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1.
Xenobiotica ; 45(2): 124-30, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25138712

RESUMO

1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Intestino Delgado/enzimologia , Fígado/enzimologia , Macaca fascicularis/metabolismo , Animais , Feminino , Masculino , Microssomos/enzimologia , Microssomos Hepáticos/enzimologia
2.
Drug Metab Dispos ; 42(5): 867-71, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24553381

RESUMO

Due to the importance of in vitro cytochrome P450 (P450) induction assay to assess the possible drug-drug interaction events, the recent US Food and Drug Administration draft guidance and European Medicines Agency guideline recommend to assess P450 induction using fresh or cryopreserved hepatocytes at mRNA level and/or enzyme activity level. Although cryopreserved hepatocytes are commercially available for P450 induction assays, feasibility and practicability of these hepatocytes have not been fully investigated. In this study, a total of 23 lots of human cryopreserved hepatocytes were treated with three typical inducers (omeprazole, phenobarbital, and rifampicin), and induction of CYP1A2, CYP2B6, and CYP3A4 enzyme activity was measured. In 8 of these 23 hepatocyte lots, induction of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA was also analyzed. The results revealed that CYP1A2, CYP2B6, and CYP3A4 were induced (>2.0-fold) by omeprazole, phenobarbital, and rifampicin, respectively, in all the hepatocyte lots tested at enzyme activity level (23 lots) and mRNA level (8 lots). In contrast, of the 8 hepatocyte lots treated with rifampicin, CYP2C8 and CYP2C9 mRNA were not induced in 5 and 2 hepatocyte lots, respectively, and CYP2C19 mRNA was not induced in any of the 8 hepatocyte lots tested. These results suggest that induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed, but evaluation for CYP2C mRNA induction might not be feasible, using commercially available human cryopreserved hepatocytes.


Assuntos
Criopreservação , Sistema Enzimático do Citocromo P-450/biossíntese , Hepatócitos/enzimologia , Técnicas de Cultura de Células , Células Cultivadas , Descoberta de Drogas , Interações Medicamentosas , Indução Enzimática , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Preparações Farmacêuticas/metabolismo , RNA Mensageiro/biossíntese
3.
Xenobiotica ; 44(9): 769-74, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24593267

RESUMO

The expression of small intestinal cytochromes P450 (P450s) has not been systematically measured in cynomolgus monkeys, which are widely used in preclinical drug studies to predict pharmacokinetics and toxicity in humans: therefore, P450 content of small intestine was quantified in 35 cynomolgus monkeys by immunoblotting using 11 selective antibodies. CYP2D, CYP2J2, CYP3A4 and CYP3A5 were detected in all 35 animals, while CYP1A and CYP2C9/19 were detected in 31 and 17 animals, respectively. CYP2C9 and CYP2C19 were detected with the same antibody. CYP1D, CYP2A, CYP2B6, CYP2C76 and CYP2E1 were not detected in any of the 35 animals examined. On analysis of pooled microsomes (35 animals), CYP3A (3A4+3A5) was most abundant (79% of total immunoquantified CYP1-3 proteins), followed by CYP2J2 (13%), CYP2C9/19 (4%), CYP1A (3%) and CYP2D (0.4%). On the analysis of individual microsome samples, each P450 content varied 2-to-6-fold between animals, and no sex differences were observed in any P450 content. These findings should help to increase the understanding of drug metabolism, especially the first-pass effect, in cynomolgus monkey small intestines.


Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Intestino Delgado/enzimologia , Microssomos/enzimologia , Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Animais , Citocromo P-450 CYP1A1/isolamento & purificação , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2C19/isolamento & purificação , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/isolamento & purificação , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2J2 , Citocromo P-450 CYP3A/isolamento & purificação , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Macaca fascicularis , Masculino
4.
Geriatr Gerontol Int ; 24 Suppl 1: 320-326, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38267253

