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The characteristics and fate of cancer cells partly depend on their environmental stiffness, i.e., the local mechanical cues they face. HepaRG progenitors are liver carcinoma cells exhibiting transdifferentiation properties; however, the underlying mechanisms remain unknown. To evaluate the impact of external physical forces mimicking the tumor microenvironment, we seeded them at very high density for 20 h, keeping the cells round and unanchored to the substrate. Applied without corticoids, spatial confinement due to very high density induced reprogramming of HepaRG cells into stable replicative stem-like cells after replating at normal density. Redifferentiation of these stem-like cells into cells very similar to the original HepaRG cells was then achieved using the same stress but in the presence of corticoids. This demonstrates that the cells retained the memory required to run the complete hepatic differentiation program, after bypassing the Hayflick limit twice. We show that physical stress improved chromosome quality and genomic stability, through greater efficiency of DNA repair and restoration of telomerase activity, thus enabling cells to escape progression to a more aggressive cancer state. We also show the primary importance of high-density seeding, possibly triggering compressive stress, in these processes, rather than that of cell roundness or intracellular tensional signals. The HepaRG-derived lines established here considerably extend the lifespan and availability of this surrogate cell system for mature human hepatocytes. External physical stress is a promising way to create a variety of cell lines, and it paves the way for the development of strategies to improve cancer prognosis.
Assuntos
Transdiferenciação Celular , Longevidade , Humanos , Diferenciação Celular , Linhagem Celular , Sinais (Psicologia)RESUMO
Drug-induced intrahepatic cholestasis is characterized by cellular accumulation of bile acids (BAs), whose mechanisms remain poorly understood. The present study aimed to analyze early and progressive alterations of BA profiles induced by cyclosporine A, chlorpromazine, troglitazone, tolcapone, trovafloxacin, and tacrolimus after 4-hour, 24-hour, and 6-day treatments of differentiated HepaRG cells. In BA-free medium, the potent cholestatic drugs cyclosporine A, chlorpromazine, and troglitazone reduced endogenous BA synthesis after 24 hours, whereas the rarely cholestatic drugs tolcapone, trovafloxacin, and tacrolimus reduced BA synthesis only after 6 days. In the presence of physiologic serum BA concentrations, cyclosporine A, chlorpromazine, and troglitazone induced early and preferential cellular accumulation of unconjugated lithocholic, deoxycholic, and chenodeoxycholic acids that increased 8- to 12-fold and 47- to 50-fold after 24 hours and 6 days, respectively. Accumulation of these hydrophobic BAs resulted from strong inhibition of amidation, and in addition, for lithocholic acid reduction of its sulfoconjugation, and was associated with variable alterations of uptake and efflux transporters. Trovafloxacin also caused BA accumulation, especially after 6 days, whereas tolcapone and tacrolimus were still without effect. However, when exogenous BAs were added to the medium at cholestatic serum concentrations, a 6-day treatment with all drugs resulted in cellular BA accumulation with higher folds of chenodeoxycholic and lithocholic acids. At the tested concentration, tolcapone had the lowest effect. These results bring the first demonstration that major cholestatic drugs can cause preferential and progressive in vitro cellular accumulation of unconjugated toxic hydrophobic BAs and bring new insights into mechanisms involved in drug-induced cellular accumulation of toxic BAs.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase/metabolismo , Amidas/metabolismo , Linhagem Celular , Ácido Quenodesoxicólico/metabolismo , Colestase/induzido quimicamente , Ácido Desoxicólico/metabolismo , Humanos , Ácido Litocólico/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Sulfatos/metabolismo , Simportadores/metabolismo , Ácido Taurocólico/metabolismoRESUMO
Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs.
Assuntos
Diferenciação Celular/fisiologia , Hepatócitos/citologia , Fígado/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , HumanosRESUMO
Intrahepatic cholestasis represents 20%-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na+-taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis.
