RESUMO
Friedreich ataxia (FRDA), the most common autosomal recessive neurodegenerative disease among Europeans and people of European descent, is characterized by an early onset (usually before the age of 25), progressive ataxia, sensory loss, absence of tendon reflexes and pyramidal weakness of the legs. We have recently identified a unique group of patients whose clinical presentations are characterized by autosomal recessive inheritance, early age of onset, FRDA-like clinical presentations and hypoalbuminemia. Linkage to the FRDA locus, however, was excluded. Given the similarities of the clinical presentations to those of the recently described ataxia with oculomotor apraxia (AOA) linked to chromosome 9p13, we confirmed that the disorder of our patients is also linked to the same locus. We narrowed the candidate region and have identified a new gene encoding a member of the histidine triad (HIT) superfamily as the 'causative' gene. We have called its product aprataxin; the gene symbol is APTX. Although many HIT proteins have been identified, aprataxin is the first to be linked to a distinct phenotype.
Assuntos
Apraxias/genética , Ataxia/genética , Proteínas de Ligação a DNA/genética , Mutação , Proteínas Nucleares/genética , Músculos Oculomotores/fisiopatologia , Albumina Sérica/metabolismo , Sequência de Aminoácidos , Animais , Apraxias/complicações , Ataxia/complicações , Mapeamento Cromossômico , Cromossomos Humanos Par 9 , Proteínas de Ligação a DNA/química , Feminino , Ligação Genética , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/química , Linhagem , Filogenia , Homologia de Sequência de AminoácidosRESUMO
Samples of the carbonaceous asteroid Ryugu were brought to Earth by the Hayabusa2 spacecraft. We analyzed 17 Ryugu samples measuring 1 to 8 millimeters. Carbon dioxide-bearing water inclusions are present within a pyrrhotite crystal, indicating that Ryugu's parent asteroid formed in the outer Solar System. The samples contain low abundances of materials that formed at high temperatures, such as chondrules and calcium- and aluminum-rich inclusions. The samples are rich in phyllosilicates and carbonates, which formed through aqueous alteration reactions at low temperature, high pH, and water/rock ratios of <1 (by mass). Less altered fragments contain olivine, pyroxene, amorphous silicates, calcite, and phosphide. Numerical simulations, based on the mineralogical and physical properties of the samples, indicate that Ryugu's parent body formed ~2 million years after the beginning of Solar System formation.
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BACKGROUND: The chemokine receptor CCR4 has been implicated in Th2 cell-mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation. OBJECTIVE: The role of CCR4 in Th1, Th2, and Th17 cell-mediated allergic airway inflammation was investigated. METHOD: We generated an allergic airway inflammation model by adoptive transfer of in vitro-polarized ovalbumin (OVA)-specific Th1, Th2, and Th17 cells. The effect of a low-molecular weight CCR4 antagonist, Compound 22, on this model was examined. RESULTS: Upon in vitro polarization of DO11.10 naïve T cells, Th1- and Th2-polarized cells dominantly expressed CXCR3 and CCR4, respectively, while Th17-polarized cells expressed CCR6 and CCR4. Intranasal OVA-challenge of mice transferred with each T cell subset induced accumulation of T cells in the lungs. Eosinophils were also massively accumulated in Th2-transferred mice, whereas neutrophils were preferentially recruited in Th1- and Th17-transferred mice. Compound 22, as well as anti-CCL17 or anti-CCL22 antibody selectively suppressed accumulation of Th2 cells and eosinophils in the lungs of Th2-transferred and OVA-challenged mice. Compound 22 also inhibited bronchial hyperresponsiveness but had little effect on goblet cell hyperplasia in Th2-transferred and OVA-challenged mice. CONCLUSIONS AND CLINICAL RELEVANCE: There were notable differences in allergic lung inflammation mediated by different T cell subsets. CCR4 blockage was selectively effective for suppression of Th2-mediated allergic inflammation by blocking infiltration of Th2 cells.
