RESUMO
Amiodarone (AMD) is an antiarrhythmic drug prescribed to treat ventricular tachycardia and fibrillation. However, it causes an unpredictable toxicity (idiosyncratic), which may depend on co-exposure to pollutants. AMD toxicity involves calcium homeostasis alteration and oxidative stress, which are also affected by cigarette smoke (CS). We investigated the interaction of CS-condensate (CSC), phenanthrene, and benzo(a)pyrene with AMD toxicity on Saccharomyces cerevisiae. AMD toxicity was reduced by CSC or phenanthrene. Benzo(a)pyrene mildly decreased AMD toxicity on the wild-type strain, but not on the catalase-CTT1 mutant. This latter and other mutants in glucose receptor-GPR1 or calcium transporter-PMR1 showed lower antagonistic effect to AMD by CSC or phenanthrene relative to the wild type, suggesting roles of oxidative stress, calcium homeostasis, and hexose-sensing in this interaction.
Assuntos
Amiodarona , Fumar Cigarros , Amiodarona/toxicidade , Saccharomyces cerevisiae/genética , Benzo(a)pireno/toxicidade , Cálcio , NicotianaRESUMO
Iris x germanica L., which belongs to the Iridaceae family, has been reported in the literature for its antioxidant properties in acellular chemical-antioxidant assays. Chlorpromazine (CPZ) is an antipsychotic drug known to cause adverse reactions in humans. Oxidative stress is among the main mechanisms by which CPZ exerts its toxicity in animal cell models as well as in the yeast Saccharomyces cerevisiae. In this study we investigated the protective effects of I. germanica L. crude extracts against CPZ toxicity. We demonstrated that methanolic extracts from rhizome (R-M), leaf (L-M) and flower (Fl-M) had potent antioxidant activity by scavenging the free radical DPPH, with half-maximal effective concentrations (EC50) 193, 107, and 174 µg/mL, respectively. R-M, L-M and Fl-M at doses up to 1000 µg/mL, didn't affect yeast cell growth. In addition, we demonstrated for the first time that L-M at 1000 µg/mL and R-M at all tested doses counteracted CPZ toxicity, probably by promoting yeast cell antioxidant agents. The R-M capacity to counteract CPZ toxicity was lost in the yeast strain mutant in catalase-encoding gene (Cta1), while strains mutant in Sod2, Skn7 and Rap1 showed mild or full R-M-induced protective effect against CPZ toxicity. Our results demonstrated that I. germanica L. R-M extract counteracted CPZ toxicity in the yeast cell model. Further studies are planned to isolate the involved bioactive compounds and identify the involved genes and the antioxidant agents.
Assuntos
Antioxidantes , Gênero Iris , Animais , Antioxidantes/farmacologia , Clorpromazina/toxicidade , Extratos Vegetais/farmacologia , Saccharomyces cerevisiae/genéticaRESUMO
Chlorpromazine (CPZ) is an antipsychotic phenothiazine which is still commonly prescribed though it causes idiosyncratic toxicity such as cholestasis. CPZ toxicity mechanisms involve oxidative stress among others. Cigarette smoke (CS) causes deleterious effects through diverse mechanisms such as oxidative stress. CS alters drug metabolizing enzymes expression and drug transporters expression and activity in animal cell models as well as in Saccharomyces cerevisiae. CS therefore alters pharmacokinetic and pharmacodynamics of many drugs including CPZ and caffeine whose toxicity is promoted by CS condensate (CSC). CSC interaction with CPZ toxicity deserves investigation. In this study, CSC exerted mild toxicity on Saccharomyces cerevisiae which resisted to this chemical stress after several hours. CPZ toxicity on yeast was dose-dependent and the cells resisted to CPZ up to 40 µM after 24 h of treatment. Yeast cells treated simultaneously with CPZ and a nontoxic CSC dose were less sensitive to CPZ. CSC probably triggers cross-resistance to CPZ. Using Sod1 mutant strain, we showed that this gene is potentially involved in the potential cross-resistance. Other genes encoding stress-related transcription factors could be involved in this process. Nicotine and cadmium chloride, which caused a dose-dependent toxicity individually, acted with CPZ in an additive or synergistic manner in terms of toxicity. Although our results cannot be extrapolated to humans, they clearly show that CSC and its components interact with CPZ toxicity.
