RESUMO
Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.
Assuntos
Técnicas Biossensoriais , Citocromo P-450 CYP1A1/isolamento & purificação , Citocromo P-450 CYP2B6/isolamento & purificação , Citocromo P-450 CYP3A/isolamento & purificação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP3A/genética , Proteínas de Fluorescência Verde/genética , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Lentivirus/genética , Taxa de Depuração Metabólica , Transgenes/genéticaRESUMO
Being able to explore the metabolism of broad metabolizing cells is of critical importance in many research fields. This article presents an original modeling solution combining metabolic network and omics data to identify modulated metabolic pathways and changes in metabolic functions occurring during differentiation of a human hepatic cell line (HepaRG). Our results confirm the activation of hepato-specific functionalities and newly evidence modulation of other metabolic pathways, which could not be evidenced from transcriptomic data alone. Our method takes advantage of the network structure to detect changes in metabolic pathways that do not have gene annotations and exploits flux analyses techniques to identify activated metabolic functions. Compared to the usual cell-specific metabolic network reconstruction approaches, it limits false predictions by considering several possible network configurations to represent one phenotype rather than one arbitrarily selected network. Our approach significantly enhances the comprehensive and functional assessment of cell metabolism, opening further perspectives to investigate metabolic shifts occurring within various biological contexts.
Assuntos
Redes e Vias Metabólicas , Metabolômica/métodos , Modelos Biológicos , Diferenciação Celular , Linhagem Celular , Humanos , Fígado/citologia , Fígado/metabolismoRESUMO
CYP2E1 activity is measured in vitro and in vivo via hydroxylation of the Chlorzoxazone (CHZ) producing the 6-hydroxychlorzoxazone (OH-CHZ) further metabolized as a glucuronide excreted in urine. Thus, the quantification of the OH-CHZ following enzymatic hydrolysis of CHZ-derived glucuronide appears to be a reliable assay to measure the CYP2E1 activity without direct detection of this glucuronide. However, OH-CHZ hydrolyzed from urinary glucuronide accounts for less than 80% of the CHZ administrated dose in humans leading to postulate the production of other unidentified metabolites. Moreover, the Uridine 5'-diphospho-glucuronosyltransferase (UGT) involved in the hepatic glucuronidation of OH-CHZ has not yet been identified. In this study, we used recombinant HepG2 cells expressing CYP2E1, metabolically competent HepaRG cells, primary hepatocytes and precision-cut human liver slices to identify metabolites of CHZ (300 µM) by high pressure liquid chromatography-UV and liquid-chromatography-mass spectrometry analyses. Herein, we report the detection of the CHZ-O-glucuronide (CHZ-O-Glc) derived from OH-CHZ in culture media but also in mouse and human urine and we identified a novel CHZ metabolite, the CHZ-N-glucuronide (CHZ-N-Glc), which is resistant to enzymatic hydrolysis and produced independently of CHZ hydroxylation by CYP2E1. Moreover, we demonstrate that UGT1A1, 1A6 and 1A9 proteins catalyze the synthesis of CHZ-O-Glc while CHZ-N-Glc is produced by UGT1A9 specifically. Together, we demonstrated that hydrolysis of CHZ-O-Glc is required to reliably quantify CYP2E1 activity because of the rapid transformation of OH-CHZ into CHZ-O-Glc and identified the CHZ-N-Glc produced independently of the CYP2E1 activity. Our results also raise the questions of the contribution of CHZ-N-Glc in the overall CHZ metabolism and of the quantification of CHZ glucuronides in vitro and in vivo for measuring UGT1A activities.