Assuntos
Canalículos Biliares/metabolismo , Colestase Intra-Hepática/metabolismo , Fígado/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Colestase Intra-Hepática/induzido quimicamente , Humanos , Fígado/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ácido Taurocólico/metabolismoRESUMO
AIM: The hepatoma-derived cell line HepaRG is regarded as an in vitro model of drug metabolism because fully differentiated HepaRG cells demonstrate functional metabolic responses comparable to those of primary human hepatocytes. Recently, it was demonstrated that the 3D culture of HepaRG cells enhanced their metabolic functions and toxicological responses. We approached the mechanisms underlying these enhancement effects. METHODS: We compared 2D-cultured HepaRG cells with 3D-cultured HepaRG spheroids in the gene expression patterns and the metabolic functions. In the present study, we performed 3D culture of HepaRG cells using functional polymers (FP). To reveal the in vivo differentiation ability, we transplanted the 3D-cultured HepaRG spheroids into TK-NOG mice. RESULTS: A comparison between 2D and 3D cultures revealed that 3D-cultured HepaRG spheroids demonstrated reductions in bile duct marker expression, accelerated expression of cytochrome P450 3A4, and increases in the ratio of albumin-expressing hepatocytes. Furthermore, catalytic activities of cytochrome P450 3A4 were modified by omeprazole and rifampicin in the 3D-cultured HepaRG spheroids. Transplantation analysis revealed that 3D-cultured HepaRG spheroids formed hepatocyte-like colonies rather than cholangiocytes in vivo. CONCLUSION: Our results indicated that the enhancement of hepatic functions in 3D-cultured HepaRG cells was induced by selective hepatocyte differentiation and accelerated hepatocyte maturation. HepaRG spheroids reproduced the metabolic responses of human hepatocytes. Therefore, FP-dependent 3D-cultured HepaRG cells may serve as an excellent in vitro model for evaluating the hepatic metabolism and toxicity.
RESUMO
BACKGROUND & AIMS: Hepatocyte-like cells, differentiated from different stem cell sources, are considered to have a range of possible therapeutic applications, including drug discovery, metabolic disease modelling, and cell transplantation. However, little is known about how stem cells differentiate into mature and functional hepatocytes. METHODS: Using transcriptomic screening, a transcription factor, liver X receptor α (NR1H3), was identified as increased during HepaRG cell hepatogenesis; this protein was also upregulated during embryonic stem cell and induced pluripotent stem cell differentiation. RESULTS: Overexpressing NR1H3 in human HepaRG cells promoted hepatic maturation; the hepatocyte-like cells exhibited various functions associated with mature hepatocytes, including cytochrome P450 (CYP) enzyme activity, secretion of urea and albumin, upregulation of hepatic-specific transcripts and an increase in glycogen storage. Importantly, the NR1H3-derived hepatocyte-like cells were able to rescue lethal fulminant hepatic failure using a non-obese diabetic/severe combined immunodeficient mouse model. CONCLUSIONS: In this study, we found that NR1H3 accelerates hepatic differentiation through an HNF4α-dependent reciprocal network. This contributes to hepatogenesis and is therapeutically beneficial to liver disease.
Assuntos
Diferenciação Celular/fisiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/fisiologia , Receptores Nucleares Órfãos/fisiologia , Células-Tronco/fisiologia , Animais , Tetracloreto de Carbono/efeitos adversos , Linhagem Celular , Transplante de Células , Modelos Animais de Doenças , Hepatócitos/citologia , Humanos , Técnicas In Vitro , Falência Hepática/induzido quimicamente , Falência Hepática/terapia , Regeneração Hepática/fisiologia , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco/citologiaRESUMO
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
Several factors are thought to be implicated in the occurrence of idiosyncratic adverse drug reactions. The present work aimed to question as to whether inflammation is a determinant factor in hepatic lesions induced by chlorpromazine (CPZ) using the human HepaRG cell line. An inflammation state was induced by a 24-hour exposure to proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß; then the cells were simultaneously treated with CPZ and/or cytokine for 24 hours or daily for 5 days. The inflammatory response was assessed by induction of C-reactive protein and IL-8 transcripts and proteins as well as inhibition of CPZ metabolism and down-regulation of cytochrome 3A4 (CYP3A4) and CYP1A2 transcripts, two major cytochrome P450 (P450) enzymes involved in its metabolism. Most effects of cotreatments with cytokines and CPZ were amplified or only observed after five daily treatments; they mainly included increased cytotoxicity and overexpression of oxidative stress-related genes, decreased Na(+)-taurocholate cotransporting polypeptide mRNA levels and activity, a key transporter involved in bile acids uptake, and deregulation of several other transporters. However, CPZ-induced inhibition of taurocholic acid efflux and pericanalicular F-actin distribution were not affected. In addition, a time-dependent induction of phospholipidosis was noticed in CPZ-treated cells, without obvious influence of the inflammatory stress. In summary, our results show that an inflammatory state induced by proinflammatory cytokines increased cytotoxicity and enhanced some cholestatic features induced by the idiosyncratic drug CPZ in HepaRG cells. These changes, together with inhibition of P450 activities, could have important consequences if extrapolated to the in vivo situation.
