RESUMO
1. Budesonide is a glucocorticoid used in the treatment of several respiratory and gastrointestinal inflammatory diseases. Glucocorticoids have been demonstrated to induce cytochrome P450 (CYP) 3A and the efflux transporter P-glycoprotein (P-gp). This study aimed to evaluate the potential of budesonide to act as a perpetrator or a victim of transporter- or CYP-mediated drug-drug interactions (DDIs). 2. In vitro studies were conducted for P-gp, breast cancer resistance protein and organic anion and cation transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT2) in transporter-transfected cells. Changes in mRNA expression in human hepatocytes and enzyme activity in human liver microsomes by budesonide were determined for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A. 3. The data indicated that budesonide is a substrate of P-gp but is not a substrate or an inhibitor of the other transporters investigated. Budesonide is neither an inducer nor an inhibitor of major CYP enzymes. The effect of P-gp on budesonide disposition is anticipated to be low owing to CYP3A-mediated clearance. 4. Collectively, our data indicate there is a low risk of budesonide perpetrating clinical DDIs mediated by the transporters or CYPs studied.
Assuntos
Budesonida/farmacologia , Budesonida/farmacocinética , Proteínas de Transporte/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/metabolismo , Microssomos Hepáticos/enzimologia , Células Cultivadas , Interações Medicamentosas , Hepatócitos/citologia , HumanosRESUMO
Silymarin, derived from the milk thistle plant Silybum marianum and widely used for self-treatment of liver diseases, is composed of six major flavonolignans including silybin A and silybin B, which are the predominant flavonolignans quantified in human plasma. The single- and multiple-dose pharmacokinetics of silymarin flavonolignans were examined in patients with nonalcoholic fatty liver disease (NAFLD) or hepatitis C virus (HCV) to determine whether the disposition of silymarin and therefore its potential efficacy vary among liver disease populations. Cohorts of eight subjects with noncirrhotic liver disease were randomized 3:1 to oral silymarin or placebo (280 or 560 mg) every 8 h for 7 days. Forty-eight-hour blood sampling was conducted after the first and final doses. In general, plasma concentrations of silybin A and silybin B were higher, whereas concentrations of conjugates were lower in NAFLD compared with HCV. After adjustment of the area under plasma concentration-time curve from 0 to 8 h (AUC(0-8 h)) for weight and dose, only silybin B and silybin B conjugates differed significantly between disease types. For NAFLD, the adjusted mean AUC(0-8 h) was higher for silybin B (p < 0.05) but lower for silybin B conjugates (p < 0.05) compared with that for HCV. At the 280-mg dose, steady-state plasma concentrations of silybin B conjugates for NAFLD subjects were characterized by 46% lower AUC(0-8 h) (p < 0.05) and 42% lower C(max) (p < 0.05) compared with HCV subjects. Evidence of enterohepatic cycling of flavonolignans was only observed in NAFLD subjects. In summary, the efficacy of silymarin may be more readily observed in NAFLD patients because of their higher flavonolignan plasma concentrations and more extensive enterohepatic cycling compared with those in HCV patients.
Assuntos
Fígado Gorduroso/metabolismo , Hepatite C Crônica/metabolismo , Silimarina/farmacocinética , Adulto , Área Sob a Curva , Feminino , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Silimarina/sangueRESUMO
This paper focuses on the study of environmental risk assessment and comprehensive index model of disaster loss for COVID-19 transmission. Considering the five environmental vectors of carrier vulnerability, environmental instability of pregnancy and disaster, intensity of disaster-causing factors, disaster prevention and mitigation capacity and emergency prevention and control capacity and its 38 indicators, the correlation coefficient matrix and principal component expressions of each vector are established by principal component analysis, respectively, and the index model of each vector is established on the basis. Then, considering the index models of these five vectors, we established the disaster loss composite index model, which was used to conduct environmental risk assessment and disaster loss composite index analysis of the transmission of COVID-19 in Hubei Province during the period of January 21, 2020 to March 18, 2020. The empirical study showed that: (1) the risk index peaked from January 21 to January 23; (2) the risk index was at a low but volatile level from January 24 to March 14; (3) the risk index rose again slightly from March 15 and rose to another peak on March 16. These fluctuating, smooth and fluctuating processes of the comprehensive index of disaster losses of COVID-19 in Hubei Province are basically stable and consistent with the actual situation of the virus outbreak in the early stage, isolation and prevention and control in the middle stage, and resumption of work and production in the late stage. The study in this paper provides a scientific decision-making reference for the prevention and control of COVID-19 as well as emergency prevention and control measures.
