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1.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805788

RESUMO

Persistent chronic liver diseases increase the scar formation and extracellular matrix accumulation that further progress to liver fibrosis and cirrhosis. Nevertheless, there is no antifibrotic therapy to date. The ketogenic diet is composed of high fat, moderate to low-protein, and very low carbohydrate content. It is mainly used in epilepsy and Alzheimer's disease. However, the effects of the ketogenic diet on liver fibrosis remains unknown. Through ketogenic diet consumption, ß-hydroxybutyrate (bHB) and acetoacetate (AcAc) are two ketone bodies that are mainly produced in the liver. It is reported that bHB and AcAc treatment decreases cancer cell proliferation and promotes apoptosis. However, the influence of bHB and AcAc in hepatic stellate cell (HSC) activation and liver fibrosis are still unclear. Therefore, this study aimed to investigate the effect of the ketogenic diet and ketone bodies in affecting liver fibrosis progression. Our study revealed that feeding a high-fat ketogenic diet increased cholesterol accumulation in the liver, which further enhanced the carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis. In addition, more severe liver inflammation and the loss of hepatic antioxidant and detoxification ability were also found in ketogenic diet-fed fibrotic mouse groups. However, the treatment with ketone bodies (bHB and AcAc) did not suppress transforming growth factor-ß (TGF-ß)-induced HSC activation, platelet-derived growth factor (PDGF)-BB-triggered proliferation, and the severity of CCl4-induced liver fibrosis in mice. In conclusion, our study demonstrated that feeding a high-fat ketogenic diet may trigger severe steatohepatitis and thereby promote liver fibrosis progression. Since a different ketogenic diet composition may exert different metabolic effects, more evidence is necessary to clarify the effects of a ketogenic diet on disease treatment.


Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Acetoacetatos/farmacologia , Colesterol/biossíntese , Dieta Cetogênica/efeitos adversos , Cirrose Hepática/metabolismo , Fígado/efeitos dos fármacos , Ácido 3-Hidroxibutírico/biossíntese , Acetoacetatos/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Becaplermina/farmacologia , Tetracloreto de Carbono/administração & dosagem , Catalase/genética , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Colesterol/sangue , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Desmina/genética , Desmina/metabolismo , Progressão da Doença , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Índice de Gravidade de Doença , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Tioacetamida/administração & dosagem , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/farmacologia
2.
Toxicol Lett ; 344: 58-68, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33727136

RESUMO

Luteolin (5,7,3',4'-tetrahydroxyflavone) belongs to the flavone subclass of flavonoids. Luteolin and its glycosides are present in many botanical families, including edible plants, fruits, and vegetables. While the beneficial properties of luteolin have been widely studied, fewer studies have investigated its toxicity. In the present study, using human lymphoblastoid TK6 cells and our newly developed TK6-derived cell lines that each stably express a single human cytochrome P450 (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C18, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), we systematically evaluated luteolin-induced cytotoxicity and genotoxicity, and the role of specific CYPs in the bioactivation of luteolin. Treatments with luteolin for 4-24 h induced cytotoxicity, apoptosis, DNA damage, and chromosome damage in a concentration-dependent manner. Subsequently, we observed that luteolin-induced cytotoxicity and genotoxicity, measured by the high-throughput micronucleus assay, were significantly increased in TK6 cells transduced with CYP1A1 and 1A2. In addition, key apoptosis and DNA damage biomarkers, including cleaved PARP-1, cleaved caspase-3, and phosphorylated histone 2AX (γH2A.X), were all significantly increased in the CYP1A1- and 1A2-expressing cells compared with the empty vector controls. Analysis by LC-MS/MS revealed that TK6 cells biotransformed the majority of luteolin into diosmetin, a less toxic O-methylated flavone, after 24 h; the presence of CYP1A1 and 1A2 partially reversed this process. Altogether, these results indicate that metabolism by CYP1A1 and 1A2 enhanced the toxicity of luteolin in vitro. Our results further support the utility of our TK6 cell system for identification of the specific CYPs responsible for chemical bioactivation and toxicity potential.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Luteolina/toxicidade , Trifosfato de Adenosina/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular , Sobrevivência Celular , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Dano ao DNA/efeitos dos fármacos , Humanos , Luteolina/química , Micronúcleos com Defeito Cromossômico , Estrutura Molecular , Mutagênicos
3.
Ecotoxicol Environ Saf ; 207: 111547, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33254406