RESUMO

AIM: To examine the actual conditions of older patients receiving home medical care after hospitalization over a period of 2 years in Japan. METHODS: The study population included 102 participants, aged ≥65 years, receiving home medical care, who consented to participate in the Osaka Home Care Registry (OHCARE) study in Japan over a period of 2 years. We investigated the actual conditions for returning home after hospitalization. RESULTS: The median age of the 102 participants was 84 years, and 61 (59.8%) were women. In the group that returned home, 42 (55.3%) of the respondents desired to recuperate in a familiar place, as in advanced care planning (ACP). During the 2-year follow-up period, the group that did not return home had significantly more deaths. A multivariate analysis showed the association in the presence of ACP (odds ratio: 4.72, 95% confidence interval: 1.60-13.86) and cardiac disease (odds ratio: 0.25, 95% confidence interval: 0.08-0.76). The lack of ACP in the medical records when the patient was admitted to the hospital may have prevented the return home. CONCLUSION: In older patients who had difficulty returning home after hospitalization, the lack of ACP in home medical care may have been an influencing factor. ACP could help continue with home medical care. Geriatr Gerontol Int 2024; 24: 320-326.


Assuntos
Serviços de Assistência Domiciliar , Humanos , Feminino , Idoso , Idoso de 80 Anos ou mais , Masculino , Japão , Hospitalização , Hospitais
5.
J Vet Med Sci ; 73(4): 487-90, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21099187

RESUMO

Cytochrome P450 (CYP) is important for metabolism of not only xenobiotics such as drugs, but also endogenous compounds including arachidonic acids. CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 have been identified in cynomolgus macaque, an animal species widely used for investigation of drug metabolism due to its evolutionary closeness to human. However, their metabolic functions have not been investigated. In this study, proteins were heterologously expressed in Escherichia coli and characterized by metabolic assays using arachidonic acids as substrates that are metabolized by CYP4 isoforms in human. The results showed that all four CYPs metabolized arachidonic acids. Therefore, cynomolgus macaque CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 are functional enzymes.


Assuntos
Ácido Araquidônico/metabolismo , Citocromo P-450 CYP4A/classificação , Macaca fascicularis/metabolismo , Animais , Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Humanos , Isoenzimas , Filogenia , Especificidade por Substrato
6.
J Vet Med Sci ; 72(2): 225-8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19940391

RESUMO

The macaque is widely used for investigation of drug metabolism due to its evolutionary closeness to the human. However, the genetic backgrounds of drug-metabolizing enzymes have not been fully investigated; therefore, identification and characterization of drug-metabolizing enzyme genes are important for understanding drug metabolism in this species. In this study, we isolated and characterized a novel cytochrome P450 2C18 (CYP2C18) cDNA in cynomolgus macaques. This cDNA was highly homologous (96%) to human CYP2C18 cDNA. Cynomolgus CYP2C18 was preferentially expressed in the liver and kidney. Moreover, a metabolic assay using cynomolgus CYP2C18 protein heterologously expressed in Escherichia coli revealed its activity toward S-mephenytoin 4'-hydroxylation. These results suggest that cynomolgus CYP2C18 could function as a drug-metabolizing enzyme in the liver.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Fígado/enzimologia , Macaca fascicularis/metabolismo , Mefenitoína/metabolismo , Filogenia , Sequência de Aminoácidos , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Sequência de Bases , Feminino , Humanos , Masculino , Dados de Sequência Molecular , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Análise de Sequência de DNA
7.
PLoS One ; 15(1): e0228039, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31968008

RESUMO

Stress is prevalent in modern society and can affect human health through its effects on appetite. Therefore, in the present study, we aimed to clarify the neural mechanisms by which acute stress affects appetite in healthy, non-obese males during fasting. In total, 22 volunteers participated in two experiments (stress and control conditions) on different days. The participants performed a stress-inducing speech-and-mental-arithmetic task under both conditions, and then viewed images of food, during which, their neural activity was recorded using magnetoencephalography (MEG). In the stress condition, the participants were told to perform the speech-and-mental-arithmetic task again subsequently to viewing the food images; however, another speech-and-mental-arithmetic task was not performed actually. Subjective levels of stress and appetite were then assessed using a visual analog scale. Electrocardiography was performed to assess the index of heart rate variability reflecting sympathetic nerve activity. The findings showed that subjective levels of stress and sympathetic nerve activity were increased in the MEG session in the stress condition, whereas appetite gradually increased in the MEG session only in the control condition. The decrease in alpha band power in the frontal pole caused by viewing the food images was greater in the stress condition than in the control condition. These findings suggest that acute stress can suppress the increase of appetite, and this suppression is associated with the frontal pole. The results of the present study may provide valuable clues to gain a further understanding of the neural mechanisms by which acute stress affects appetite. However, since the stress examined in the present study was related to the expectation of forthcoming stressful event, our present findings may not be generalized to the stress unrelated to the expectation of forthcoming stressful event.