Assuntos
Clorpromazina/efeitos adversos , Colestase/metabolismo , Inflamação/metabolismo , Actinas/genética , Actinas/metabolismo , Ácidos e Sais Biliares/genética , Ácidos e Sais Biliares/metabolismo , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Linhagem Celular , Colestase/induzido quimicamente , Colestase/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação para Baixo/genética , Humanos , Inflamação/genética , Interleucinas/genética , Interleucinas/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Estresse Oxidativo/genética , RNA Mensageiro/genética , Simportadores/genética , Simportadores/metabolismo , Ácido Taurocólico/genética , Ácido Taurocólico/metabolismoRESUMO
Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated by H. Yamazaki et al. ( ( 2013 ) Chem. Res. Toxicol. 26 , 486 - 489 ) using the model substrate midazolam in various in vitro and in vivo models. Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pretreatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting the activation of upstream transcription factors involved in detoxication, e.g., the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of coregulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 coregulator-derived nuclear receptor-interaction motifs and coregulator and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5-hydroxythalidomide displayed significant modulation of coregulator interaction with PXR and CAR ligand-binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Hepatócitos/metabolismo , Receptores de Esteroides/metabolismo , Talidomida/farmacologia , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biotransformação , Linhagem Celular , Células Cultivadas , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Humanos , Receptor de Pregnano X , Análise Serial de Proteínas , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
1. Humanized-liver mice, in which the liver has been repopulated with human hepatocytes, have been used to study aspects of human liver physiology such as drug metabolism, toxicology and hepatitis infection. However, the procurement of human hepatocytes is a major problem in producing humanized-liver mice because of the finite nature of the patient-derived resource. 2. In order to overcome this limitation, the human hepatic cell line HepaRG® were evaluated as promising donor cells for liver reconstitution in the TK-NOG mouse model. 3. We demonstrate that, in vivo, transplanted confluent culture or differentiated HepaRG® cells proliferated and differentiated toward both hepatocyte-like and biliary-like cells within the recipient liver. In contrast, proliferative HepaRG® cells could engraft TK-NOG mouse liver but could differentiate only toward biliary-like cells. The differentiation to hepatocyte-like cells was characterized by the detection of human albumin in the recipient mouse serum and was confirmed by immunohistochemical staining for human leukocyte antigen, human albumin, cytochrome P450 3A4, and multidrug resistance-associated protein 2. Biliary-like cells were characterized by positive staining for cytokeratin-19. 4. These results indicated that the differentiated HepaRG® cells are a possible cell source for generating humanized-liver mice, which are a useful model for in vivo studies of liver physiology.