Assuntos
Regulação para Baixo/imunologia , Receptores CCR4/antagonistas & inibidores , Hipersensibilidade Respiratória/tratamento farmacológico , Células Th2/imunologia , Transferência Adotiva , Animais , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Células Caliciformes/imunologia , Células Caliciformes/patologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores CCR4/genética , Receptores CCR4/imunologia , Receptores CCR6/antagonistas & inibidores , Receptores CCR6/genética , Receptores CCR6/imunologia , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologia , Células Th2/patologiaRESUMO
Ovarian hyperstimulation syndrome (OHSS) commonly occurs as a complication of ovarian stimulation with gonadotrophins. Spontaneous OHSS is an extremely rare event, but can occur as a result of stimulation with pregnancy-derived hCG. We herein report a case of quadruplet pregnancy complicated by OHSS with spontaneous ovulation. The patient had previously undergone ovarian stimulation with clomiphene citrate plus FSH. After that, she conceived spontaneously and developed OHSS after three weeks of amenorrhea. The OHSS was managed by conservative treatment and improved at six weeks of gestation. However, a quadruplet pregnancy became apparent on ultrasound examination. The patient therefore elected to have an induced abortion. Besides the conception in the cycle without administration of exogenous gonadotrophins, the symptoms in this case had the same kinetics as iatrogenic OHSS caused by ovarian stimulation.
Assuntos
Síndrome de Hiperestimulação Ovariana/complicações , Síndrome de Hiperestimulação Ovariana/fisiopatologia , Ovulação , Complicações na Gravidez , Gravidez de Quadrigêmeos , Dor Abdominal , Aborto Induzido , Clomifeno/administração & dosagem , Feminino , Hormônio Foliculoestimulante/administração & dosagem , Humanos , Síndrome de Hiperestimulação Ovariana/diagnóstico , Indução da Ovulação/efeitos adversos , Indução da Ovulação/métodos , Gravidez , Complicações na Gravidez/diagnóstico por imagem , UltrassonografiaRESUMO
Mucosal tissues of mice are enriched in T cells that express the gamma/delta T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in gamma/delta T cell receptor-deficient (TCRdelta-/-) mice versus control mice of the same genetic background. The frequency of intestinal immunoglobulin (Ig) A plasma cells as well as IgA levels in serum, bile, saliva, and fecal samples were markedly reduced in TCRdelta-/- mice. The TCRdelta-/- mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toxoid plus cholera toxin as adjuvant. Conversely, the antigen-specific IgM and IgG antibody responses were comparable to orally immunized control mice. Direct assessment of the cells forming antibodies against the tetanus toxoid and cholera toxin antigens indicated that significantly lower numbers of IgA antibody-producing cells were present in the intestinal lamina propria and Peyer's patches of TCRdelta-/- mice compared with the orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of gamma/delta T cells suggests a specialized role for gamma/delta cells in mucosal immunity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação a DNA , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Proteínas/genética , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Diferenciação Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Timo/citologia , Timo/imunologiaRESUMO
The development of a safe and effective mucosal vaccine adjuvant is a crucial step for the development of vaccines against human immunodeficiency virus type-1 (HIV). We have previously reported that a mutant tumor necrosis factor-alpha (TNF-alpha), mTNF-K90R, possessed strong mucosal vaccine adjuvant activities in mice. Here, we evaluated the potential of mTNF-K90R as a mucosal vaccine adjuvant for the induction of systemic and mucosal immune responses against HIV. Nasal immunization of BALB/c mice with 5 microg of an HIV gp120 env protein immunogen together with mTNF-K90R induced higher serum anti-HIV gp120 protein immunoglobulin G (IgG) responses than gp120 alone. Furthermore, mTNF-K90R induced anti-gp120 IgA responses in nasal as well as vaginal washes from immunized mice, although these were not administration sites. Again, responses with mTNF-K90R were higher than with gp120 alone. These results indicate that mTNF-K90R may be applicable as amucosal adjuvant for HIV vaccination to induce both systemic and mucosal immune responses.