Assuntos
Regulação do Apetite/fisiologia , Encéfalo/fisiologia , Fadiga Mental/psicologia , Estresse Psicológico , Adulto , Jejum/psicologia , Voluntários Saudáveis , Frequência Cardíaca/fisiologia , Humanos , Magnetoencefalografia/métodos , Masculino , Adulto Jovem
8.
Drug Metab Dispos ; 37(1): 14-7, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18824522

RESUMO

The monkey CYP2C76 gene does not correspond to any of the human CYP2C genes, and its enzyme is at least partly responsible for the species difference occasionally seen in drug metabolism between monkeys and humans. To establish a line and/or lines of monkeys that are expected to show metabolic patterns highly similar to humans, we set out to find monkeys that lacked CYP2C76 activity. By genetic screening of 73 monkeys and a database search of expressed sequence tags, we found a total of 10 nonsynonymous genetic variants in the coding region of CYP2C76, including a null genotype (c.449TG>A). Some of the variants were differently distributed between two animal groups originating from different geographical regions (Indochina and Indonesia). After screening 170 additional genomic samples, we identified a total of eight animals (six males and two females) that were heterozygous for c.449TG>A, which could be used for establishing a homozygous line. If the homozygotes show drug-metabolizing properties more similar to humans than wild-type monkeys, the homozygotes may serve as a better animal model for drug metabolism. The data presented in this article provide the essential genetic information to perform a successful study by using cynomolgus monkeys and present a possible tool to generate a better animal model for drug metabolism.


Assuntos
Alelos , Sistema Enzimático do Citocromo P-450/genética , Modelos Animais , Farmacocinética , Animais , Macaca fascicularis , Reação em Cadeia da Polimerase
9.
J Vet Med Sci ; 71(12): 1653-6, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20046035

RESUMO

Cytochrome P450 2B6 (CYP2B6), an important drug-metabolizing enzyme, is involved in the metabolism of prescribed drugs in humans. Despite its importance, cDNA for a CYP2B6 ortholog has not been identified and characterized in cynomolgus macaques, which are frequently used in preclinical studies. In this study, cDNA highly homologous to human CYP2B6 was cloned from the cynomolgus macaque liver. This cDNA contained an open reading frame of 491 amino acids and functional domains characteristic for CYP protein, such as substrate recognition sites and a heme-binding region. Cynomolgus CYP2B6 was expressed predominantly in the liver with some extra-hepatic expression among 10 tissues. Moreover, cynomolgus CYP2B6 revealed activities toward testosterone 16beta-hydroxylation and bupropion hydroxylation. These results suggest that cynomolgus CYP2B6 has a functional role in the liver.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , DNA Complementar/genética , Macaca fascicularis/genética , Oxirredutases N-Desmetilantes/genética , Sequência de Aminoácidos , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2B6 , Regulação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Fígado/metabolismo , Dados de Sequência Molecular , Oxirredutases N-Desmetilantes/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Distribuição Tecidual
10.
Sci Rep ; 9(1): 4000, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850665

RESUMO

Fatigue is a health problem prevalent in modern societies. Fatigue sensation plays an important role as a biological alarm urging rest to maintain homeostasis, and clarifying the neural mechanisms related to fatigue sensations by which we decide to engage in rest is therefore essential. This study enrolled healthy male volunteers and showed that the decrease in alpha-band power as assessed by magnetoencephalography of the left Brodmann's area (BA) 6 before perception of fatigue when a button-press based on the level of fatigue was required was smaller than that before perception of the intention to move when a voluntary button-press was required. In addition, the decrease of alpha-band power in the left BA 6 before the perception of fatigue was not altered compared with that in the right BA 6 when a button-press based on the level of fatigue was required. These results suggest that the button-press based on the perception of fatigue is not prepared before the perception of fatigue. These findings will advance the understanding of the neural mechanisms related to subjective feelings such as fatigue sensation.