Assuntos
Clorzoxazona/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronídeos/metabolismo , Hepatócitos/metabolismo , Animais , Clorzoxazona/análogos & derivados , Clorzoxazona/farmacocinética , Clorzoxazona/urina , Cromatografia Líquida de Alta Pressão , Meios de Cultura/análise , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Hidroxilação , Masculino , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Hepatic steatosis (i.e. lipid accumulation) and steatohepatitis have been related to diverse etiologic factors, including alcohol, obesity, environmental pollutants. However, no study has so far analyzed how these different factors might interplay regarding the progression of liver diseases. The impact of the co-exposure to the environmental carcinogen benzo[a]pyrene (B[a]P) and the lifestyle-related hepatotoxicant ethanol, was thus tested on in vitro models of steatosis (human HepaRG cell line; hybrid human/rat WIF-B9 cell line), and on an in vivo model (obese zebrafish larvae). Steatosis was induced prior to chronic treatments (14, 5 or 7 days for HepaRG, WIF-B9 or zebrafish, respectively). Toxicity and inflammation were analyzed in all models; the impact of steatosis and ethanol towards B[a]P metabolism was studied in HepaRG cells. Cytotoxicity and expression of inflammation markers upon co-exposure were increased in all steatotic models, compared to non steatotic counterparts. A change of B[a]P metabolism with a decrease in detoxification was detected in HepaRG cells under these conditions. A prior steatosis therefore enhanced the toxicity of B[a]P/ethanol co-exposure in vitro and in vivo; such a co-exposure might favor the appearance of a steatohepatitis-like state, with the development of inflammation. These deleterious effects could be partly explained by B[a]P metabolism alterations.
Assuntos
Benzo(a)pireno/efeitos adversos , Etanol/efeitos adversos , Fígado Gorduroso/patologia , Fígado/patologia , Animais , Biomarcadores/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Poluentes Ambientais/efeitos adversos , Fígado Gorduroso/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Larva/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Ratos , Peixe-ZebraRESUMO
Human exposure to bisphenol A (BPA) could favor obesity and related metabolic disorders such as hepatic steatosis. Investigations in rodents have shown that these deleterious effects are observed not only when BPA is administered during the adult life but also with different protocols of perinatal exposure. Whether perinatal BPA exposure could pose a risk in human is currently unknown, and thus appropriate in vitro models could be important to tackle this major issue. Accordingly, we determined whether long-term BPA treatment could induce steatosis in human HepaRG cells by using a protocol mimicking perinatal exposure. To this end, the kinetics of expression of seven proteins differentially expressed during liver development was determined during a 4-week period of cell culture required for proliferation and differentiation. By analogy with data reported in rodents and humans, our results indicated that the period of cell culture around day 15 and day 18 after seeding could be considered as the "natal" period. Consequently, HepaRG cells were treated for 3 weeks with BPA (from 0.2 to 2000 nM), with a treatment starting during the proliferating period. BPA was able to induce steatosis with a nonmonotonic dose response profile, with significant effects on neutral lipids and triglycerides observed for the 2 nM concentration. However, the expression of many enzymes involved in lipid and carbohydrate homeostasis was unchanged in exposed HepaRG cells. The expression of other potential BPA targets and enzymes involved in BPA biotransformation was also determined, giving answers as well as new questions regarding the mechanisms of action of BPA. Hence, HepaRG cells provide a valuable model that can prove useful for the toxicological assessment of endocrine disruptors on hepatic metabolisms, in particular in the developing liver. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1024-1036, 2017.
Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Exposição Ambiental , Fígado Gorduroso/induzido quimicamente , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Fenóis/toxicidade , Linhagem Celular , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/embriologia , Fígado/enzimologia , Fígado/metabolismo , Triglicerídeos/metabolismoRESUMO
Obesity and nonalcoholic fatty liver disease (NAFLD) can increase susceptibility to hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are poorly understood. For acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during NAFLD. The first aim of our study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, human HepaRG cells were incubated for one week with stearic acid or oleic acid, in the presence of different concentrations of insulin. Although cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids, CYP2E1 activity was significantly increased only by stearic acid. CYP2E1 activity was reduced by insulin and this effect was reproduced in cultured primary human hepatocytes. Next, APAP cytotoxicity was assessed in HepaRG cells with or without lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations showed that the loss of ATP and glutathione was almost always greater in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole, recovery of ATP was significantly higher in the presence of stearate with low (2.5mM) or high (20mM) concentrations of APAP. Levels of APAP-glucuronide were significantly enhanced by insulin. Hence, HepaRG cells can be used as a valuable model of NAFLD to unveil important metabolic and hormonal factors which can increase susceptibility to drug-induced hepatotoxicity.
Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismo , Linhagem Celular , Células Cultivadas , Indutores do Citocromo P-450 CYP2E1/toxicidade , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , HumanosRESUMO
The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay.
Assuntos
Linhagem Celular Tumoral , Dano ao DNA , Histonas/análise , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Aflatoxina B1/toxicidade , Benzo(a)pireno/toxicidade , Ensaio Cometa , DNA/efeitos dos fármacos , Endossulfano/toxicidade , Células Hep G2 , Histonas/metabolismo , Humanos , Testes para Micronúcleos , Fosforilação , Pirazóis/toxicidadeRESUMO
Xenobiotics, such as contaminants and drugs, can be converted to potentially toxic reactive metabolites by phase 1 oxidizing enzymes. These metabolites are further detoxified by phase 2 conjugating enzymes and eliminated from cells by phase 3 transporters. Moreover, many of these xenobiotics are also able to induce or inhibit these enzymes, potentially modulating their own toxicity or that of other chemicals. The present review is focused on bisphenol A, a synthetic monomer used for many industrial applications and exhibiting xenoestrogen properties. The impact of this contaminant on all major classes of metabolizing enzymes (i.e., cytochromes P450, glutathione-S-transferases, sulfotransferases, UDP-glucuronyltransferases, and transporters) was reviewed, with a highlight on the modulation of cytochromes P450 involved in steroid metabolism. Interestingly, most of the studies reported in this review show that BPA is able to induce or inhibit metabolizing enzymes at high doses but also at doses compatible with human exposure.
Assuntos
Compostos Benzidrílicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Glutationa Transferase/metabolismo , Fenóis/metabolismo , Sulfotransferases/metabolismo , Animais , Compostos Benzidrílicos/química , Humanos , Modelos Animais , Fenóis/química , Xenobióticos/metabolismoRESUMO
Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation.
Assuntos
Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Endossulfano/metabolismo , Metoxicloro/metabolismo , Linhagem Celular Tumoral , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP3A/genética , Células Hep G2 , Humanos , Microssomos Hepáticos/metabolismo , Praguicidas/metabolismo , Receptor de Pregnano X , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismoRESUMO
Although acetaminophen (APAP) is usually considered as a safe drug, this painkiller can lead to acute liver failure after overdoses. Moreover, there is evidence that the maximum recommended dosage can induce hepatic cytolysis in some individuals. Several predisposing factors appear to enhance the risk and severity of APAP-induced liver injury including chronic alcoholic liver disease and nonalcoholic fatty liver disease (NAFLD), which refers to a large spectrum of hepatic lesions linked to obesity. In contrast, obesity by itself does not seem to be associated with a higher risk of APAP-induced liver injury. Since 1987, seven studies dealt with APAP-induced hepatotoxicity in rodent models of NAFLD and five of them found that this liver disease was associated with higher APAP toxicity. Unfortunately, these studies did not unequivocally established the mechanism(s) whereby NAFLD could favour APAP hepatotoxicity, although some investigations suggested that pre-existent induction of hepatic cytochrome P450 2E1 (CYP2E1) could play a significant role by increasing the generation of N-acetyl-p-benzoquinone imine (NAPQI), the toxic metabolite of APAP. Moreover, pre-existent mitochondrial dysfunction associated with NAFLD could also be involved. In contrast, some investigations suggested that factors that could reduce the risk and severity of APAP hepatotoxicity in obesity and NAFLD include higher hepatic APAP glucuronidation, reduced CYP3A4 activity and increased volume of body distribution. Thus, the occurrence and the outcome of APAP-induced liver injury in an obese individual with NAFLD might depend on a delicate balance between metabolic factors that can be protective and others that favour large hepatic levels of NAPQI.