RESUMO
Silymarin, a mixture of polyphenolic flavonoids extracted from milk thistle (Silybum marianum), is composed mainly of silychristin, silydianin, silybin A, silybin B (SB(B)), isosilybin A (ISB(A)), and isosilybin B. In this study, the plasma concentrations of free (unconjugated), conjugated (sulfated and glucuronidated), and total (free and conjugated) silymarin flavonolignans were measured using liquid chromatography-electrospray ionization-mass spectrometry, after a single oral dose of 600 mg of standardized milk thistle extracts to three healthy volunteers. Pharmacokinetic analysis indicated that silymarin flavonolignans were rapidly eliminated with short half-lives (1-3 and 3-8 h for free and conjugated, respectively). The AUC(0-->infinity) values of the conjugated silymarin flavonolignans were 4- to 30-fold higher than those of their free fractions, with SB(B) (mean AUC(0-->infinity) = 51 and 597 microg x h/l for free and conjugated, respectively) and ISB(A) (mean AUC(0-->infinity) = 30 and 734 microg x h/l for free and conjugated, respectively) exhibiting higher AUC(0-->infinity) values in comparison with other flavonolignans. Near the plasma peak times (1-3 h), the free, sulfated, and glucuronidated flavonolignans represented approximately 17, 28, and 55% of the total silymarin, respectively. In addition, the individual silymarin flavonolignans exhibited quite different plasma profiles for both the free and conjugated fractions. These data suggest that, after oral administration, silymarin flavonolignans are quickly metabolized to their conjugates, primarily forming glucuronides, and the conjugates are primary components present in human plasma.
Assuntos
Silybum marianum/química , Silimarina/sangue , Silimarina/farmacocinética , Administração Oral , Área Sob a Curva , Biotransformação , Cromatografia Líquida de Alta Pressão , Composição de Medicamentos , Humanos , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/normas , Silimarina/isolamento & purificação , Silimarina/metabolismo , Silimarina/normas , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Silymarin, an extract from seeds of Silybum marianum, is used by 8 to 33% of patients to self-treat chronic viral hepatitis C in the United States. Studies in humans and rodents suggest that biliary excretion of glucuronide and sulfate conjugates is the major route for silymarin's elimination. To determine the role of multidrug resistance-associated protein 2 (Mrp2) (Abcc2) in the biliary excretion of silymarin, the hepatobiliary disposition of the six major silymarin flavonolignans was studied using isolated perfused livers (IPRLs) from wild-type (WT) and Mrp2-deficient (TR(-)) Wistar rats. For all the flavonolignans, approximately 96% of the dose was cleared from perfusate within 30 min in both WT and TR(-) livers, and <5% of parent was recovered in bile or perfusate by the end of the perfusion. In WT livers, the percentage of dose excreted as conjugates into bile varied for each flavonolignan (silychristin, 51.6 +/- 9.3%; silydianin, 101.5 +/- 28.3%; silybin A, 21.0 +/- 8.3%; silybin B, 31.7 +/- 13.2%; isosilybin A, 50.5 +/- 23.6%; isosilybin B, 42.8 +/- 19.3%). Among the flavonolignans, only silydianin was primarily glucuronidated and almost completely recovered in bile. In TR(-) livers, biliary excretion of flavonolignan conjugates was reduced 80 to 92%, with 30 to 83% of each flavonolignan conjugate recovered in perfusate compared with only 5 to 30% at 90 min. Biliary excretion of glucuronide and sulfate conjugates of all the flavonolignans were reduced by 94 to 98% and 73 to 84%, respectively, in TR(-) IPRLs. These data indicate a primary role for Mrp2 in the biliary elimination of silymarin flavonolignan conjugates.