RESUMO

In this paper, earthworms (Eisenia fetida) were exposed to sublethal doses of dichlorvos (spiked concentration of 0.1, 1.0, 10 mg/kg) in soil for 14 days, the metabolomics and activities of cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP2C9 and CYP3A4) of earthworms were analyzed aiming to identify sensitive biomarkers and reveal possible mode of toxic action. The results showed that CYP1A2 and CYP2C9 activity appeared to be more sensitive than CYP3A4 activity in response to dichlorvos, and that metabolic responses based on the metabolomics depended on both of the length of exposure and exposure dose. Malate, ornithine, glucose, inosine, myo-inositol and some amino acids (glutamine, tryptophan, phenylalanine, tyrosine, leucine, histidine, glutamate, lysine) and CYP isozenzymes may be biomarkers to reveal the toxic effect of dichlorvos on earthworms. Compared to controls, when dichlorvos dose reached 1.0 and 10 mg/kg on day 14, glucose and ornithine increased significantly, malate and some amino acids (glutamine, tryptophan, phenylalanine, tyrosine, leucine) decreased significantly, and activities of CYP1A2 and CYP2C9 were inhibited significantly. The current results suggested that 1.0 and 10 mg/kg dichlorvos for 14 days of exposure blocked energy metabolism, disordered Krebs cycle, interfered amino acids metabolism and evoked toxic effects on earthworms.


Assuntos
Diclorvós/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Animais , Biomarcadores/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1A2/farmacologia , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Isoenzimas/metabolismo , Metabolômica/métodos , Oligoquetos/efeitos dos fármacos , Solo/química , Poluentes do Solo/análise
4.
Encephale ; 46(3S): S14-S34, 2020 Jun.
Artigo em Francês | MEDLINE | ID: mdl-32376004

RESUMO

The 2019-20 coronavirus pandemic (SARS-CoV-2; severe acute respiratory syndrome coronavirus 2) has dramatic consequences on populations in terms of morbidity and mortality and in social terms, the general confinement of almost half of the world's population being a situation unprecedented in history, which is difficult today to measure the impact at the individual and collective levels. More specifically, it affects people with various risk factors, which are more frequent in patients suffering from psychiatric disorders. Psychiatrists need to know: (i) how to identify, the risks associated with the prescription of psychotropic drugs and which can prove to be counterproductive in their association with COVID-19 (coronavirus disease 2019), (ii) how to assess in terms of benefit/risk ratio, the implication of any hasty and brutal modification on psychotropic drugs that can induce confusion for a differential diagnosis with the evolution of COVID-19. We carried out a review of the literature aimed at assessing the specific benefit/risk ratio of psychotropic treatments in patients suffering from COVID-19. Clinically, symptoms suggestive of COVID-19 (fever, cough, dyspnea, digestive signs) can be caused by various psychotropic drugs and require vigilance to avoid false negatives and false positives. In infected patients, psychotropic drugs should be used with caution, especially in the elderly, considering the pulmonary risk. Lithium and Clozapine, which are the reference drugs in bipolar disorder and resistant schizophrenia, warrant specific attention. For these two treatments the possibility of a reduction in the dosage - in case of minimal infectious signs and in a situation, which does not allow rapid control - should ideally be considered taking into account the clinical response (even biological; plasma concentrations) observed in the face of previous dose reductions. Tobacco is well identified for its effects as an inducer of CYP1A2 enzyme. In a COVID+ patient, the consequences of an abrupt cessation of smoking, particularly related with the appearance of respiratory symptoms (cough, dyspnea), must therefore be anticipated for patients receiving psychotropics metabolized by CYP1A2. Plasma concentrations of these drugs are expected to decrease and can be related to an increase risk of relapse. The symptomatic treatments used in COVID-19 have frequent interactions with the most used psychotropics. If there is no curative treatment for infection to SARS-CoV-2, the interactions of the various molecules currently tested with several classes of psychotropic drugs (antidepressants, antipsychotics) are important to consider because of the risk of changes in cardiac conduction. Specific knowledge on COVID-19 remains poor today, but we must recommend rigor in this context in the use of psychotropic drugs, to avoid adding, in patients suffering from psychiatric disorders, potentially vulnerable in the epidemic context, an iatrogenic risk or loss of efficiency.