Assuntos
Transplante de Células/métodos , Hepatócitos/fisiologia , Fígado/citologia , Fígado/fisiologia , Albuminas/análise , Animais , Testes de Carcinogenicidade , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Feminino , Hepatócitos/citologia , Hepatócitos/transplante , Humanos , Inativação Metabólica/genética , Masculino , Camundongos , Camundongos Transgênicos , Quimeras de TransplanteRESUMO
In this article, we present a liver-kidney co-culture model in a micro fluidic biochip. The liver was modeled using HepG2/C3a and HepaRG cell lines and the kidney using MDCK cell lines. To demonstrate the synergic interaction between both organs, we investigated the effect of ifosfamide, an anticancerous drug. Ifosfamide is a prodrug which is metabolized by the liver to isophosforamide mustard, an active metabolite. This metabolism process also leads to the formation of chloroacetaldehyde, a nephrotoxic metabolite and acrolein a urotoxic one. In the biochips of MDCK cultures, we did not detect any nephrotoxic effects after 72 h of 50 µM ifosfamide exposure. However, in the liver-kidney biochips, the same 72 h exposure leads to a nephrotoxicity illustrated by a reduction of the number of MDCK cells (up to 30% in the HepaRG-MDCK) when compared to untreated co-cultures or treated MDCK monocultures. The reduction of the MDCK cell number was not related to a modification of the cell cycle repartition in ifosfamide treated cases when compared to controls. The ifosfamide biotransformation into 3-dechloroethylifosfamide, an equimolar byproduct of the chloroacetaldehyde production, was detected by mass spectrometry at a rate of apparition of 0.3 ± 0.1 and 1.1 ± 0.3 pg/h/biochips in HepaRG monocultures and HepaRG-MDCK co-cultures respectively. Any metabolite was detected in HepG2/C3a cultures. Furthermore, the ifosfamide treatment in HepaRG-MDCK co-culture system triggered an increase in the intracellular calcium release in MDCK cells on contrary to the treatment on MDCK monocultures. As 3-dechloroethylifosfamide is not toxic, we have tested the effect of equimolar choloroacetaldehyde concentration onto the MDCK cells. At this concentration, we found a quite similar calcium perturbation and MDCK nephrotoxicity via a reduction of 30% of final cell numbers such as in the ifosfamide HepaRG-MDCK co-culture experiments. Our results suggest that ifosfamide nephrotoxicity in a liver-kidney micro fluidic co-culture model using HepaRG-MDCK cells is induced by the metabolism of ifosfamide into chloroacetaldehyde whereas this pathway is not functional in HepG2/C3a-MDCK model. This study demonstrates the interest in the development of systemic organ-organ interactions using micro fluidic biochips. It also illustrated their potential in future predictive toxicity model using in vitro models as alternative methods.
Assuntos
Técnicas de Cocultura/métodos , Ifosfamida/toxicidade , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Técnicas Analíticas Microfluídicas/métodos , Análise Serial de Tecidos/métodos , Acetaldeído/análogos & derivados , Acetaldeído/toxicidade , Animais , Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Cães , Corantes Fluorescentes , Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Rim/citologia , Fígado/citologia , Células Madin Darby de Rim Canino , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.
Assuntos
Proliferação de Células , Hepatócitos/enzimologia , Regeneração Hepática , Fígado/enzimologia , Regiões Promotoras Genéticas , Telomerase/genética , Ativação Transcricional , Animais , Sítios de Ligação , Diferenciação Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Fator de Transcrição E2F2/metabolismo , Fator de Transcrição E2F7/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Hepatectomia , Humanos , Óperon Lac , Fígado/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Interferência de RNA , Elementos Reguladores de Transcrição , Proteína do Retinoblastoma/metabolismo , Telomerase/metabolismo , Fatores de TempoRESUMO
Fatty acid microsomal ω-oxidation involves cytochrome P450 enzymes. Some of them belonging to the CYP4F3 family are mainly expressed in the liver, making this organ a major player in energy homeostasis and lipid metabolism. To study this important regulation pathway, we used HepaRG cells, which gradually undergo a complete differentiation process. Even at the early stage of the differentiation process, CYP4F3B generated by alternative splicing of the CYP4F3 gene represented the prevalent isoform in HepaRG cells as in the liver. Its increasing expression associated with hepatocyte differentiation status suggested a hepatic-specific control of this isoform. As in liver microsomes, the catalytic hydroxylation of the CYP4F3B substrate [1-¹4C]Z9(10)-epoxystearic acid led to major production of 18-hydroxy-9(10)-epoxystearic acid. When treated with saturated, monounsaturated, or polyunsaturated fatty acids, CYP4F3B and CYP4A11 expression remained unchanged whereas CYP4F2 and CYP4F12 expression was transiently up-regulated. A 24-h exposure of differentiated HepaRG cells to various polyunsaturated fatty acids and derivatives induced microvesicular steatosis; down-regulation of lipid metabolism gene regulators such as sterol regulatory element-binding protein-1c, fatty acid synthase, peroxisome proliferator-activated receptor γ (PPARγ), PPARα, and decreased expression of glucose-dependent metabolism genes, which could limit de novo lipogenesis. Docosahexaenoic acid seemed to be the most effective compound. These results suggest that a PPARα-independent pathway could participate to limit lipogenesis and emphasize the role of hepatocytes in the fatty acid ω-hydroxylation pathway. They also give insights on the use of HepaRG hepatocytes to open new avenues of investigations on factors mediating the lipid metabolic pathway and finding new hypolipidemic molecules.