Assuntos
Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Adjuvantes Imunológicos , Imunidade nas Mucosas/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Proteína gp120 do Envelope de HIV/imunologia , Imunização , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Ovalbumina/imunologiaRESUMO
The near-Earth asteroid (162173) Ryugu is thought to be a primitive carbonaceous object that contains hydrated minerals and organic molecules. We report sample collection from Ryugu's surface by the Hayabusa2 spacecraft on 21 February 2019. Touchdown images and global observations of surface colors are used to investigate the stratigraphy of the surface around the sample location and across Ryugu. Latitudinal color variations suggest the reddening of exposed surface material by solar heating and/or space weathering. Immediately after touchdown, Hayabusa2's thrusters disturbed dark, fine grains that originate from the redder materials. The stratigraphic relationship between identified craters and the redder material indicates that surface reddening occurred over a short period of time. We suggest that Ryugu previously experienced an orbital excursion near the Sun.
RESUMO
A new type of carbonaceous chondrite, the Tagish Lake meteorite, exhibits a reflectance spectrum similar to spectra observed from the D-type asteroids, which are relatively abundant in the outer solar system beyond the main asteroid belt and have been inferred to be more primitive than any known meteorite. Until the Tagish Lake fall, these asteroids had no analog in the meteorite collections. The Tagish Lake meteorite is a carbon-rich (4 to 5 weight %), aqueously altered carbonaceous chondrite and contains high concentrations of presolar grains and carbonate minerals, which is consistent with the expectation that the D-type asteroids were originally made of primitive materials and did not experience any extensive heating.
Assuntos
Meteoroides , Planetas Menores , Canadá , Carbono/análise , Carbonatos/análise , Análise EspectralRESUMO
Reflectance spectra (0.3 to 2.6 micrometers) of 14 C, G, B, and F asteroids and 21 carbonaceous chondrite powders are compared in detail. Only three thermally metamorphosed CM-Cl chondrites that have a weak ultraviolet absorption are shown to have close counterparts among those asteroids. Reflectance spectra of heated Murchison CM2 chondrite are compared with the average C and G type asteroid spectra. Murchison heated at 600 degrees to 1000 degrees C exhibits a similar weak ultraviolet absorption and provides the best analog for those spectra. Comparison of ultraviolet absorption strengths between 160 C, G, B, and F asteroids and carbonaceous chondrites suggests that surface minerals of most of those asteroids are thermally metamorphosed at temperatures around 600 degrees to 1000 degrees C.
RESUMO
The near-Earth asteroid 162173 Ryugu, the target of the Hayabusa2 sample-return mission, is thought to be a primitive carbonaceous object. We report reflectance spectra of Ryugu's surface acquired with the Near-Infrared Spectrometer (NIRS3) on Hayabusa2, to provide direct measurements of the surface composition and geological context for the returned samples. A weak, narrow absorption feature centered at 2.72 micrometers was detected across the entire observed surface, indicating that hydroxyl (OH)-bearing minerals are ubiquitous there. The intensity of the OH feature and low albedo are similar to thermally and/or shock-metamorphosed carbonaceous chondrite meteorites. There are few variations in the OH-band position, which is consistent with Ryugu being a compositionally homogeneous rubble-pile object generated from impact fragments of an undifferentiated aqueously altered parent body.
RESUMO
The near-Earth carbonaceous asteroid 162173 Ryugu is thought to have been produced from a parent body that contained water ice and organic molecules. The Hayabusa2 spacecraft has obtained global multicolor images of Ryugu. Geomorphological features present include a circum-equatorial ridge, east-west dichotomy, high boulder abundances across the entire surface, and impact craters. Age estimates from the craters indicate a resurfacing age of [Formula: see text] years for the top 1-meter layer. Ryugu is among the darkest known bodies in the Solar System. The high abundance and spectral properties of boulders are consistent with moderately dehydrated materials, analogous to thermally metamorphosed meteorites found on Earth. The general uniformity in color across Ryugu's surface supports partial dehydration due to internal heating of the asteroid's parent body.