Assuntos
Betacoronavirus , Infecções por Coronavirus , Transtornos Mentais/tratamento farmacológico , Pandemias , Pneumonia Viral , Psicotrópicos/uso terapêutico , Fatores Etários , Antivirais/efeitos adversos , Antivirais/uso terapêutico , Biotransformação , Doenças Cardiovasculares/induzido quimicamente , Comorbidade , Continuidade da Assistência ao Paciente , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Citocromo P-450 CYP1A2/metabolismo , Interações Medicamentosas , Febre/induzido quimicamente , França/epidemiologia , Gastroenteropatias/induzido quimicamente , Humanos , Transtornos Mentais/induzido quimicamente , Transtornos Mentais/epidemiologia , Preparações Farmacêuticas/provisão & distribução , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/epidemiologia , Psicotrópicos/administração & dosagem , Psicotrópicos/efeitos adversos , Psicotrópicos/farmacocinética , Transtornos Respiratórios/induzido quimicamente , Medição de Risco , Abandono do Hábito de Fumar , Avaliação de Sintomas
5.
PLoS One ; 15(5): e0233010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396581

RESUMO

Methamphetamine use has increased over the past decade and the first use of methamphetamine is most often when women are of reproductive age. Methamphetamine accumulates in the liver; however, little is known about the effect of methamphetamine use on hepatic drug metabolism. Methamphetamine was administered on 3 occassions to female Dunkin Hartley guinea pigs of reproductive age, mimicking recreational drug use. Low doses of test drugs caffeine and midazolam were administered after the third dose of methamphetamine to assess the functional activity of cytochrome P450 1A2 and 3A, respectively. Real-time quantitative polymerase chain reaction was used to quantify the mRNA expression of factors involved in glucocorticoid signalling, inflammation, oxidative stress and drug transporters. This study showed that methamphetamine administration decreased hepatic CYP1A2 mRNA expression, but increased CYP1A2 enzyme activity. Methamphetamine had no effect on CYP3A enzyme activity. In addition, we found that methamphetamine may also result in changes in glucocorticoid bioavailability, as we found a decrease in 11ß-hydroxysteroid dehydrogenase 1 mRNA expression, which converts inactive cortisone into active cortisol. This study has shown that methamphetamine administration has the potential to alter drug metabolism via the CYP1A2 metabolic pathway in female guinea pigs. This may have clinical implications for drug dosing in female methamphetamine users of reproductive age.


Assuntos
Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metanfetamina/administração & dosagem , Animais , Estimulantes do Sistema Nervoso Central/administração & dosagem , Estimulantes do Sistema Nervoso Central/sangue , Estimulantes do Sistema Nervoso Central/toxicidade , Citocromo P-450 CYP1A2/genética , Feminino , Cobaias , Humanos , Taxa de Depuração Metabólica , Redes e Vias Metabólicas/efeitos dos fármacos , Metanfetamina/sangue , Metanfetamina/toxicidade , Modelos Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
J Food Sci ; 85(6): 1956-1962, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32406939

RESUMO

We evaluated the influence of pine bark extract (PBE) on organs, the cytochrome-P450 (CYP) activities in liver and estrogenic effects in normal and ovariectomized (OVX) female mice. The PBE did not affect organ weights and liver-function indexes (activities of alkaline phosphatase, aspartate amino transferase, and alanine amino transferase) at doses; 0.04%, 0.4%, and 2.0% PBE in the diet, in normal and OVX female mice. In the OVX mice, CYP1A1 activity was significantly higher in the 0.4% and 2.0% PBE groups than in the OVX control group, and in the 0.4% and 2.0% PBE groups were significantly higher than in the 0.04% PBE group. CYP1A2 and 3A4 activities were significantly higher in the 2.0% PBE group than in all other groups. The PBE did not affect uterine weight and femoral bone mineral density at all PBE doses. These results showed that the dose of PBE at the recommended human intake, had no toxic and estrogenic effects in normal female and OVX mice, however, it may need attention to use the excess intake of PBE with some drugs in postmenopausal women.


Assuntos
Osteoporose Pós-Menopausa/tratamento farmacológico , Pinus/química , Casca de Planta/química , Extratos Vegetais/administração & dosagem , Animais , Densidade Óssea/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Feminino , Fêmur/química , Fêmur/crescimento & desenvolvimento , Humanos , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/fisiopatologia , Ovariectomia , Ovário/metabolismo , Ovário/cirurgia , Extratos Vegetais/efeitos adversos
7.
Food Chem ; 319: 126578, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32187570

RESUMO

For clementine juice, previous data indicate a possible food-drug interaction with substrates of key enzymes responsible for drug metabolism (i.e. cytochrome P450 [CYP] 3A4, CYP1A2). However, which compounds in clementine juice are responsible for these effects are unknown. Therefore, we aimed to identify the compounds in clementine juice provoking metabolic enzyme inhibition or induction. The results demonstrated that the flavonoid fraction of clementine juice provoked induction of several genes and inhibition of both CYP3A4 and CYP1A2, matching effects observed with whole clementine juice. CYP1A2 inhibition and induction can most likely be attributed to nobiletin, sinensetin, and tangeretin. Tangeretin was the only compound causing CYP3A4 induction while CYP3A4 inhibition was most likely the result of additive or synergistic effects caused by several compounds. Thus, whenever evaluating the clinical relevance of clementine interactions, flavonoid contents should be reported because these might explain differences between cultivars and harvests.