Assuntos
Sistema Biliar/metabolismo , Flavonolignanos/metabolismo , Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Silimarina/metabolismo , Animais , Bile/metabolismo , Disponibilidade Biológica , Bovinos , Flavonolignanos/farmacocinética , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Perfusão/métodos , Ratos , Ratos Transgênicos , Ratos Wistar , Silimarina/farmacocinéticaRESUMO
Silymarin, used by 30 to 40% of liver disease patients, is composed of six major flavonolignans, each of which may contribute to silymarin's hepatoprotective properties. Previous studies have only described the pharmacokinetics for two flavonolignans, silybin A and silybin B, in healthy volunteers. The aim of this study was to determine the pharmacokinetics of the major silymarin flavonolignans in liver disease patients. Healthy volunteers and three patient cohorts were administered a single, 600-mg p.o. dose of milk thistle extract, and 14 blood samples were obtained over 24 h. Silybin A and B accounted for 43% of the exposure to the sum of total silymarin flavonolignans in healthy volunteers and only 31 to 38% in liver disease cohorts as a result of accumulation of silychristin (20-36%). Area under the curve (AUC(0-24h)) for the sum of total silymarin flavonolignans was 2.4-, 3.3-, and 4.7-fold higher for hepatitis C virus (HCV) noncirrhosis, nonalcoholic fatty liver disease (p Assuntos
Caspase 3/sangue
, Caspase 7/sangue
, Fígado Gorduroso/metabolismo
, Hepatite C/metabolismo
, Silimarina/farmacocinética
, Adolescente
, Adulto
, Idoso
, Apoptose
, Área Sob a Curva
, Estudos de Coortes
, Feminino
, Meia-Vida
, Humanos
, Masculino
, Pessoa de Meia-Idade
, Estresse Oxidativo
, Silimarina/sangue
RESUMO
Proteasomes are multicatalytic proteinase complexes within cells that selectively degrade ubiquitinated proteins. We have recently demonstrated that fatty acids, major components of cell membranes, are able to regulate the proteasomal degradation of tyrosinase, a critical enzyme required for melanin biosynthesis, in contrasting manners by relative increases or decreases in the ubiquitinated tyrosinase. In the present study, we show that altering the intracellular composition of fatty acids affects the post-Golgi degradation of tyrosinase. Incubation with linoleic acid (C18:2) dramatically changed the fatty acid composition of cultured B16 melanoma cells, i.e. the remarkable increase in polyunsaturated fatty acids such as linoleic acid and arachidonic acid (C20:4) was compensated by the decrease in monounsaturated fatty acids such as oleic acid (C18:1) and palmitoleic acid (C16:1), with little effect on the proportion of saturated to unsaturated fatty acid. When the composition of intracellular fatty acids was altered, tyrosinase was rapidly processed to the Golgi apparatus from the ER (endoplasmic reticulum) and the degradation of tyrosinase was increased after its maturation in the Golgi. Retention of tyrosinase in the ER was observed when cells were treated with linoleic acid in the presence of proteasome inhibitors, explaining why melanin synthesis was decreased in cells treated with linoleic acid and a proteasome inhibitor despite the abrogation of tyrosinase degradation. These results suggest that the intracellular composition of fatty acid affects the processing and function of tyrosinase in connection with the ubiquitin-proteasome pathway and suggest that this might be a common physiological approach to regulate protein degradation.
Assuntos
Ácido Linoleico/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Ácido Palmítico/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Leupeptinas , Camundongos , Transporte ProteicoRESUMO
A 21-year-old woman with a short stature presented with primary amenorrhoea and a 45X karyotype, and comparative genomic hybridization revealed 1p36 deletion and abnormal genes in multiple chromosomes to support the diagnosis of Turner syndrome and monosomy 1p36 deletion syndrome. The main clinical features of this condition include microsomia, poor sexual development, menoschesis, gigantorectum, absence of internal genitalia, sometimes with thyropenia and low intelligence. This disease can be easily diagnosed for its heterogeneous clinical manifestations.