Assuntos
Acetaminofen/efeitos adversos , Benzoquinonas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Iminas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade Abdominal/metabolismo , Acetaminofen/metabolismo , Animais , Benzoquinonas/toxicidade , Citocromo P-450 CYP3A/metabolismo , Humanos , Iminas/toxicidade , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade Abdominal/complicações , RatosRESUMO
Drinking water can be contaminated with pharmaceuticals. However, it is uncertain whether this contamination can be harmful for the liver, especially during obesity. Hence, the goal of our study was to determine whether chronic exposure to low doses of pharmaceuticals could have deleterious effects on livers of lean and obese mice. To this end, lean and ob/ob male mice were treated for 4 months with a mixture of 11 drugs provided in drinking water at concentrations ranging from 10 to 106 ng/l. At the end of the treatment, some liver and plasma abnormalities were observed in ob/ob mice treated with the cocktail containing 106 ng/l of each drug. For this dosage, a gene expression analysis by microarray showed altered expression of circadian genes (e.g. Bmal1, Dbp, Cry1) in lean and obese mice. RT-qPCR analyses carried out in all groups of animals confirmed that expression of 8 different circadian genes was modified in a dose-dependent manner. For some genes, a significant modification was observed for dosages as low as 10²-10³ ng/l. Drug mixture and obesity presented an additive effect on circadian gene expression. These data were validated in an independent study performed in female mice. Thus, our study showed that chronic exposure to trace pharmaceuticals disturbed hepatic expression of circadian genes, particularly in obese mice. Because some of the 11 drugs can be found in drinking water at such concentrations (e.g. acetaminophen, carbamazepine, ibuprofen) our data could be relevant in environmental toxicology, especially for obese individuals exposed to these contaminants.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Proteínas Circadianas Period/metabolismo , Preparações Farmacêuticas/administração & dosagem , Poluentes Químicos da Água/administração & dosagem , Fatores de Transcrição ARNTL/agonistas , Fatores de Transcrição ARNTL/antagonistas & inibidores , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Criptocromos/agonistas , Criptocromos/antagonistas & inibidores , Criptocromos/genética , Criptocromos/metabolismo , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/induzido quimicamente , Obesidade/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Circadianas Period/agonistas , Proteínas Circadianas Period/antagonistas & inibidores , Proteínas Circadianas Period/genética , Testes de Toxicidade Crônica , Fatores de Transcrição/agonistas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Poluentes Químicos da Água/toxicidadeRESUMO
The worldwide epidemic of obesity and insulin resistance favors nonalcoholic fatty liver disease (NAFLD). Insulin resistance (IR) in the adipose tissue increases lipolysis and the entry of nonesterified fatty acids (NEFAs) in the liver, whereas IR-associated hyperinsulinemia promotes hepatic de novo lipogenesis. However, several hormonal and metabolic adaptations are set up in order to restrain hepatic fat accumulation, such as increased mitochondrial fatty acid oxidation (mtFAO). Unfortunately, these adaptations are usually not sufficient to reduce fat accumulation in liver. Furthermore, enhanced mtFAO without concomitant up-regulation of the mitochondrial respiratory chain (MRC) activity induces reactive oxygen species (ROS) overproduction within different MRC components upstream of cytochrome c oxidase. This event seems to play a significant role in the initiation of oxidative stress and subsequent development of nonalcoholic steatohepatitis (NASH) in some individuals. Experimental investigations also pointed to a progressive reduction of MRC activity during NAFLD, which could impair energy output and aggravate ROS overproduction by the damaged MRC. Hence, developing drugs that further increase mtFAO and restore MRC activity in a coordinated manner could ameliorate steatosis, but also necroinflammation and fibrosis by reducing oxidative stress. In contrast, physicians should be aware that numerous drugs in the current pharmacopoeia are able to induce mitochondrial dysfunction, which could aggravate NAFLD in some patients.