Assuntos
Citrus/química , Flavonas/farmacocinética , Flavonoides/farmacocinética , Interações Alimento-Droga , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Inibidores do Citocromo P-450 CYP1A2/farmacologia , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Flavonoides/análise , Frutas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo
8.
Chemosphere ; 248: 126036, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32045972

RESUMO

Aflatoxin B1 (AFB1) and microcystin-LR (MC-LR) co-existed in food and water, and were associated with hepatocellular carcinoma (HCC). AFB1 induced HCC by activating oxidative stress and generating AFB1-DNA adducts, while MC-LR could promote HCC progression. However, whether they have co-effects in HCC progression remains uncertain. In this study, we found the antagonistic effects of MC-LR on AFB1 induced HCC when they were exposed simultaneously. Compared with single exposure to AFB1, co-exposed to MC-LR significantly repressed the AFB1 induced malignant transformation of human hepatic cells and the glutathione S-transferase Pi positive foci formation in rat livers. MC-LR inhibited AFB1 induced upregulation of cytochrome P450 family 1 subfamily A member 2 (CYP1A2) and reduced the AFB1-DNA adducts generation in both human hepatic cells and rat livers. These results suggest that when co-exposure with AFB1, MC-LR might repress hepatocarcinogenicity of AFB1, which might be associated with its repression on AFB1 induced CYP1A2 upregulation and activation.


Assuntos
Aflatoxina B1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Adutos de DNA/metabolismo , Microcistinas/toxicidade , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Masculino , Toxinas Marinhas , Estresse Oxidativo , Ratos
9.
Biomed Res Int ; 2020: 9872936, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31998804

RESUMO

Background and Objective: Clozapine is a second-generation antipsychotic drug that is considered the most effective treatment for refractory schizophrenia. Several clozapine population pharmacokinetic models have been introduced in the last decades. Thus, a systematic review was performed (i) to compare published pharmacokinetics models and (ii) to summarize and explore identified covariates influencing the clozapine pharmacokinetics models. Methods: A search of publications for population pharmacokinetic analyses of clozapine either in healthy volunteers or patients from inception to April 2019 was conducted in PubMed and SCOPUS databases. Reviews, methodology articles, in vitro and animal studies, and noncompartmental analysis were excluded. Results: Twelve studies were included in this review. Clozapine pharmacokinetics was described as one-compartment with first-order absorption and elimination in most of the studies. Significant interindividual variations of clozapine pharmacokinetic parameters were found in most of the included studies. Age, sex, smoking status, and cytochrome P450 1A2 were found to be the most common identified covariates affecting these parameters. External validation was only performed in one study to determine the predictive performance of the models. Conclusions: Large pharmacokinetic variability remains despite the inclusion of several covariates. This can be improved by including other potential factors such as genetic polymorphisms, metabolic factors, and significant drug-drug interactions in a well-designed population pharmacokinetic model in the future, taking into account the incorporation of larger sample size and more stringent sampling strategy. External validation should also be performed to the previously published models to compare their predictive performances.


Assuntos
Antipsicóticos , Clozapina , Citocromo P-450 CYP1A2/metabolismo , Modelos Biológicos , Esquizofrenia , Adulto , Antipsicóticos/farmacocinética , Antipsicóticos/uso terapêutico , Clozapina/farmacocinética , Clozapina/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Esquizofrenia/patologia
10.
Environ Res ; 182: 109074, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31923849

RESUMO

Xenobiotic metabolism at menopause is an under-investigated topic, albeit women spend one-third of their life in the postmenopausal period. The present study examined the effect of menopause on the in vivo activities of CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyltransferase-2 (NAT2) xenobiotic metabolizing enzymes. Enzyme activity was determined in 152 non-smoking volunteers following oral intake of a single dose of 200 mg caffeine and subsequent determination of caffeine metabolite ratios (CMRs) in a 6-h urine sample as follows: CYP1A2: (AFMU+1U+1X)/17U, CYP2A6: 17U/(17U + 17X), XO: 1U/(1U+1X) and NAT2: AFMU/(AFMU+1U+1X). CMRs among groups were analyzed using one-way ANOVA. Significantly lower CYP1A2 and higher CYP2A6 CMRs were observed in postmenopausal compared to premenopausal women and age-matched men. These changes could be attributed to menopause rather than chronological aging since an age-related effect was not observed in premenopausal women or men of any age group. XO CMRs were higher in postmenopausal women and men>50 compared to premenopausal women and men<50, respectively, suggesting an age-related increase in XO activity. No significant alterations were discerned in NAT2 CMRs, in either slow- or rapid-acetylators, indicating that menopause exerts minimal modulation of xenobiotics metabolized by this enzyme. This study provides evidence that the transition to menopause induces significant alterations in xenobiotic-metabolizing enzymes independent of chronological aging suggesting altered metabolism of pharmaceutical and environmental agents.