Assuntos
Diferenciação Celular/fisiologia , Sistema Enzimático do Citocromo P-450/biossíntese , Ácidos Graxos/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Processamento Alternativo , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450 , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Regulação Enzimológica da Expressão Gênica , Hepatócitos/enzimologia , Humanos , Hidroxilação , Metabolismo dos Lipídeos , Lipogênese , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Isoformas de Proteínas , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Current developments in tissue engineering and microtechnology fields allow the use of microfluidic biochip as microtools for in vitro investigations. In the present study, we describe the behavior of HepG2/C3a cells cultivated in a poly(dimethylsiloxane) (PDMS) microfluidic biochip coupled to a perfusion system. Cell culture in the microfluidic biochip for 96 h including 72 h of perfusion provoked a 24 h delay in cell growth compared to plate cultures. Inside the microfluidic biochip, few apoptosis, and necrosis were detected along the culture and 3D cell organization was observed. Regarding the hepatic metabolism, glucose and glutamine consumptions as well as albumin synthesis were maintained. A transcriptomic analysis performed at 96 h of culture using Affymetrix GeneChip demonstrated that 1,025 genes with a fold change above 1.8 were statistically differentially expressed in the microfluidic biochip cultures compared to plate cultures. Among those genes, phase I enzymes involved in the xenobiotic's metabolism such as the cytochromes P450 (CYP) 1A1/2, 2B6, 3A4, 3A5, and 3A7 were up-regulated. The CYP1A1/2 up-regulation was associated with the appearance of CYP1A1/2's activity evidenced by using EROD biotransformation assay. Several phase II enzymes such as sulfotransferases (SULT1A1 and SULT1A2), UDP-glucuronyltransferase (UGT1A1, UGT2B7) and phase III transporters (such as MDR1, MRP2) were also up-regulated. In conclusion, microfluidic biochip could and provide an important insight to exploring the xenobiotic's metabolism. Altogether, these results suggest that this kind of biochip could be considered as a new pertinent tool for predicting cell toxicity and clearance of xenobiotics in vitro.
Assuntos
Hepatócitos/fisiologia , Microfluídica/métodos , Engenharia Tecidual/métodos , Albuminas/metabolismo , Morte Celular , Linhagem Celular , Sobrevivência Celular , Dimetilpolisiloxanos , Perfilação da Expressão Gênica , Glucose/metabolismo , Glutamina/metabolismo , Hepatócitos/metabolismo , Humanos , NylonsRESUMO
BACKGROUND & AIMS: Liver resection includes temporal vascular inflow occlusion resulting in ischemia/reperfusion injury in the remnant liver. Here, we developed a rat model of selective lobe occlusion to isolate reperfusion stress from ischemia and to analyze its effect on liver regeneration. METHODS: Left lateral and median lobes of liver were either mobilized or subjected twice for 10min to ischemia followed by 5min reperfusion prior to resection while the regenerative lobes were only subjected to reperfusion. RESULTS: Although intermittent reperfusion stress induced higher levels of serum transaminases, analysis of cell cycle regulators revealed accelerated regenerative response compared to standard partial hepatectomy. The G0/G1 transition occurred before tissue resection, as evidenced by c-fos, junB, and IL-6 induction. Following hepatectomy, Cyclin D1 up-regulation, G1/S transition, and cell division occurred earlier than normal. Unexpectedly, liver mobilization, a component of the clamping procedure, also resulted in earlier G1/S transition. The shortened G1-phase was driven by the c-Jun N-terminal Kinase pathway and was associated with an oxidative stress response as evidenced by the expression of inducible nitric oxide synthase. CONCLUSION: Intermittent selective clamping of lobes to be resected induced reperfusion stress on remnant liver that was beneficial for liver regeneration, suggesting this procedure could be applied in clinical practice.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Regeneração Hepática/fisiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Estresse Fisiológico/fisiologia , Animais , Divisão Celular/fisiologia , Ciclina D1/genética , Fase G1/fisiologia , Expressão Gênica/fisiologia , Heme Oxigenase-1/metabolismo , Hepatectomia/métodos , Hepatócitos/citologia , Hepatócitos/metabolismo , Interleucina-6/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Ratos , Ratos Sprague-Dawley , Fase de Repouso do Ciclo Celular/fisiologia , Fase S/fisiologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Superóxido Dismutase/metabolismo , Instrumentos CirúrgicosRESUMO