Assuntos
Encéfalo/fisiologia , Fadiga/fisiopatologia , Movimento/fisiologia , Percepção/fisiologia , Sensação/fisiologia , Adulto , Mapeamento Encefálico/métodos , Emoções/fisiologia , Voluntários Saudáveis , Humanos , Magnetoencefalografia/métodos , Masculino , Adulto Jovem
11.
Sci Rep ; 9(1): 11044, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363158

RESUMO

It has been reported that physical activity not only increases energy expenditure, but also affects appetite. However, little remains known about the effects of physical activity-induced fatigue sensation on appetite. In the present study, classical conditioning related to fatigue sensation was used to dissociate fatigue sensation from physical activity. The participants were 20 healthy male volunteers. After overnight fasting, on day 1, the participants performed hand-grip task trials for 10 min with listening to a sound. The next day, they viewed food images with (target task) and without (control task) listening to the sound identical to that used on day 1, and their neural activity during the tasks were recorded using magnetoencephalography. The subjective levels of appetite and fatigue sensation were assessed using a visual analog scale. The subjective level of fatigue increased and that of appetite for fatty foods showed a tendency toward increase in the target task while the subjective level of fatigue and that of appetite for fatty foods were not altered in the control task. In the target task, the decrease of theta (4-8 Hz) band power in the supplementary motor area (SMA), which was observed in the control task, was suppressed, and the suppression was positively correlated with appetite for fatty foods, suggesting hand grip activity-induced fatigue sensation may increase the appetite for fatty food; this increase could be related to neural activity in the SMA. These findings are expected to contribute to the understanding of the neural mechanisms of appetite in relation to fatigue.


Assuntos
Apetite/fisiologia , Condicionamento Clássico/fisiologia , Força da Mão/fisiologia , Fadiga Muscular/fisiologia , Humanos , Magnetoencefalografia , Masculino , Adulto Jovem
12.
Sci Rep ; 8(1): 3119, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29449657

RESUMO

Obesity is a major public health problem in modern society. Appetitive behavior has been proposed to be partially driven by unconscious decision-making processes and thus, targeting the unconscious cognitive processes related to eating behavior is essential to develop strategies for overweight individuals and obese patients. Here, we presented food pictures below the threshold of awareness to healthy male volunteers and examined neural activity related to appetitive behavior using magnetoencephalography. We found that, among participants who did not recognize food pictures during the experiment, an index of heart rate variability assessed by electrocardiography (low-frequency component power/high-frequency component power ratio, LF/HF) just after picture presentation was increased compared with that just before presentation, and the increase in LF/HF was negatively associated with the score for cognitive restraint of food intake. In addition, increased LF/HF was negatively associated with increased alpha band power in Brodmann area (BA) 47 caused by food pictures presented below the threshold of awareness, and level of cognitive restraint was positively associated with increased alpha band power in BA13. Our findings may provide valuable clues to the development of methods assessing unconscious regulation of appetite and offer avenues for further study of the neural mechanisms related to eating behavior.


Assuntos
Apetite/fisiologia , Comportamento Apetitivo/fisiologia , Comportamento Alimentar/fisiologia , Adolescente , Conscientização/fisiologia , Córtex Cerebral/fisiologia , Tomada de Decisões/fisiologia , Ingestão de Alimentos/fisiologia , Eletrocardiografia , Comportamento Alimentar/psicologia , Alimentos , Voluntários Saudáveis , Frequência Cardíaca/fisiologia , Humanos , Magnetoencefalografia/métodos , Masculino , Obesidade , Estimulação Luminosa/métodos , Dados Preliminares , Adulto Jovem
13.
Exp Anim ; 56(5): 385-8, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18075200

RESUMO

The DBA/2J mouse strain is a standard laboratory strain that is widely used for biomedical research. This strain, however, suffers from poor reproductive performance. In addition, the conditions for reliable embryo transfer (ET) of this strain have not been elucidated. The intention of this study was to determine the optimal number of embryos for transfer that allow the effective production of DBA/2J offspring. In the experiment, 7 to 15 embryos per oviduct were transferred into pseudopregnant ICR females. A relatively high success rate for pup production was observed when a large number of DBA/2J embryos (30 embryos per female) were transferred. This result shows that the ET efficiency of the DBA/2J strain can be improved by increasing the number of transferred embryos.