Assuntos
Adaptação Fisiológica/fisiologia , Fígado Gorduroso/fisiopatologia , Mitocôndrias Hepáticas/fisiologia , Animais , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Humanos , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Hepatopatia Gordurosa não AlcoólicaRESUMO
UNLABELLED: Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with an altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F-actin. Inhibition of [3H]-taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N-acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ-induced bile acid (BA) accumulation. Moreover, 24-hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na+ -dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance-associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down-regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up-regulated BSEP and MDR3 in the absence of oxidative stress. CONCLUSION: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ-induced intrahepatic cholestasis in human hepatocytes.
Assuntos
Carcinoma Hepatocelular/patologia , Clorpromazina/efeitos adversos , Colestase/induzido quimicamente , Colestase/fisiopatologia , Neoplasias Hepáticas/patologia , Estresse Oxidativo/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Actinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Clorpromazina/farmacologia , Colestase/metabolismo , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ácido Taurocólico/metabolismoRESUMO
Clinical investigations suggest that hepatotoxicity after acetaminophen (APAP) overdose could be more severe in the context of obesity and nonalcoholic fatty liver disease. The pre-existence of fat accumulation and CYP2E1 induction could be major mechanisms accounting for such hepatic susceptibility. To explore this issue, experiments were performed in obese diabetic ob/ob and db/db mice. Preliminary investigations performed in male and female wild-type, ob/ob, and db/db mice showed a selective increase in hepatic CYP2E1 activity in female db/db mice. However, liver triglycerides in these animals were significantly lower compared with ob/ob mice. Next, APAP (500 mg/kg) was administered in female wild-type, ob/ob, and db/db mice, and investigations were carried out 0.5, 2, 4, and 8 h after APAP intoxication. Liver injury 8 h after APAP intoxication was higher in db/db mice, as assessed by plasma transaminases, liver histology, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. In db/db mice, however, the extent of hepatic glutathione depletion, levels of APAP-protein adducts, c-Jun N-terminal kinase activation, changes in gene expression, and mitochondrial DNA levels were not greater compared with the other genotypes. Furthermore, in the db/db genotype plasma lactate and ß-hydroxybutyrate were not specifically altered, whereas the plasma levels of APAP-glucuronide were intermediary between wild-type and ob/ob mice. Thus, early APAP-induced hepatotoxicity was greater in db/db than ob/ob mice, despite less severe fatty liver and similar basal levels of transaminases. Hepatic CYP2E1 induction could have an important pathogenic role when APAP-induced liver injury occurs in the context of obesity and related metabolic disorders.
Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Ácido 3-Hidroxibutírico/sangue , Acetaminofen/sangue , Animais , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citocromo P-450 CYP2E1/metabolismo , DNA Mitocondrial/metabolismo , Fígado Gorduroso/sangue , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Glucuronídeos/sangue , Glutationa/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Ácido Láctico/sangue , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Obesidade/sangue , Obesidade/patologia , Sulfatos/sangue , Triglicerídeos/sangueRESUMO
A frequent mechanism for drug-induced liver injury (DILI) is the formation of reactive metabolites that trigger hepatitis through direct toxicity or immune reactions. Both events cause mitochondrial membrane disruption. Genetic or acquired factors predispose to metabolite-mediated hepatitis by increasing the formation of the reactive metabolite, decreasing its detoxification, or by the presence of critical human leukocyte antigen molecule(s). In other instances, the parent drug itself triggers mitochondrial membrane disruption or inhibits mitochondrial function through different mechanisms. Drugs can sequester coenzyme A or can inhibit mitochondrial ß-oxidation enzymes, the transfer of electrons along the respiratory chain, or adenosine triphosphate (ATP) synthase. Drugs can also destroy mitochondrial DNA, inhibit its replication, decrease mitochondrial transcripts, or hamper mitochondrial protein synthesis. Quite often, a single drug has many different effects on mitochondrial function. A severe impairment of oxidative phosphorylation decreases hepatic ATP, leading to cell dysfunction or necrosis; it can also secondarily inhibit ß-oxidation, thus causing steatosis, and can also inhibit pyruvate catabolism, leading to lactic acidosis. A severe impairment of ß-oxidation can cause a fatty liver; further, decreased gluconeogenesis and increased utilization of glucose to compensate for the inability to oxidize fatty acids, together with the mitochondrial toxicity of accumulated free fatty acids and lipid peroxidation products, may impair energy production, possibly leading to coma and death. Susceptibility to parent drug-mediated mitochondrial dysfunction can be increased by factors impairing the removal of the toxic parent compound or by the presence of other medical condition(s) impairing mitochondrial function. New drug molecules should be screened for possible mitochondrial effects.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Respiração Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , DNA Mitocondrial/biossíntese , DNA Mitocondrial/efeitos dos fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Sistema Imunitário/metabolismo , Fígado/patologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Mitocôndrias Hepáticas/ultraestrutura , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/efeitos dos fármacos , Oxirredução , Preparações Farmacêuticas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24h exposure of these hepatocyte models to 0.05 and 0.25µM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds.