UNLABELLED: Liver regeneration is a unique process to restore hepatic homeostasis through rapid and synchronous proliferation of differentiated hepatocytes. Previous studies have shown that hepatocyte proliferation is characterized by high expression levels of the "mitotic" cyclin-dependent kinase 1 (Cdk1) during S-phase compared to other mammalian cells. In the light of findings showing that Cdk1 compensates for the loss of Cdk2 and drives S-phase in Cdk2-deficient cells derived from Cdk2 knockout mice, we took advantage of the models of liver regeneration following partial hepatectomy and primary cultures of normal rat hepatocytes to further examine the involvement of Cdk1 during DNA replication in hepatocytes and to dissect specific cell cycle regulation in hepatocytes compared to control human foreskin fibroblasts. In hepatocytes, Cdk1 exhibited a biphasic activation pattern correlating S-phase and G(2)/M transition, bound to cyclin A or B1 and localized to the nucleus during DNA replication. Importantly, small interfering RNA (siRNA)-mediated silencing of Cdk1 led to a strong decrease in DNA synthesis without affecting centrosome duplication. Furthermore, in hepatocytes arrested by the iron chelator O-Trensox in early S-phase prior to DNA replication, Cdk1/cyclin complexes were active, while replication initiation components such as the minichromosome maintenance 7 (Mcm7) protein were loaded onto DNA. Moreover, Mcm7 expression and loading onto DNA were not modified by Cdk1 silencing. Conversely, in fibroblasts, Cdk1 expression and activation were low in S-phase and its silencing did not reduce DNA synthesis. CONCLUSION: Cdk1 is essential for DNA replication downstream formation of replication initiation complexes in hepatocytes but not in fibroblasts and, as such, our data exemplify crucial differences in the cell cycle regulation between various mammalian cell types.
Assuntos
Proteína Quinase CDC2/fisiologia , Replicação do DNA , Regeneração Hepática , Animais , Ciclo Celular , Células Cultivadas , Ciclina A/fisiologia , Ciclina B1/fisiologia , Fibroblastos/enzimologia , Hepatócitos/enzimologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Xenobióticos/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular Transformada , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Omeprazol/farmacologia , Fenobarbital/farmacologia , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Reprodutibilidade dos Testes , Rifampina/farmacologia , Fatores de TempoRESUMO
Human exposure to heterocyclic aromatic amines (HAA) usually occurs through mixtures rather than individual compounds. However, the toxic effects and related mechanisms of co-exposure to HAA in humans remain unknown. We compared the effects of two of the most common HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), individually or in combination, in the metabolically competent human hepatoma HepaRG cells. Various endpoints were measured including cytotoxicity, apoptosis, oxidative stress and DNA damage by the comet assay. Moreover, the effects of PhIP and/or MeIQx on mRNA expression and activities of enzymes involved in their activation and detoxification pathways were evaluated. After a 24h treatment, PhIP and MeIQx, individually and in combination, exerted differential effects on apoptosis, oxidative stress, DNA damage and cytochrome P450 (CYP) activities. Only PhIP induced DNA damage. It was also a stronger inducer of CYP1A1 and CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx exposure resulted in a significant induction of CYP1A2 activity. The combination of PhIP with MeIQx induced an oxidative stress and showed synergistic effects on apoptosis. However, PhIP-induced genotoxicity was abolished by a co-exposure with MeIQx. Such an inhibitory effect could be explained by a significant decrease in CYP1A2 activity which is responsible for PhIP genotoxicity. Our findings highlight the need to investigate interactions between HAA when assessing risks for human health and provide new insights in the mechanisms of interaction between PhIP and MeIQx.