RESUMO
We have studied the local structure of LaO0.5F0.5BiS2-x Se x by Bi L1-edge extended x-ray absorption fine structure (EXAFS). We find a significant effect of Se substitution on the local atomic correlations with a gradual elongation of average in-plane Bi-S bondlength. The associated mean square relative displacement, measuring average local distortions in the BiS2 plane, hardly shows any change for small Se substitution, but decreases significantly for [Formula: see text]. The Se substitution appears to suppress the local distortions within the BiS2 plane that may optimize in-plane orbital hybridization and hence the superconductivity. The results suggest that the local structure of the BiS2-layer is one of the key ingredients to control the physical properties of the BiS2-based dichalcogenides.
RESUMO
The tissue distributions of four isoforms (CYP2D1/5, 2D2, 2D3 and 2D4/18) in rat CYP2D subfamily were investigated. Twelve kinds of tissue (liver, kidney, brain, lung, heart, spleen, adrenal gland, small intestine mucosa, bladder, testis, ovary and gonecystis) were removed from Sprague-Dawley male and female rats. The expression of CYP2D mRNA in these tissues was detected by RT-PCR. Specific primers were designed to recognize the four isoforms individually. In liver, kidney and small intestine mucosa, the mRNA expression of all four CYP2D isoforms was detected as high-intensity PCR products. mRNA of CYP2D1/5 was expressed in all tissues used in this study except the brain, although the intensity of PCR products varied among tissues. mRNAs of CYP2D2 and CYP2D3 were mainly expressed in liver, kidney and small intestine mucosa, which were exposed to xenobiotics such as drugs, food components and environmental contaminations. mRNA of CYP2D4/18 was expressed in liver, kidney, small intestine mucosa and brain. In brain, only mRNA of CYP2D4/18 was expressed. CYP2D4/18 mRNA was also expressed in ovary, testis and gonecystis. The tissue distributions help to clarify the differences in physiological and pharmacological functions between CYP2D isoforms.
Assuntos
Citocromo P-450 CYP2D6/genética , Isoenzimas/genética , Família Multigênica , RNA Mensageiro/metabolismo , Animais , Citocromo P-450 CYP2D6/biossíntese , Primers do DNA , Feminino , Isoenzimas/biossíntese , Masculino , Especificidade de Órgãos/genética , Reação em Cadeia da Polimerase/métodos , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
A way of fragmentation of Clostridium botulinum neurotoxin was carried out to elucidate the structure-function relationship of neurotoxin. The hitherto only plausible fragment was isolated from the trypsin-treated heavy chain of botulinum type E neurotoxin. In the presence of 4 M urea, one protein peak emerged from QAE-Sephadex column loaded with the heavy chain mildly treated with trypsin by elution with 0.1 M sodium chloride. Although many protein bands were detected in SDS-PAGE of the treated heavy chain, the eluted protein migrated in a single band to the position of 41,000 Da. The recovery of the 41,000-Da fragment was 28.6%, but with a 2 M urea-containing buffer as eluant, the recovery was less than 12%. The 41,000-Da fragment bound to gangliosides GD1a, GT1b, and GQ1b, to which neurotoxin and the heavy chain bound. The 41,000-Da fragment partially interfered with the binding of 125I-labeled neurotoxin to mouse brain synaptosomes. We have proposed a three-fragment structure (L.H-1.H-2) for botulinum type E neurotoxin. The characters of the 41,000-Da fragment described in this paper seem to substantiated our proposal that type E neurotoxin consists of three fragments, L.H-1.H-2, and that the ganglioside-binding fragment is H-2.