Assuntos
Transtornos Cromossômicos/diagnóstico , Hibridização Genômica Comparativa , Síndrome de Turner/diagnóstico , Anormalidades Múltiplas , Deleção Cromossômica , Cromossomos Humanos Par 1 , Erros de Diagnóstico , Feminino , Humanos , Cariótipo , Adulto JovemRESUMO
Silymarin, derived from the milk thistle plant Silybum marianum, is widely used for self-treatment of liver diseases, including hepatitis C virus (HCV), and its antiviral activity has been demonstrated in vitro and in HCV patients administered an intravenous formulation of the major silymarin flavonolignans, silybin A and silybin B. The safety and dose-exposure relationships of higher than customary oral doses of silymarin and its acute effects on serum HCV RNA were evaluated in noncirrhotic HCV patients. Four cohorts of 8 patients with well-compensated, chronic noncirrhotic HCV who failed interferon-based therapy were randomized 3:1 to silymarin or placebo. Oral doses of 140, 280, 560, or 700 mg silymarin were administered every 8 hours for 7 days. Steady-state exposures for silybin A and silybin B increased 11-fold and 38-fold, respectively, with a 5-fold increase in dose, suggesting nonlinear pharmacokinetics. No drug-related adverse events were reported, and no clinically meaningful reductions from baseline serum transaminases or HCV RNA titer were observed. Oral doses of silymarin up to 2.1 g per day were safe and well tolerated. The nonlinear pharmacokinetics of silybin A and silybin B suggests low bioavailability associated with customary doses of silymarin may be overcome with doses above 700 mg.
Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Cirrose Hepática , Silimarina/administração & dosagem , Administração Oral , Adulto , Estudos de Coortes , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Hepatite C Crônica/sangue , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/sangueRESUMO
Phosphatidylserine (PtdSer), major acidic phospholipids in neuronal membranes, participate in important cell signaling processes. The PtdSer in brain is highly enriched with docosahexaenoic acid (DHA; 22:6n-3), and the DHA status or ethanol exposure has been shown to influence the PtdSer level. This study shows that ethanol exposure during prenatal and developmental period significantly attenuates microsomal PtdSer biosynthetic activities and reduces PtdSer, particularly 18:0, 22:6-PtdSer, in developing rat brain cortices. Brain microsomes were incubated with deuterium labeled exogenous substrates in vitro and the products formed were detected by reversed phase HPLC-electrospray ionization mass spectrometry (ESI-MS). These in vitro bioassays showed that 1-stearoyl-2-docosahexaenoyl (18:0, 22:6) species is the best substrate for PtdSer synthesis from both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). The PtdSer biosynthetic activity of brain, especially for 18:0, 22:6-PtdSer production, was hampered significantly by maternal exposure to ethanol. PtdSer levels were consistently reduced significantly in brain cortices of the pups from ethanol-exposed dams, due mainly to the depletion of 18:0, 22:6-PtdSer. The mRNA expression of PtdSer synthase 1 (PSS1) and PtdSer synthase 2 (PSS2) was not reduced by ethanol. Similarly, the PSS1 enzyme level did not change after ethanol exposure but PSS2 could not be probed with the antibody available currently. Degradation of PtdSer by mitochondrial PtdSer decarboxylation was not enhanced but also inhibited. Taken together, attenuated PtdSer biosynthetic activities are largely responsible for the PtdSer reduction observed in developing rat brains after maternal exposure to ethanol.