Assuntos
Arilamina N-Acetiltransferase , Menopausa , Xenobióticos , Arilamina N-Acetiltransferase/metabolismo , Cafeína , Estudos Transversais , Citocromo P-450 CYP1A2/metabolismo , Feminino , Humanos , Masculino , Xenobióticos/metabolismo
11.
Diabetologia ; 63(1): 162-178, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31776611

RESUMO

AIMS/HYPOTHESIS: Exposure to environmental pollution has been consistently linked to diabetes incidence in humans, but the potential causative mechanisms remain unclear. Given the critical role of regulated insulin secretion in maintaining glucose homeostasis, environmental chemicals that reach the endocrine pancreas and cause beta cell injury are of particular concern. We propose that cytochrome P450 (CYP) enzymes, which are involved in metabolising xenobiotics, could serve as a useful biomarker for direct exposure of islets to pollutants. Moreover, functional CYP enzymes in islets could also impact beta cell physiology. The aim of this study was to determine whether CYP1A enzymes are activated in islets following direct or systemic exposure to environmental pollutants. METHODS: Immortalised liver (HepG2) and rodent pancreatic endocrine cell lines (MIN6, ßTC-6, INS1, α-TC1, α-TC3), as well as human islets, were treated in vitro with known CYP1A inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3-MC). In addition, mice were injected with either a single high dose of TCDD or multiple low doses of TCDD in vivo, and islets were isolated 1, 7 or 14 days later. RESULTS: CYP1A enzymes were not activated in any of the immortalised beta or alpha cell lines tested. However, both 3-MC and TCDD potently induced CYP1A1 gene expression and modestly increased CYP1A1 enzyme activity in human islets after 48 h. The induction of CYP1A1 in human islets by TCDD was prevented by cotreatment with a cytokine mixture. After a systemic single high-dose TCDD injection, CYP1A1 enzyme activity was induced in mouse islets ~2-fold, ~40-fold and ~80-fold compared with controls after 1, 7 and 14 days, respectively, in vivo. Multiple low-dose TCDD exposure in vivo also caused significant upregulation of Cyp1a1 in mouse islets. Direct TCDD exposure to human and mouse islets in vitro resulted in suppressed glucose-induced insulin secretion. A single high-dose TCDD injection resulted in lower plasma insulin levels, as well as a pronounced increase in beta cell death. CONCLUSIONS/INTERPRETATION: Transient exposure to TCDD results in long-term upregulation of CYP1A1 enzyme activity in islets. This provides evidence for direct exposure of islets to lipophilic pollutants in vivo and may have implications for islet physiology.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Poluentes Ambientais/toxicidade , Animais , Glicemia/efeitos dos fármacos , Linhagem Celular , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dibenzodioxinas Policloradas/toxicidade , Reação em Cadeia da Polimerase em Tempo Real
12.
Biochem Pharmacol ; 171: 113697, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31706844

RESUMO

The methylation of adenosines at the N6 position (m6A formation) is the most prevalent type of RNA modification in humans. This modification is mediated by methyltransferase like 3 (METTL3)-METTL14 complex, and the methyl group can be removed by RNA demethylases including fat mass and obesity-associated (FTO) and AlkB homolog 5. The formed m6A is recognized by reader proteins such as members of the YT521-B homology (YTH) family, resulting in changes in the splicing, nuclear export, and decay of RNA or translation. In this study, we examined the impact of m6A modification on the expression of drug-metabolizing P450 isoforms. By treatment with 3-deazaadenosine, an inhibitor of RNA methylation, CYP1A2, CYP2B6, and CYP2C8 levels were significantly increased (1.6-fold, 2.2-fold, and 2.7-fold, respectively) in HepaRG cells. In subsequent experiments, we focused on CYP2C8, which showed the largest increase. Consistent with the increase in the mRNA level, CYP2C8 protein level and activity were significantly increased by treatment with 3-deazaadenosine. The CYP2C8 expression levels and activities in HepaRG and Huh-7 cells were increased by knockdown of METTL3/14, whereas they were decreased by knockdown of FTO, suggesting that m6A modification downregulates CYP2C8 expression. With an RNA immunoprecipitation assay using an anti-m6A antibody, it was revealed that the adenosines in the 5'-UTR and the last exon of CYP2C8 are methylated in HepaRG cells and human liver samples. It was demonstrated that YTHDC2, which is known to degrade m6A-containing mRNA, downregulates CYP2C8 expression. In conclusion, we found a novel post-transcriptional regulation mechanism in which the YTHDC2 promotes CYP2C8 mRNA degradation via recognizing the m6A in CYP2C8 mRNA, which is installed by METTL3/14 and removed by FTO.