Assuntos
Toxinas Botulínicas/isolamento & purificação , Clostridium botulinum/metabolismo , Gangliosídeos/metabolismo , Animais , Sítios de Ligação , Toxinas Botulínicas/química , Toxinas Botulínicas/metabolismo , Camundongos , Modelos Moleculares , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Relação Estrutura-Atividade , Sinaptossomos/metabolismo , TripsinaRESUMO
The existence of cytochrome P450 2D isoforms in the brain has been demonstrated, although their physiological functions remain to be elucidated. In this study we demonstrated that recombinant rat cytochrome P450 2D1 and 2D4 and human cytochrome P450 2D6 possess progesterone 6 beta- and 16 alpha- hydroxylation activities; 2 beta- and 21-hydroxylation activities; and 2 beta-, 6 beta-, 16 alpha- and 21-hydroxylation activities, respectively. Cytochrome P450 2D4 had the lowest K(m) value and the highest maximum velocity value toward these activities. Progesterone 2 beta- and 21-hydroxylation activities were also detected in rat brain microsomes, and these activities were completely inhibited by anticytochrome P450 2D antibodies. The presence of endogenous 2 beta- and 21-hydroxyprogesterones in rat brain tissues was also demonstrated. The mRNAs of cytochrome P450 2D4, CYP11A, and 3 beta-hydroxysteroid dehydrogenase were detected in the rat brain, suggesting that progesterone was generated from cholesterol by CYP11A and 3 beta-hydroxysteroid dehydrogenase and then underwent hydroxylation to hydroxyprogesterones by cytochrome P450 2D4 in rat brain. Collectively, our findings support the idea that cytochrome P450 2D may be involved in the regulation (metabolism and/or synthesis) of endogenous neuroactive steroids, such as progesterone and its derivatives, in brain tissues.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Encéfalo/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Oxigenases de Função Mista/metabolismo , Progesterona/metabolismo , Oxirredutases do Álcool , Animais , Catálise/efeitos dos fármacos , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Família 2 do Citocromo P450 , Desoxicorticosterona/metabolismo , Enzimas/metabolismo , Humanos , Hidroxilação , Masculino , Sistema Nervoso/metabolismo , Oxirredução , Progesterona/análogos & derivados , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Esteroide 16-alfa-Hidroxilase , Esteroides/metabolismo , Esteroides/farmacologiaRESUMO
PURPOSE: Immunologic characterization of IgA-committed B-1 and B-2 cells, and unique subsets of T cells isolated from the murine lacrimal gland (LG), the primary exocrine tissue for the ocular surface, which is considered to be a part of the mucosal immune system. METHODS: Single cells were obtained from LGs of C57BL/6 mice by the enzyme dissociation method using collagenase type IV. Samples underwent flow cytometric analysis to characterize the unique subsets of T and B cells. To test the effectiveness of ocular vaccination, mice were immunized ocularly or nasally with cholera toxin (CT; 10 microg/mouse) suspended in phosphate-buffered saline. Antigen-specific immune responses were determined by isotype and CT-specific enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. RESULTS: When mononuclear cells (MC) isolated from LG samples were examined by flow cytometry, approximately 28% of cells were characterized as B220+ B cells. Because surface IgA+ (sIgA+) B cells develop from B-1 and B-2 lineages, it was important to examine which subset of B cells gives rise to LG sIgA+ B cells. Examination of the MC isolated from LG samples showed that approximately 4% of cells were sIgA+ B cells. Furthermore, nearly all these sIgA+ B cells (97.5%) belonged to the B-1 lineage, especially the B-1a cell line (B220low, CD5+). Of the isolated CD3+ T cells, 75% were alpha(beta) and 25% were gamma(delta)T-cell receptor positive. The proportion of NK1.1+ alpha(beta) T cells was higher (3%) in LG samples than in submandibular gland samples (0.5%). Ocular immunization with CT-induced antigen-specific mucosal (e.g., found in tear-wash and saliva samples) and systemic (e.g., serum) immune responses. The magnitude of antigen-specific antibody responses was comparable to those induced by nasal immunization. CONCLUSIONS: These results show that LG contains unique subsets of B (e.g., sIgA+ B-1 cells) and T (e.g., NK1.1+ alpha(beta)T cells) cells. Furthermore, as a part of the mucosal immune barrier, the LG is an important immunologic tissue for the ocular surface.