Assuntos
Córtex Cerebral/metabolismo , Etanol/farmacologia , Fosfatidilserinas/biossíntese , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Animais , Depressores do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Ácidos Graxos/metabolismo , Feminino , Microssomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transferases de Grupos Nitrogenados/efeitos dos fármacos , Transferases de Grupos Nitrogenados/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Estatísticas não ParamétricasRESUMO
Bevirimat [BVM, PA-457, 3-O-(3',3'-dimethylsuccinyl)-betulinic acid], a new anti-human immunodeficiency virus drug candidate, is metabolized to two monoglucuronides [mono-BVMG (I) and mono-BVMG (II)] and one diglucuronide (di-BVMG) both in vivo and in vitro. UDP-glucuronosyltransferase (UGT) reaction screening, enzyme kinetics, and species differences for the glucuronidation of BVM in vitro were investigated with pooled human liver microsomes (HLMs) and human intestinal microsomes (HIMs), animal liver microsomes, and 12 recombinant human UGT isoforms. Glucuronidation of BVM with HLMs predominantly involved the formation of mono-BVMG (I) (V(max) = 61 pmol/min/mg protein, K(m) = 27 microM) and mono-BVMG (II) (V(max) = 48 pmol/min/mg protein, K(m) = 16 microM). Di-BVMG was also observed but was a minor metabolite. HIMs mainly revealed glucuronidation to form mono-BVMG (II) (V(max) = 90 pmol/min/mg of protein, K(m) = 8.3 microM). UGT1A3 predominantly formed mono-BVMG (I) (V(max) = 65 pmol/min/mg of protein, K(m) = 13 microM), whereas UGT1A4 is a less active isoform (V(max) = 1.8 pmol/min/mg of protein, K(m) = 5.6 microM). UGT2B7 was involved in the formation of both mono-BVMG (I) (V(max) = 6.1 pmol/min/mg of protein, K(m) = 6.0 microM) and mono-BVMG (II) (V(max) = 6.5 pmol/min/mg of protein, K(m) = 7.8 microM). Among the animal liver microsomes examined, all species (rat, mouse, dog, and marmoset) demonstrated conjugation to form both mono-BVMG (I) and mono-BVMG (II), with dog liver microsomes exhibiting a higher formation rate for mono-BVMG (I), whereas marmoset liver microsomes showed a higher formation rate for mono-BVMG (II). The data suggest a primary role of UGT1A3 for the glucuronidation of BVM.
Assuntos
Fármacos Anti-HIV/metabolismo , Glucuronosiltransferase/metabolismo , Succinatos/metabolismo , Triterpenos/metabolismo , Animais , Cães , Glucuronídeos/metabolismo , Glucuronosiltransferase/genética , Humanos , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Camundongos , Microssomos/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Especificidade da EspécieRESUMO
Etoposide, an important anticancer agent, undergoes glucuronidation both in vitro and in vivo. In this study, three isomeric glucuronides of etoposide, including one phenolic (EPG) and two alcoholic glucuronides (EAG1 and EAG2), were biosynthesized in vitro with human liver microsomes (HLMs), and identified by liquid chromatography-electrospray ionization-mass spectrometry and confirmed by beta-glucuronidase cleavage. In vitro UDP-glucuronosyltransferase (UGT) reaction screening with 12 recombinant human UGTs demonstrated that etoposide glucuronidation is mainly catalyzed by UGT1A1. Although UGT1A8 and 1A3 also catalyzed the glucuronidation of etoposide, their activities were approximately 10 and 1% of UGT1A1. Enzyme kinetic study indicated that the predominant form of etoposide glucuronide in HLMs and human intestinal microsomes (HIMs) was EPG, whereas EAG1 and EAG2 were the minor metabolites, with approximately an 8 to 10% glucuronidation rate of EPG. For the formation of EPG, the V(max) of HLMs (110 pmol/min/mg protein) was very similar to that of recombinant UGT1A1 (124 pmol/min/mg protein), whereas the V(max) of HIMs (54.4 pmol/min/mg protein) was 2-fold lower than those of HIMs and UGT1A1. The K(m) values of HLMs (530 microM) and HIMs (608 microM) were 2-fold higher than that of UGT1A1 (285 microM). The V(max)/K(m) values for the formation of EPG were 0.21 and 0.09 microl/min/mg protein for HLMs and HIMs, respectively. The data indicated that UGT1A1 is principally responsible for the formation of etoposide glucuronides, mainly in the form of phenolic glucuronide, suggesting that etoposide can be used as a highly selective probe substrate for human UGT1A1 in vitro.