Assuntos
Imidazóis/toxicidade , Quinoxalinas/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Dano ao DNA , Relação Dose-Resposta a Droga , Humanos , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da PolimeraseRESUMO
Synthesis of alternating pyridine-pyrrole molecular strands composed of two electron-rich pyrrole units (donors) sandwiched between three pyridinic cores (acceptors) is described. The envisioned strategy was a smooth electrosynthesis process involving ring contraction of corresponding tripyridyl-dipyridazine precursors. 2,6-Bis[6-(pyridazin-3-yl)]pyridine ligands 2a-c bearing pyridine residues at the terminal positions were prepared in suitable quantities by a Negishi metal cross-coupling procedure. The yields of heterocyclic coupling between 2-pyridyl zinc bromide reagents 12a-c and 2,6-bis(6-trifluoromethanesulfonylpyridazin-3-yl)pyridine increased from 68 to 95% following introduction of electron-donating methyl groups on the metallated halogenopyridine units. Favorable conditions for preparative electrochemical reduction of tripyridyl-dipyridazines 2b,c were established in THF/acetate buffer (pH 4.6)/acetonitrile to give the targeted 2,6-bis[5-(pyridin-2-yl)pyrrol-2-yl]pyridines 1b and 1c in good yields. The absorption behavior of the donor-acceptor tripyridyl-dipyrrole ligands was evaluated and compared to theoretical calculations. Highly fluorescent properties of these chromophores were found (ν(em)≈2 × 10(4) cm(-1) in MeOH and CH(2)Cl(2)), and both pyrrolic ligands exhibit a remarkable quantum yield in CH(2)Cl(2) (φ(f)=0.10). Structural studies in the solid state established the preferred cis conformation of the dipyrrolic ligands, which adopting a planar arrangement with an embedded molecule of water having a complexation energy exceeding 10 kcal mol(-1). The ability of the tripyridyl-dipyrrole to complex two copper(II) ions in a pentacoordinate square was investigated.
Assuntos
Antineoplásicos/síntese química , Cobre/química , Nitrogênio/química , Piridinas/síntese química , Pirróis/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Catálise , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas Eletroquímicas , Feminino , Humanos , Ligantes , Masculino , Modelos Químicos , Estrutura Molecular , Piridinas/química , Piridinas/farmacologia , Pirróis/química , Pirróis/farmacologiaRESUMO
Many chemical carcinogens require metabolic activation to form genotoxic compounds in human. Standard in vitro genotoxicity assays performed with activation systems, such as rat liver S9, are recognised to lead to a high number of false positives. The aim of this study was to evaluate the suitability of differentiated human hepatoma HepaRG cells as an in vitro model system for the detection of DNA damage induced by promutagens using the comet and the cytokinesis-block micronucleus assays. Several promutagens were tested, including aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), acrylamide, N-nitrosodimethylamine (NDMA), cyclophosphamide (CPA), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Cytotoxicity of these compounds was assessed by measuring lactate dehydrogenase leakage. A 24 h exposure was generally needed to obtain an obvious positive response in differentiated HepaRG cells in the comet and in the cytokinesis-block micronucleus assays. Comet formation was observed with all compounds except IQ. B[a]P, CPA and AFB1 showed a dose-dependent increase in micronucleated cells, whereas no increase was observed with PhIP, IQ and acrylamide. These preliminary data on genotoxicity in differentiated HepaRG cells are promising but more chemicals must be tested to determine the ability of HepaRG cells to assess genotoxicity of chemicals in humans.