Assuntos
Antígenos/metabolismo , Subpopulações de Linfócitos B/imunologia , Imunidade nas Mucosas/imunologia , Imunoglobulina A Secretora/imunologia , Aparelho Lacrimal/imunologia , Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Células Produtoras de Anticorpos/imunologia , Antígenos Ly , Antígenos de Superfície , Linhagem da Célula , Toxina da Cólera/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Lectinas Tipo C , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Receptores de Antígenos de Linfócitos T gama-delta/imunologiaRESUMO
Significant sex differences exist among cases of bladder cancer in humans as well as in experimental animals such as rats. Aromatic amines such as benzidine and 2-naphthylamine are known to induce bladder cancer. These carcinogenic amines are activated to genotoxic substances by cytochrome P 450 CYP4B1, which is present in bladder mucosa. In this study, regulation of CYP4B1 was investigated to elucidate sex difference in bladder carcinogenesis. Competitive reverse transcription-polymerase chain reaction was used to investigate the expression of rat CYP4B1 mRNA occurring in small amounts of tissue such as bladder tissue. Expression of CYP4B1 in the bladder of male rats increased with development but not in that of female rats. Moreover, mature male rats exhibited higher expression of CYP4B1 in the bladder than did mature female rats. Castration of male rats decreased CYP4B1 levels and treatment with testosterone led to a partial recovery of CYP4B1 levels. These results indicate that CYP4B1 levels in the rat bladder are partly regulated by androgens. Furthermore, the present findings suggest that the sex difference observed in bladder carcinogenesis was due to sex-different expression of CYP4B1 in bladder tissue.
Assuntos
Androgênios/fisiologia , Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Bexiga Urinária/metabolismo , Fatores Etários , Animais , Carcinógenos/metabolismo , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/genética , Feminino , Masculino , Mucosa/enzimologia , Mucosa/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Sexuais , Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/etiologiaRESUMO
The dose dependence of the promoting effects of the alpha-isomer of benzene hexachloride (alpha-BHC) on hepatocarcinogenesis was investigated in a medium-term rat liver bioassay (Ito test). A total of 195 F344 male rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at the start of the experiment and subjected to two-thirds partial hepatectomy at week 3. Two weeks after the administration of DEN, alpha-BHC were fed to rats at doses of 0, 0.01, 0.1, 0.5, 1, 2, 4, 7.5, 15, 30, 60, 125 and 500 ppm in diet for 6 weeks. All surviving animals were killed at week 8, and their livers were examined immunohistochemically for detection of glutathione S-transferase placental form (GST-P)-positive foci, surrogate preneoplastic lesions. Quantitative values for numbers and areas were dose-dependently increased in rats given alpha-BHC at 0.5-500 ppm. However, those for groups treated with 0.01 and 0.1 ppm were decreased, albeit not significantly in comparison to the controls. Cytochrome P450 3A2 (CYP3A2) protein levels and activities showed a good correlation to the number and area of GST-P-positive foci. These results support evidence of hormesis and indicate a no-observed effect level for alpha-BHC promoting potentials may exist regarding rat liver carcinogenesis, which correlates with expression of CYP3A2 in the liver.