Assuntos
Antineoplásicos Fitogênicos/metabolismo , Etoposídeo/metabolismo , Glucuronídeos/biossíntese , Glucuronosiltransferase/metabolismo , Álcoois/metabolismo , Bilirrubina/farmacologia , Estradiol/farmacologia , Etinilestradiol/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Humanos , Mucosa Intestinal/metabolismo , Cinética , Fígado/metabolismo , Microssomos/metabolismo , Fenóis/metabolismoRESUMO
PA-457 [3-O-(3',3'-dimethylsuccinyl)-betulinic acid] represents a new class of anti-HIV drug candidates termed maturation inhibitors. After oral administration to rats, PA-457 was metabolized to several glucuronide conjugates and mainly eliminated into rat bile. Liquid chromatography-electrospray ionization-mass spectrometry analysis showed that the glucuronidation products of PA-457 were acyl glucuronides including one di-glucuronide, di-PA-457G, and two mono-glucuronides, referred to as mono-PA-457G (I) and mono-PA-457G (II), respectively. In-source fragmentation of MS spectra supported the conclusion that mono-PA-457G (I) was glucuronidated at the C-28 carboxyl of PA-457, whereas mono-PA-457G (II) was conjugated at the dimethylsuccinic acid side chain of the C-3 position. Quantification demonstrated that the predominant glucuronide of PA-457 in rat bile was mono-PA-457G (I) with lower amounts of mono-PA-457G (II) and di-PA-457G. In vitro stability indicated that the mono-acyl glucuronides of PA-457 were not degraded after incubation with 0.1 M phosphate buffer (pH 4, 7.4 and 9), plasma (human, rat, and mouse), and UDP-glucuronosyltransferase reaction media (without uridine 5'-diphosphoglucuronic acid) with microsomes (human, rat, and mouse liver microsomes), respectively, whereas the minor diglucuronide was unstable in rodent liver microsomes. All glucuronides of PA-457 could be hydrolyzed both by beta-glucuronidase and alkaline (1 M NaOH). Minor putative acyl migration products were slowly formed at pH 9, suggesting that the acyl glucuronides of PA-457 have relatively high in vitro stability.
Assuntos
Fármacos Anti-HIV/farmacocinética , Bile/metabolismo , Glucuronídeos/metabolismo , Succinatos/farmacocinética , Triterpenos/farmacocinética , Animais , Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/química , Biotransformação , Cromatografia Líquida/métodos , Estabilidade de Medicamentos , Glucuronídeos/química , Humanos , Fígado/metabolismo , Masculino , Camundongos , Microssomos Hepáticos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Succinatos/sangue , Succinatos/química , Triterpenos/sangue , Triterpenos/químicaRESUMO
Prenatal and postnatal ethanol exposure induces abnormal cell death in the nervous system. We have previously reported that docosahexaenoic acid (DHA; 22:6n-3) prevents neuronal apoptosis through promoting phosphatidylserine (PS) accumulation. Previously, we have shown in C6 glioma cells that ethanol inhibits the accumulation of PS caused by DHA supplementation. In this report, we demonstrate that in vitro or in vivo exposure to ethanol inhibits DHA-dependent PS accumulation and neuronal survival. We found that Neuro 2A cells exposed to ethanol accumulated considerably less PS in response to the DHA enrichment and were less effective at phosphorylating Akt and suppressing caspase-3 activity under serum-starved or staurosporine-treated conditions. The in vivo paradigm correlated well with the in vitro findings. We found that the total PS and DHA contents in the fetal hippocampus were slightly but significantly lowered by the prenatal ethanol exposure. Fetal hippocampal cultures obtained at embryonic day 18 from ethanol-treated pregnant rats contained significantly higher apoptotic cells after 7 days in vitro under basal conditions and exhibited particular susceptibility to cell death induced by trophic factor removal in comparison with the pair-fed control group. The reduction of PS and the resulting neuronal cell death inappropriately enhanced during development may contribute to the defects in brain function often observed in fetal alcohol syndrome.
