acyl-CoAs. Medium- to long-chain fatty acyl-CoAs, including oleoyl-CoAs, at a physiologic level (around 15 microM) markedly enhanced M-3-G formation catalyzed by rat liver microsomes. A separate experiment indicated that 15 microM oleoyl-CoA enhanced (14)C-UDP-glucuronic acid (UDPGA) uptake by microsomes. The activation by acyl-CoAs disappeared or was greatly reduced by either pre-treating microsomes with detergent or freezing/thawing the rat liver before preparation. Many of the microsomes prepared from frozen human livers (N=14) resisted oleoyl-CoA-mediated activation of UDP-glucuronosyltransferase (UGT) activity, including M-6-G formation, which is highly specific to humans. In sharp contrast, the activity of M-6-G and M-3-G formation in freshly-prepared hepatic microsomes from chimeric mice with humanized liver was potently activated by oleoyl-CoA. Thus, acyl-CoAs activate morphine glucuronidation mediated by human as well as rat UGTs. This activation is assumed to be due to the acyl-CoA-facilitated transportation of UDPGA, and microsomes need to maintain the intact conditions required for the activation. The function of UGT appears to be dynamically changed depending on the cellular acyl-CoA level in many species.


Assuntos
Acil Coenzima A/farmacologia , Glucuronosiltransferase/metabolismo , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Morfina/metabolismo , Animais , Criopreservação , Feminino , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Derivados da Morfina , Ratos , Quimeras de Transplante/metabolismo
15.
J Clin Invest ; 120(1): 115-26, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19955657

RESUMO

Pancreatic beta cell failure is thought to underlie the progression from glucose intolerance to overt diabetes, and ER stress is implicated in such beta cell dysfunction. We have now shown that the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) accumulated in the islets of diabetic animal models as a result of ER stress before the onset of hyperglycemia. Transgenic overexpression of C/EBPbeta specifically in beta cells of mice reduced beta cell mass and lowered plasma insulin levels, resulting in the development of diabetes. Conversely, genetic ablation of C/EBPbeta in the beta cells of mouse models of diabetes, including Akita mice, which harbor a heterozygous mutation in Ins2 (Ins2WT/C96Y), and leptin receptor-deficient (Lepr-/-) mice, resulted in an increase in beta cell mass and ameliorated hyperglycemia. The accumulation of C/EBPbeta in pancreatic beta cells reduced the abundance of the molecular chaperone glucose-regulated protein of 78 kDa (GRP78) as a result of suppression of the transactivation activity of the transcription factor ATF6alpha, thereby increasing the vulnerability of these cells to excess ER stress. Our results thus indicate that the accumulation of C/EBPbeta in pancreatic beta cells contributes to beta cell failure in mice by enhancing susceptibility to ER stress.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/fisiologia , Células Secretoras de Insulina/metabolismo , Fator 6 Ativador da Transcrição , Animais , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Insulina/metabolismo , Secreção de Insulina , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Receptores para Leptina/fisiologia , Transativadores/fisiologia
19.
Drug Metab Pharmacokinet ; 24(2): 153-60, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19430171

RESUMO

Chimeric mice, constructed by transplanting human hepatocytes, are useful for predicting the human metabolism of drug candidates. In this study, we investigated whether these mice show similar metabolic profile to humans by examining the hydroxylation of S-warfarin reported to be mainly metabolized to S-7-hydroxywarfarin (7-OH-warfarin), catalyzed by CYP2C9, in humans. When S-(3)H-warfarin was administered to chimeric mice and control (uPA(+/+)/SCID(wt/wt)) mice, the blood concentration-time curve was higher in chimeric than control mice. Plasma protein binding of S-(3)H-warfarin of chimeric and control mice amounted to 98.1 and 92.1%, respectively. When S-(3)H-warfarin was administered to these mice, radioactivity was mainly recovered in urine (81.7% in chimeric mice and 65.9% in control mice). After S-(3)H-warfarin was administered to these mice, the radioactivity was recovered in the bile of chimeric and control mice at 5.1 and 17.9%, respectively. The main urinary metabolite in chimeric mice was 7-OH-warfarin. the main urinary metabolite in control mice was S-4'-hydroxywarfarin. These results show that mass balance, metabolic disposition of S-(3)H-warfarin in chimeric mice with humanized liver were similar to reported human data.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Quimera/metabolismo , Varfarina/análogos & derivados , Animais , Bile/metabolismo , Citocromo P-450 CYP2C9 , Humanos , Masculino , Camundongos , Camundongos SCID , Varfarina/metabolismo
20.
Biomed Res ; 30(6): 325-30, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20051640

RESUMO

We analyzed the effect of low birth weight on pancreatic beta cell mass. We used pregnant C57BL6J mice, and we reduced their food supply by 30% during the late gestational period and examined the changes in the metabolism and pancreatic beta cell mass. Pancreatic beta cell mass at birth was greatly decreased in the mice of the food restriction group (RG) as compared to the mice of the control group (CG). The body weight of RG mice exhibited a "catch-up growth" pattern and became equivalent to that of CG mice 7 days after birth, and thereafter exceeded that of CG mice; however, the pancreatic beta cell mass in RG mice remained lower than that in CG mice at the age of 4 weeks. A high-fat diet significantly increased the pancreatic beta cell mass in RG mice as compared to that in CG mice at 12 weeks of age. However, RG mice fed on high-fat diets tended to exhibit a decrease in the pancreatic beta cell mass at approximately 20 weeks of age. The plasma insulin concentrations also tended to be decreased in RG mice after 24 weeks of age as compared to those of CG mice. These results thus indicate that the growth of pancreatic beta cells is insufficient in RG mice, and pancreatic beta cell failure can easily develop as a consequence of insulin resistance.


Assuntos
Transtornos da Nutrição Fetal/metabolismo , Células Secretoras de Insulina/metabolismo , Desnutrição/metabolismo , Animais , Animais Recém-Nascidos , Restrição Calórica , Dieta , Gorduras na Dieta , Feminino , Transtornos da Nutrição Fetal/patologia , Feto/anatomia & histologia , Feto/fisiologia , Idade Gestacional , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/patologia , Desnutrição/patologia , Camundongos , Camundongos Endogâmicos C57BL , Gravidez
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