Assuntos
Adenosina/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica/genética , Fígado/metabolismo , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C8/genética , Citocromo P-450 CYP2C8/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/genética , Metiltransferases/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tubercidina/farmacologia
13.
Basic Clin Pharmacol Toxicol ; 126(3): 296-303, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31608602

RESUMO

BACKGROUND: Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is the most toxic congener of a family of structurally and mechanistically related persistent organic pollutants whose effects are mediated through the aryl hydrocarbon receptor (AhR). Induction of CYP1A1/2 by TCDD through the AhR depends on the magnitude and the duration of exposure. We aimed to assess CYP1A2 activity after acute and chronic exposure to TCDD. The Maincy cohort is a sample population from Melun in the Val-de-Seine region in France that lived for at least 5 years close to a waste incinerator emitting polluted vapours (1974-2002) with high concentrations of dioxins (up to 2000 times the maximal recommended values). Acute exposure to TCDD (Viktor Yushchenko) has been described elsewhere by Sorg et al (Toxicol. Sci. 2012; 125:310-317). Both are rare cases of well-identified source of chronic and acute exposure to TCDD. METHODS: All subjects underwent a full medical history and physical examination and had a cutaneous examination, and a retro-auricular skin biopsy was taken. A questionnaire was designed and used regarding demographic, personal, environmental and occupational characteristics. CYP1A2 activity was assessed 2 hours after the ingestion of a drink containing caffeine through measurement of the metabolic ratio of paraxanthine (17X) over caffeine (137X) by LC-MS/MS or LC-UV. CYP1A1 expression in skin biopsies was determined by immunohistochemical analysis. RESULTS: Forty-seven exposed subjects (age 11-78) and 31 controls were included in the study. Eleven exposed subjects had a history of thyroid disease (23.4%), and 7 (14.8%) had a cancer vs none and 1, respectively, in controls. Nodular skin lesions were found in 13 exposed subjects (27.7%) vs none in controls. Mean CYP1A2 activity of the exposed population was modestly elevated as compared to controls (17X/137X metabolic ratio of 0.475 vs 0.374, P = .051). CYP1A2 was, however, induced (17X/137X, metabolic ratio >0.5) in 27.6% of the exposed cases vs 6.4% of the controls. In contrast, acute dioxin exposure was associated with a strong induction (mean 17X/137X, metabolic ratio of 1.9) still present 29 months after the acute exposure. CYP1A1 was expressed in 59.6% of the skin biopsies (highly expressed in 31.9%) of the Maincy cohort. No correlation between CYP1A2 activity, CYP1A1 expression and clinical manifestations (thyroid disease, cancer, skin lesions) could be demonstrated. CONCLUSION: Higher frequencies of dysthyroidism and cancer were detected in the population exposed chronically to dioxins from a waste incinerator. CYP1A2 was induced in 27.6% of the exposed population, while the magnitude of induction was fourfold higher after acute exposure in the case of Yushchenko.


Assuntos
Citocromo P-450 CYP1A2/metabolismo , Neoplasias/epidemiologia , Dibenzodioxinas Policloradas/toxicidade , Doenças da Glândula Tireoide/epidemiologia , Adolescente , Adulto , Idoso , Criança , Estudos de Coortes , Citocromo P-450 CYP1A1/metabolismo , Exposição Ambiental/efeitos adversos , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/toxicidade , Feminino , França/epidemiologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Dibenzodioxinas Policloradas/administração & dosagem , Inquéritos e Questionários , Adulto Jovem
14.
Xenobiotica ; 50(4): 380-388, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31233374