Assuntos
Biomarcadores Tumorais/metabolismo , Glutationa Transferase/metabolismo , Hexaclorocicloexano/toxicidade , Neoplasias Hepáticas/induzido quimicamente , Lesões Pré-Cancerosas/induzido quimicamente , Animais , Carcinógenos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Dietilnitrosamina , Relação Dose-Resposta a Droga , Interações Medicamentosas , Indução Enzimática , Isomerismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Hepáticas/enzimologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Lesões Pré-Cancerosas/enzimologia , Ratos , Ratos Endogâmicos F344 , Esteroide Hidroxilases/metabolismoRESUMO
The binding of [3H]1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl propyl) piperazine (GBR-12935), an antagonist of the dopamine transporter, to human P450s expressed in yeast cells was investigated. Among the ten forms of human P450 tested (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4), [3H]GBR-12935 bound most strongly to CYP2D6. The calculated Kd of [3H]GBR-12935 binding to CYP2D6 was 42.2 nM, indicating that GBR-12935 has a high affinity for CYP2D6. The characteristics of [3H]GBR-12935 binding to CYP2D6 were investigated by competitive studies using several chemicals. The binding of [3H]GBR-12935 to CYP2D6 was not changed by dopamine, suggesting that these binding sites are not dopamine-sensitive binding sites. The binding of [3H]GBR-12935 to CYP2D6 was decreased partially by substrates or inhibitors of CYP2D isoforms (quinine, quinidine, propranolol, bufuralol, imipramine, and desipramine). By means of binding studies using several forms of expressed human P450, we demonstrated that the CYP2D isoform is one GBR-12935 binding site that is insensitive to dopamine.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Citocromo P-450 CYP2D6/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Piperazinas/metabolismo , Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina , Expressão Gênica , Humanos , Cinética , Mitocôndrias/enzimologia , Quinidina/farmacologia , Quinina/farmacologia , Saccharomyces cerevisiae/enzimologiaRESUMO
Bufuralol hydroxylation activities of liver microsomal cytochrome P450 (P450) enzymes were studied in the rat; the reaction has been used widely in determining levels of liver microsomal P450 2D6, which shows debrisoquine-type genetic polymorphism in humans. Liver microsomes catalyzed the conversion of bufuralol to 1'-hydroxybufuralol and a structurally unidentified metabolite (termed here as M-1) in the presence of an NADPH-generating system and molecular oxygen. Bufuralol 1'-hydroxylation activities catalyzed by the liver microsomes were not increased in rats treated with several P450 inducers, whereas beta-naphthoflavone treatment (and to a lesser extent that of isosafrole) caused a significant induction of M-1 formation. The major role of P450 1A1/2 in M-1 formation was confirmed by catalytic inhibition with anti-P450 antibodies and alpha-naphthoflavone in liver microsomes of beta-naphthoflavone-treated rats, and by reconstitution experiments containing P450 1A1 and 1A2. Among nine forms of purified rat P450 enzymes studied in the reconstituted system, P450 2C11 displayed the highest activities for bufuralol 1'-hydroxylation, followed by P450 1A1 and P450 2D1. A female-specific form of P450 2C12 did not catalyze bufuralol 1'-hydroxylation. In liver microsomes of male rats, however, P450 2D1 was the dominant enzyme because only anti-P450 2D1 antibodies, and not anti-P450 2C11 and anti-P450 1A1, inhibited the bufuralol hydroxylation activities, and a specific P450 2D1 inhibitor, quinine, caused a dramatic decrease in the hydroxylation activities. The major contribution of P450 2D1 in the bufuralol 1'-hydroxylation activities was also supported by a kinetic analysis of the reconstituted system; P450 2D1 enzyme had a very low Km value (8.4 microM) as compared with those of P450 2C11 (Km = 83 microM) and P450 1A1 (Km = 230 microM). Thus, the present results suggested that different P450 enzymes are involved in the hydroxylation of bufuralol in rat liver microsomes, and the kinetic analysis, as well as immunoinhibition and chemical inhibition experiments, may be of great importance for determining the major roles of P450 enzymes in drug hydroxylation reactions.