Assuntos
Aflatoxina B1/toxicidade , Dano ao DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Aflatoxina B1/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Reparo do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Hepatócitos/patologia , Humanos , Mutagênicos/administração & dosagem , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although carbon tetrachloride (CCl(4))-induced acute and chronic hepatotoxicity have been extensively studied, little is known about the very early in vivo effects of this organic solvent on oxidative stress and mitochondrial function. In this study, mice were treated with CCl(4) (1.5 ml/kg ie 2.38 g/kg) and parameters related to liver damage, lipid peroxidation, stress/defense and mitochondria were studied 3 h later. Some CCl(4)-intoxicated mice were also pretreated with the cytochrome P450 2E1 inhibitor diethyldithiocarbamate or the antioxidants Trolox C and dehydroepiandrosterone. CCl(4) induced a moderate elevation of aminotransferases, swelling of centrilobular hepatocytes, lipid peroxidation, reduction of cytochrome P4502E1 mRNA levels and a massive increase in mRNA expression of heme oxygenase-1 and heat shock protein 70. Moreover, CCl(4) intoxication induced a severe decrease of mitochondrial respiratory chain complex IV activity, mitochondrial DNA depletion and damage as well as ultrastructural alterations. Whereas DDTC totally or partially prevented all these hepatic toxic events, both antioxidants protected only against liver lipid peroxidation and mitochondrial damage. Taken together, our results suggest that lipid peroxidation is primarily implicated in CCl(4)-induced early mitochondrial injury. However, lipid peroxidation-independent mechanisms seem to be involved in CCl(4)-induced early hepatocyte swelling and changes in expression of stress/defense-related genes. Antioxidant therapy may not be an efficient strategy to block early liver damage after CCl(4) intoxication.
Assuntos
Intoxicação por Tetracloreto de Carbono/metabolismo , Hepatócitos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Animais , Antioxidantes/farmacologia , Tetracloreto de Carbono , Cromanos/farmacologia , Inibidores do Citocromo P-450 CYP2E1 , Desidroepiandrosterona/farmacologia , Ditiocarb/farmacologia , Masculino , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacosRESUMO
There has been growing evidence that phase I metabolizing enzymes cytochromes P450 (CYPs) are not only located in the endoplasmic reticulum but also in other subcellular compartments and particularly in mitochondria. The presence of CYPs in these organelles raises questions regarding their metabolic role and their possible deleterious effects on the respiratory chain complexes and mitochondrial DNA. This review will focus on one particular CYP, CYP2E1, which represents a significant source of reactive oxygen species and is involved in the metabolism of small molecule substrates including ethanol, drugs and carcinogens. Since hepatic CYP2E1 expression is increased in different physiopathological situations such as type 2 diabetes, obesity and ethanol intoxication, the presence of significant levels of this CYP within the mitochondria could have major deleterious effects. This review recalls the main data that brought to the fore the presence of CYP2E1 in mitochondria and the mechanism of its targeting in this organelle. The potential pathological consequences linked to the presence of CYP2E1 in mitochondria will be subsequently discussed.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Citocromo P-450 CYP2E1/metabolismo , Etanol/toxicidade , Mitocôndrias/metabolismo , Obesidade/fisiopatologia , Estresse Oxidativo , Animais , Anti-Infecciosos Locais/toxicidade , Humanos , Transporte Proteico , Espécies Reativas de OxigênioRESUMO