Assuntos
Transferência Embrionária/veterinária , Camundongos Endogâmicos DBA/fisiologia , Animais , Transferência Embrionária/métodos , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Masculino , Camundongos , Camundongos Endogâmicos ICR
14.
Bone ; 39(1): 27-34, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16459153

RESUMO

Some cancers frequently affect the skeleton, and the bone microenvironment supports growth of certain cancer cells. After tumors metastasize to bone, they stimulate osteoclastogenesis and expand in the bone tissue. Hepatocyte growth factor (HGF), which was originally identified as a potent mitogen for hepatocytes, promotes tumor growth, invasion and metastasis. HGF is mainly produced by cells of mesenchymal origin, and osteoblasts/osteocytes and bone marrow stromal cells originate from mesenchymal cells. However, it is not clear what effect HGF has on tumor progression in bone metastasis. In the present study, we investigated the roles of HGF in bone metastasis using the mouse mammary cancer cell line BALB/c-MC. Cancer cells injected into hearts of mice metastasized to bone in their hind limbs. HGF immunoreactivity was detected in the stroma surrounding the tumor nests, and blood vessels expressing CD31 (a marker of endothelial cells) were observed in the HGF-positive area. To identify the cells producing HGF, we measured concentration of HGF in culture media. HGF concentration was elevated in osteoblast cultures (3.13+/-0.25 ng/ml), whereas HGF was undetectable (<0.4 ng/ml) in BALB/c-MC and bone marrow cell cultures. HGF concentration in osteoblast cultures increased 2.5-fold in response to 10(-6) M PGE(2). Addition of HGF to BALB/c-MC cultures caused doubling of the cell number. Moreover, Western blot analysis revealed expression of c-Met/HGF receptor by BALB/c-MC. In the Matrigel invasion chamber assay, addition of HGF to the bottom well increased the rate at which BALB/c-MC invaded the bottom well through the membrane. Furthermore, when osteoblasts were cultured in the bottom well, the number of BALB/c-MC cells that invaded the bottom well through the membrane increased 3.7-fold, compared to assays without osteoblasts. Addition of NK4, an inhibitor of HGF, completely abolished the enhancement of the invasive potential of the BALB/c-MC cells in the presence of osteoblasts. These findings suggest that HGF produced by osteoblasts induces migration of cancer cells from sinusoidal capillaries to bone marrow space and stimulates growth of cancer cells in the bone microenvironment. Thus, osteoblasts appear to promote bone metastasis of some cancers via HGF-c-Met signaling.


Assuntos
Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Animais , Animais Recém-Nascidos , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/química , Dinoprostona/farmacologia , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Invasividade Neoplásica/diagnóstico por imagem , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Transplante de Neoplasias , Osteoblastos/citologia , Gravidez , Proteínas Proto-Oncogênicas c-met/metabolismo , Crânio/citologia , Tíbia/diagnóstico por imagem , Tíbia/patologia , Tomografia Computadorizada por Raios X
15.
Anticancer Res ; 26(6B): 4335-41, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17201152

RESUMO

Several trifluoromethyl ketones (TF1-4) and related non-fluorinated ketones (TF5 and 6) were tested for their relative cytotoxicity on four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4 and promyelocytic leukemia HL-60) and three normal human cells [gingival fibroblasts (HGF), pulp cells (HPC) and periodontal ligament fibroblasts (HPLF)]. Trifluoromethylated a-diketone (TF1, CF3COCOPh) and alpha-hydroxy ketones (TF2, CF3CH(OH)COPh; TF3, CF3CH(OH)COCH2Ph) showed higher tumor-specific cytotoxic activity than the corresponding non-fluorinated analogs (TF5, CH3COCOPh; TF6, CH3CH(OH)COPh), while the anti-tumor potency of trifluoromethyl ketone (TF4, CF3COCH2Ph) was lower. Among four tumor cell lines, HL-60 cells were the most sensitive to TF1-4, followed by HSC-2 and HSC-3 cells. HSC-4 cells were the most resistant in most cases. Agarose gel electrophoresis showed that TF1-3 did not induce intemucleosomal DNA fragmentation nor activated caspase-3. The cytotoxic activities of TF1-3 were not significantly affected by FeCl3. Electron microscopy of TF2- or 3-treated HL-60 cells showed the development of autophagosomes in HL-60 cells, without the production of an apoptotic body, or affecting the mitochondria and cell surface microvilli. The autophagy inhibitor, 3-methyladenine (3-MA), partially inhibited the TF2- or 3-induced cytotoxicity. These data suggest the induction of non-apoptotic cell death by TF2 or 3.