RESUMO

1. Glycyrol is a coumestan derivative that is isolated from roots of Glycyrrhiza uralensis. Glycyrol exhibits several biological effects, including anti-oxidative and anti-inflammatory effects.2. Herein, we characterized glycyrol metabolism by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM), human liver cytosol, human intestinal microsomes, or human recombinant cDNA-expressed CYPs and UGTs. The analysis was conducted using high resolution mass spectroscopy (HR-MS) on a Q ExactiveTM HF Hybride Quadrupole-Orbitrap mass spectrometer.3. NADPH-supplemented HLM generated six glycyrol metabolites (M1-M6) via hydroxylation, oxidation, and hydration; both NADPH- and UDPGA-supplemented liver microsomes generated three glucuronides (M7-M9). Reaction phenotyping revealed that CYP1A2 is the primary enzyme responsible for phase I metabolism, with minor involvement of the CYP3A4/5, CYP2D6, and CYP2E1 enzymes. Glucuronidation of glycyrol was primarily mediated by UGT1A1, UGT1A3, UGT1A9, and UGT2B7.4. In conclusion, glycyrol undergoes the efficient metabolic hydroxylation and glucuronidation reactions in human liver microsomes, which are predominantly catalyzed by CYP1A2, UGT1A1/3/9, and UGT2B7.


Assuntos
Flavonoides/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Microssomos/metabolismo , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem
15.
Sci Rep ; 9(1): 15901, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685846

RESUMO

Cardiovascular disease (CVD) remains the leading cause of death in chronic kidney disease (CKD) patients despite treatment of traditional risk factors, suggesting that non-traditional CVD risk factors are involved. Trimethylamine-N-oxide (TMAO) correlates with atherosclerosis burden in CKD patients and may be a non-traditional CVD risk factor. Serum TMAO concentrations are significantly increased in CKD patients, which may be due in part to increased hepatic flavin monooxygenase (FMO)-mediated TMAO formation. The objective of this work was to elucidate the mechanism of increased FMO activity in CKD. In this study, FMO enzyme activity experiments were conducted in vitro with liver microsomes isolated from experimental CKD and control rats. Trimethylamine was used as a probe substrate to assess FMO activity. The FMO activator octylamine and human uremic serum were evaluated. FMO gene and protein expression were also determined. FMO-mediated TMAO formation was increased in CKD versus control. Although gene and protein expression of FMO were not changed, metabolic activation elicited by octylamine and human uremic serum increased FMO-mediated TMAO formation. The findings suggest that metabolic activation of FMO-mediated TMAO formation is a novel mechanism that contributes to increased TMAO formation in CKD and represents a therapeutic target to reduce TMAO exposure and CVD.


Assuntos
Metilaminas/metabolismo , Oxigenases de Função Mista/metabolismo , Insuficiência Renal Crônica/patologia , Ativação Metabólica , Animais , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Modelos Animais de Doenças , Cinética , Masculino , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/metabolismo , Especificidade por Substrato
16.
Biochem J ; 476(23): 3661-3685, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31750875

RESUMO

In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane. Incorporation of CYP2E1 results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol and Chlorzaxozone. The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3-0.4 nmol/mg protein (or ∼50% CYP2E1 in the P450 pool). The incorporated CYP2E1 becomes a fully functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1. Enrichment of HLM with CYP2E1 results in pronounced changes in the metabolism of 7-ethoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2 suggesting an increase in the involvement of the latter in its metabolism. This effect goes together with an augmentation of the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin. Furthermore, probing the interactions of CYP2E1 with model microsomes containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2.


Assuntos
Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Microssomos Hepáticos/metabolismo , Membrana Celular/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Remoção de Radical Alquila , Escherichia coli/metabolismo , Feminino , Humanos , Fígado/citologia , Masculino , Espectrometria de Massas , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxazinas/metabolismo , Oxirredução , Espectrometria de Fluorescência , Especificidade por Substrato , Doadores de Tecidos
17.
Genes (Basel) ; 10(10)2019 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-31569378

RESUMO

The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with ß-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and ß-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M, while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.


Assuntos
Perfilação da Expressão Gênica/normas , Fígado/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Adulto , Idoso , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Dimetil Sulfóxido/farmacologia , Feminino , Humanos , Fígado/efeitos dos fármacos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Padrões de Referência , Rifampina/farmacologia , Transcriptoma , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo , beta-Naftoflavona/farmacologia
18.
J Pharm Sci ; 108(12): 3903-3910, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31472121

RESUMO

Cytochrome P450 (CYP) downregulation is a mechanism of drug-drug interaction encountered in pharmaceutical development which is difficult to evaluate in vitro because of the scarcity of evidence. A previous clinical study of obeticholic acid (OCA) with caffeine suggested that OCA may be a useful positive control to establish a method to evaluate CYP1A2 downregulation and to investigate the mechanism of its downregulation. In the present study, we investigated the ability of OCA to downregulate CYP1A2 in human hepatocytes. OCA suppressed CYP1A2 mRNA expression and CYP1A2 enzyme activity without causing direct inhibition of CYP1A2 or cytotoxicity, suggesting that OCA downregulates CYP1A2 in vitro. OCA significantly suppressed the induction of CYP1A2 mRNA expression by omeprazole in a concentration-dependent manner, suggesting that a combination of inducers and new chemical entities would be helpful to investigate the mechanism of CYP1A2 downregulation and to evaluate the potential of new chemical entities for downregulation and investigate their downregulation mechanism. We also showed that CYP1A2 was transcriptionally downregulated by OCA and that a reduction in aryl hydrocarbon receptor mRNA expression is a possible mechanism of CYP1A2 downregulation by OCA. These results indicate that OCA would be a suitable positive control for studies of CYP1A2 downregulation.