UNLABELLED: Drug-induced liver injury occurs in general after several weeks and is often unpredictable. It is characterized by a large spectrum of lesions that includes steatosis and phospholipidosis. Many drugs such as amiodarone and tetracycline have been reported to cause phospholipidosis and/or steatosis. In this study, acute and chronic hepatic effects of these two drugs were investigated using well-differentiated human hepatoma HepaRG cells. Accumulation of typical lipid droplets, labeled with Oil Red O, was observed in hepatocyte-like HepaRG cells after repeat exposure to either drug. Amiodarone caused the formation of additional intracytoplasmic vesicles that did not stain in all HepaRG cells. At the electron microscopic level, these vesicles appeared as typical lamellar bodies and were associated with an increase of phosphatidylethanolamine and phosphatidylcholine. A dose-dependent induction of triglycerides (TG) was observed after repeat exposure to either amiodarone or tetracycline. Several genes known to be related to lipogenesis were induced after treatment by these two drugs. By contrast, opposite deregulation of some of these genes (FASN, SCD1, and THSRP) was observed in fat HepaRG cells induced by oleic acid overload, supporting the conclusion that different mechanisms were involved in the induction of steatosis by drugs and oleic acid. Moreover, several genes related to lipid droplet formation (ADFP, PLIN4) were up-regulated after exposure to both drugs and oleic acid. CONCLUSION: Our results show that amiodarone causes phospholipidosis after short-term treatment and, like tetracycline, induces vesicular steatosis after repeat exposure in HepaRG cells. These data represent the first demonstration that drugs can induce vesicular steatosis in vitro and show a direct relationship between TG accumulation and enhanced expression of lipogenic genes.
Assuntos
Amiodarona/farmacologia , Carcinoma Hepatocelular/metabolismo , Fígado Gorduroso/metabolismo , Lipogênese/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Tetraciclina/farmacologia , Regulação para Cima/efeitos dos fármacos , Amiodarona/efeitos adversos , Carcinoma Hepatocelular/patologia , Proteínas de Transporte , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação para Baixo/fisiologia , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/patologia , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipogênese/genética , Lipogênese/fisiologia , Neoplasias Hepáticas/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ácido Oleico/farmacologia , Perilipina-2 , Perilipina-4 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Tetraciclina/efeitos adversos , Triglicerídeos/metabolismo , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
The role of hepatocyte apoptosis in the physiopathology of obstructive cholestasis is still controversial. Although some data have strongly suggested that hepatocellular cholestatic injury is due to Fas-mediated hepatocyte apoptosis, some others concluded that necrosis, rather than apoptosis, represents the main type of hepatocyte death in chronic cholestasis. Moreover, it has also been suggested that the reduced liver injury observed in the absence of Fas receptor after bile duct ligation was not due to lower hepatocyte apoptosis but to the indirect role of this receptor in non-hepatocytic cells such as cholangiocytes and inflammatory cells. The aim of this work was therefore to determine whether a protection against cell death limited to hepatocytes could be sufficient to reduce liver injury and delay cholestatic fibrosis. With this purpose, we performed bile duct ligation in transgenic mice overexpressing Bcl-2 in hepatocytes and in wild-type littermates. We found that, compared with necrosis, apoptosis was negligible in this model. Our results also showed that hepatocyte Bcl-2 expression protected hepatocytes against liver injury only in the early steps of the disease. This protection was correlated with reduced mitochondrial dysfunction and lipid peroxidation. However, in contrast to Fas receptor-deficient lpr mice, fibrosis progression was not hampered and liver inflammatory response was not reduced by Bcl-2 overexpression. These results therefore comfort the hypothesis that Fas-mediated apoptotic hepatocyte pathway is not a significant contributing factor to the clinical features observed in cholestasis. Moreover, in the absence of a blunted inflammatory response in transgenic mice, Bcl-2 protection against hepatocyte mitochondrial dysfunction and lipid peroxidation was not sufficient to block fibrosis progression.