Assuntos
Carcinoma de Células Escamosas/patologia , Cetonas/farmacologia , Neoplasias Bucais/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel de Ágar , Células HL-60 , Humanos , Microscopia Eletrônica
16.
Free Radic Res ; 39(3): 225-35, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15788227

RESUMO

Eicosapentaenoic acid (EPA) induced apoptosis of rat basophilic leukemia cells (RBL2H3 cells), whereas 100 microM linoleic acid (LA) had no significant effect. Cytochrome c was released at 4 h. Apoptosis was detected at 6 h after exposure to EPA and docosahexaenoic acid (DHA), and preceded the activation of caspase-3. Liberation of apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus were observed at 4 h. A broad-specificity caspase inhibitor, z-VAD-fmk, failed to suppress the apoptosis, suggesting that EPA induced caspase-independent apoptosis. On other hand, a poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor that blocks AIF translocation to the nucleus suppressed EPA-induced apoptosis. The level of hydroperoxide in the cells and mitochondria increased at the early phase of apoptosis within 2 h. On the contrary, elevation of hydroperoxide in mitochondria was not observed after treatment with LA. The EPA-induced apoptosis was abolished by prevention of the hydroperoxide elevation in mitochondria via overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). Neither cytochrome c nor AIF were released from mitochondria in the mitochondrial PHGPx-overexpressing cells. EPA also induced apoptosis in HeLa cells, but not in L929 or RAW264.7 cells. Enhancement of the hydroperoxide level in mitochondria was found in the EPA-sensitive HeLa cells after treatment with EPA, whereas no such enhancement was observed in the apoptosis-resistant L929 and RAW264.7 cells. These results suggest that the generation of hydroperoxide in mitochondria induced by EPA is associated with AIF release from mitochondria and the induction of apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Animais , Fator de Indução de Apoptose , Caspase 3 , Caspases/metabolismo , Núcleo Celular/metabolismo , Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Flavoproteínas/metabolismo , Glutationa Peroxidase/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte Proteico , Ratos
17.
Free Radic Res ; 39(10): 1083-9, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16298733

RESUMO

Eicosapentaenoic acid (EPA) was previously shown to induce caspase-independent apoptosis in rat basophilic leukemia cells (RBL2H3 cells) by translocation of apoptosis-inducing factor (AIF) [Free Radic Res (2005) 39, 225-235]. Here, we attempted to investigate the mechanism of EPA-induced apoptosis. A rapid and sustained increase in calcium was observed in mitochondria at 2 h after the addition of EPA prior to apoptosis. Coincidently, hydroperoxide was generated in the mitochondria after exposure to EPA. Production of mitochondrial hydroperoxide was significantly reduced by ruthenium red, an inhibitor of mitochondrial calcium uniporter, and BAPTA-AM, a cytoplasmic calcium chelator, indicating that generation of hydroperoxide is triggered by an accumulation of calcium in the mitochondria. The production of mitochondrial hydroperoxide was markedly attenuated by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the mitochondria. Apoptosis was therefore, significantly prevented through inhibition of mitochondrial hydroperoxide generation with mitochondrial PHGPx, ruthenium red or BAPTA-AM. However, accumulation of calcium in the mitochondria was not prevented by mitochondrial PHGPx although apoptosis was blocked, indicating that elevated calcium does not directly induce apoptosis. Taken together, our results show that calcium-dependent hydroperoxide accumulation in the mitochondria is critical in EPA-induced apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Cálcio/farmacologia , Ácido Eicosapentaenoico/farmacologia , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Regulação Neoplásica da Expressão Gênica , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Mitocôndrias/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Ratos , Rutênio Vermelho/farmacologia
18.
Anticancer Res ; 23(6C): 4797-803, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14981928