Assuntos
Ácido Quenodesoxicólico/análogos & derivados , Citocromo P-450 CYP1A2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Interações Medicamentosas/fisiologia , Hepatócitos/efeitos dos fármacos , Cafeína/farmacologia , Células Cultivadas , Ácido Quenodesoxicólico/farmacologia , Hepatócitos/metabolismo , Humanos , Omeprazol/farmacologia , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transcrição Genética/efeitos dos fármacos
19.
Complement Ther Med ; 46: 87-94, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31519293

RESUMO

BACKGROUND: caffeine is a major constituent in numerous foods, beverages, dietary supplements and medications.Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav, and Salvia miltiorrhiza Bunge are traditional medicines commonly used in Asia. OBJECTIVES: to compare the pharmacokinetics of caffeine in humans before and after consuming an aqueous extract of A. dahurica or S. miltiorrhiza, and to propose a mechanistic explanation for in vivo caffeine metabolism inhibition based on in vitro data obtained with human liver microsomes. METHODS: Each of the four human volunteers was given a single oral dose of caffeine before and after consuming an A. dahurica or S. miltiorrhiza extract. Saliva samples were collected from the volunteers at pre-determined time points after receiving caffeine. The saliva samples were analyzed for unchanged caffeine using liquid chromatography. RESULTS: A. dahurica and S. miltiorrhiza extracts were capable of inhibiting caffeine metabolism in the human volunteers. In a separate study, cytochrome (CYP) 1A2-mediated caffeine demethylase activity was studied in incubation containing human liver microsomes, ß-nicotinamide adenine dinucleotide phosphate, and an herbal extract (or a pure bioactive chemical from the herbs). In all cases, CYP1A2 activity was decreased with an increasing inhibitor concentration, confirming the inhibition of caffeine metabolism in vivo. Caffeine metabolism inhibition most likely involved the competitive and/or non-competitive mechanism. CONCLUSION: Because a high level of caffeine in the plasma may result in adverse health effects in humans, care must be exercised when caffeine is consumed together with A. dahurica or S. miltiorrhiza.


Assuntos
Angelica/química , Cafeína/metabolismo , Medicamentos de Ervas Chinesas/farmacocinética , Extratos Vegetais/farmacocinética , Salvia miltiorrhiza/química , Adulto , Citocromo P-450 CYP1A2/metabolismo , Medicamentos de Ervas Chinesas/química , Feminino , Humanos , Masculino , Extratos Vegetais/química , Adulto Jovem
20.
Nutrients ; 11(8)2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430927

RESUMO

Caffeine is commonly taken via the daily dietary consumption of caffeine-containing foods. The absorbed caffeine is metabolized to yield various metabolites by drug-metabolizing enzymes, and measuring the levels of each caffeine metabolite can provide useful information for evaluating the phenotypes of those enzymes. In this study, the urinary concentrations of caffeine and its 13 metabolites were determined, and the phenotypes of drug metabolic enzymes were investigated based on the caffeine metabolite ratios. Human urine samples were pretreated using solid phase extraction, and caffeine and its metabolites were analyzed using liquid chromatography-tandem mass spectrometry. Based on the urinary caffeine metabolite concentrations, the caffeine metabolite ratios were calculated for six human subjects at specified time points after caffeine intake. Variations in urinary metabolite levels among individuals and time points were reported. In addition, the resultant enzyme activities showed different patterns, depending on the metabolite ratio equations applied. However, some data presented a constant metabolite ratio range, irrespective of time points, even at pre-dose. This suggests the possibility of urinary caffeine metabolite analysis for routine clinical examination. These findings show that urinary caffeine and the metabolite analysis would be useful in evaluating metabolic phenotypes for personalized medicine.


Assuntos
Arilamina N-Acetiltransferase/metabolismo , Cafeína/metabolismo , Cafeína/urina , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2A6/metabolismo , Xantina Oxidase/metabolismo , Adulto , Biomarcadores/metabolismo , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Urinálise , Adulto Jovem
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