Assuntos
Apoptose/efeitos dos fármacos , Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Neurônios/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Animais , Western Blotting/métodos , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Ácidos Graxos/metabolismo , Feminino , Hipocampo/citologia , Marcação In Situ das Extremidades Cortadas/métodos , Espectrometria de Massas/métodos , Camundongos , Neuroblastoma/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , Soro/fisiologiaRESUMO
Phosphatidylinositol 3-kinase [PI (3)K]/Akt signaling is a critical pathway in cell survival. Here, we demonstrate a mechanism where membrane alteration by the n-3 fatty acid status affects Akt signaling, impacting neuronal survival. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid highly enriched in neuronal membranes, promotes neuronal survival by facilitating membrane translocation/activation of Akt through its capacity to increase phosphatidylserine (PS), the major acidic phospholipid in cell membranes. The activation of PI (3)K and phosphatidylsinositol triphosphate formation were not affected by DHA, indicating that membrane interaction of Akt is the event responsible for the DHA effect. Docosapentaenoic acid, which replaces DHA during n-3 fatty acid deficiency, was less effective in accumulating PS and translocating Akt and thus less effective in preventing apoptosis. Consistently, in vivo reduction of DHA by dietary depletion of n-3 fatty acids decreased hippocampal PS and increased neuronal susceptibility to apoptosis in cultures. This mechanism may contribute to neurological deficits associated with n-3 fatty acid deficiency and support protective effects of DHA in pathological models such as brain ischemia or Alzheimer's disease.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos Ômega-3/metabolismo , Camundongos , Modelos Neurológicos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
It has been suggested that hippocampus-related cognitive processes are especially sensitive to ethanol. To provide an insight into the biochemical mechanisms underlying the hippocampus-related functional deficits associated with prenatal ethanol exposure, we investigated the effects of chronic ethanol exposure on the phospholipid profile in developing rat hippocampi. High-performance liquid chromatography/electrospray ionization-mass spectrometry analysis revealed that ethanol lowered the levels of total phosphatidylserine (PS) by 15-20% at all ages examined, primarily owing to the reduction in 1-stearoyl-2-docosahexaenoyl-PS (18:0,22:6n-3-PS) species. Ethanol exposure also led to a decrease in phosphatidylcholine (PC) and an increase in phosphatidylethanolamine (PE), but the total phospholipid content was not significantly changed. At the fatty acid level, ethanol exposure significantly decreased the 22:6n-3 content at postnatal days 0 and 21, with a slight increase in 22:5n-6, without changing the total fatty acid content significantly. In conclusion, ethanol depleted PS, especially 22:6-containing species, and PC from hippocampal membranes with concomitant increase in PE. Alteration of the phospholipid profile in the hippocampus resulting from exposure to ethanol during prenatal and developmental stages may have significant implications with respect to the cognitive dysfunction observed in fetal alcohol syndrome.
Assuntos
Etanol/farmacologia , Hipocampo/metabolismo , Fosfolipídeos/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Animais , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Feminino , Alimentos Formulados , Hipocampo/química , Hipocampo/efeitos dos fármacos , Mães/estatística & dados numéricos , Fosfatidilcolinas/análise , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/análise , Fosfatidilserinas/metabolismo , Fosfolipídeos/análise , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
The fatty acid (FA) docosahexaenoic acid (DHA, 22: 6n-3) is highly enriched in membrane phospholipids of the central nervous system and retina. Loss of DHA because of n-3 FA deficiency leads to suboptimal function in learning, memory, olfactory-based discrimination, spatial learning, and visual acuity. G protein-coupled receptor (GPCR) signal transduction is a common signaling motif in these neuronal pathways. Here we investigated the effect of n-3 FA deficiency on GPCR signaling in retinal rod outer segment (ROS) membranes isolated from rats raised on n-3-adequate or -deficient diets. ROS membranes of second generation n-3 FA-deficient rats had approximately 80% less DHA than n-3-adequate rats. DHA was replaced by docosapentaenoic acid (22:5n-6), an n-6 FA. This replacement correlated with desensitization of visual signaling in n-3 FA-deficient ROS, as evidenced by reduced rhodopsin activation, rhodopsin-transducin (G(t)) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-G(t) complex relative to n-3 FA-adequate ROS. ROS membranes from n-3 FA-deficient rats exhibited a higher degree of phospholipid acyl chain order relative to n-3 FA-adequate rats. These findings reported here provide an explanation for the reduced amplitude and delayed response of the electroretinogram a-wave observed in n-3 FA deficiency in rodents and nonhuman primates. Because members of the GPCR family are widespread in signaling pathways in the nervous system, the effect of reduced GPCR signaling due to the loss of membrane DHA may serve as an explanation for the suboptimal neural signaling observed in n-3 FA deficiency.