RESUMO

Possible changes in the intracellular concentrations of polyamines were investigated during the apoptosis of human promyelocytic leukemic HL-60 cells. Treatment of HL-60 cells with gallic acid and epigallocatechin gallate (EGCG) resulted in the rapid decline of the intracellular concentration of putrescine, whereas that of spermidine and spermine was not significantly changed during the first 3 hours after treatments. Irradiation with UVB also selectively reduced the intracellular concentration of putrescine. On the other hand, cytotoxic concentrations of anticancer agents, such as etoposide and doxorubicin, only marginally reduced the intracellular concentration of putrescine during the first 3 hours. A significant decline of putrescine was observed at later stages when DNA fragmentation became more prominent. Three normal human cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) and human tumor cell lines (squamous cell carcinoma, submandibular carcinoma, malignant malanoma, hepatoma), which showed higher resistance to apoptosis inducers, had significantly higher putrescine concentrations than HL-60 cells. These data suggest that the intracellular concentration of putrescine may be a useful marker for the apoptosis induction or the sensitivity of the cells to apoptosis inducers.


Assuntos
Apoptose/fisiologia , Catequina/análogos & derivados , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ácido Gálico/farmacologia , Poliaminas/metabolismo , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Fragmentação do DNA/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Gengiva/efeitos dos fármacos , Células HL-60 , Humanos , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Putrescina/metabolismo , Espermidina/metabolismo , Espermina/metabolismo , Células Tumorais Cultivadas , Raios Ultravioleta
19.
Drug Metab Pharmacokinet ; 28(6): 510-3, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23648676

RESUMO

Cynomolgus macaques, used in drug metabolism studies due to their evolutionary closeness to humans, are mainly bred in Asian countries, including Cambodia, China, and Indonesia. Cytochromes P450 (P450s) are important drug-metabolizing enzymes, present in the liver and small intestine, major drug metabolizing organs. Previously, our investigation did not find statistically significant differences in hepatic P450 metabolic activities measured in cynomolgus macaques bred in Cambodia (MacfaCAM) and China (MacfaCHN). In the present study, P450 metabolic activity was investigated in the small intestine of MacfaCAM and MacfaCHN, and cynomolgus macaques bred in Indonesia (MacfaIDN) using P450 substrates, including 7-ethoxyresorufin, coumarin, bupropion, paclitaxel, diclofenac, S-mephenytoin, bufuralol, chlorzoxazone, and testosterone. The results indicated that P450 metabolic activity of the small intestine was not statistically significantly different (<2.0-fold) in MacfaCAM, MacfaCHN, and MacfaIDN. In addition, statistically significant sex differences were not observed (<2.0-fold) in any P450 metabolic activity in MacfaCAM as supported by mRNA expression results. These results suggest that P450 metabolic activity of the small intestine does not significantly differ statistically among MacfaCAM, MacfaCHN, and MacfaIDN.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Intestino Delgado/enzimologia , Macaca fascicularis/metabolismo , Animais , Camboja , China , Feminino , Indonésia , Masculino , Especificidade da Espécie
20.
Drug Metab Pharmacokinet ; 27(3): 307-16, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22166892

RESUMO

Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (>2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (>2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (>2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (<1.5-fold) between mfCAM and mfCHN. These results suggest that gene expression profiles, including drug-metabolizing enzyme genes such as P450 genes, are similar in mfCAM, mfCHN, and mfIDN.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Expressão Gênica , Macaca fascicularis/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Camboja , China , Sistema Enzimático do Citocromo P-450/genética , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Indonésia , Fígado/enzimologia , Fígado/metabolismo , Macaca fascicularis/crescimento & desenvolvimento , Macaca mulatta/crescimento & desenvolvimento , Macaca mulatta/metabolismo , Masculino , Microssomos Hepáticos/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , RNA Mensageiro/metabolismo , Caracteres Sexuais , Especificidade da Espécie , Regulação para Cima
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