Assuntos
Clorpromazina , Saccharomyces cerevisiae , Animais , Clorpromazina/toxicidade , Humanos , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Fumaça/efeitos adversos , FumarRESUMO
Amiodarone (AMD) is an antiarrhythmic drug that induces idiosyncratic toxicity. Environmental pollutants, including heavy metals, could interact with its toxicity by affecting pharmacokinetics and pharmacodynamics. Other levels of interaction could exist in yeast, such as oxidative stress and the general stress response. In this study, we investigated the interaction of mercury chloride (HgCl2) and cadmium chloride (CdCl2) with AMD toxicity on Saccharomyces cerevisiae. Interaction type - synergistic, additive, or antagonistic - was determined by median drug effect analysis using "CompuSyn". HgCl2 potentiated AMD toxicity at high doses (≥ 71.4 µm, which yielded more than 60% inhibition). CdCl2 acted similarly at high doses (≥ 57.9 µm). An antagonistic effect appeared at lower doses with both heavy metals (≤ 49.4 µm for HgCl2 and AMD; ≤ 18.9 µm for CdCl2 and AMD). The threshold concentrations (HgCl2 or CdCl2 combined with AMD) that switched the interaction from antagonistic to additive, and then to synergistic, were decreased in the yeast strain mutant in catalase (CTT1), suggesting an important role for this enzyme. Moreover, mutation of the nutrient sensing receptor gene GPR1 caused the synergistic interaction of CdCl2, but not HgCl2, with AMD to occur at the lowest tested concentrations (1.2 µm). The reverse was obtained with the mutant strain in calcium-manganese transporter gene PMR1, where the synergistic interaction of HgCl2 with AMD occurred at concentrations (20.7 µm) lower than that of the wild type (71.4 µm). These results demonstrated a dose-dependent interaction between the two heavy metals with AMD toxicity, and the involvement of oxidative stress, calcium homeostasis, and nutrient sensing in the observed interaction.
Assuntos
Amiodarona , Mercúrio , Metais Pesados , Amiodarona/toxicidade , Cádmio/toxicidade , Cálcio , Mercúrio/toxicidade , Metais Pesados/toxicidade , Saccharomyces cerevisiae/genéticaRESUMO
Diclofenac (DCF) adverse reactions involve diverse mechanisms in different models. We recently demonstrated that DCF-induced toxicity in HepaRG decreases as they express DCF-metabolizing enzymes. DCF metabolism promotes toxicity in Saccharomyces cerevisiae expressing heterologous cytochromes-P450. N-Acetylcysteine (NAC) is used to treat diverse medical conditions due to its multiple properties (antioxidant, metal chelator, thiol-disulfide disruption). The latter property accounts for its mucolytic effects and broadens its potential molecular targets to signal transduction proteins, ABC transporters and others. Interaction of NAC with DCF effects depends on the experimental model. This study aims to investigate NAC/DCF interaction and the involvement of ABC transporters in wild type and mutant Saccharomyces cerevisiae. DCF inhibited yeast growth in a dose- and time-dependent manner and the cells started adapting to DCF 24-h post-treatment. NAC potentiated DCF-induced toxicity if added prior or parallel to DCF. Pretreatment with NAC increased its potentiation effect and compromised cells adaption to DCF. Post-treatment with NAC potentiated DCF toxicity without compromising adaptation. Moreover, mutant strains in ABC transporters Pdr5, Yor1, Bpt1 or Pdr15, were more sensitive to DCF; while mutant strains in Pdr5, Vmr1 or Pdr12 were more sensitive to NAC/DCF interaction. DCF ± NAC elicited on the mutant strain in Yap1, an oxidative stress-related protein, the same effects as on the wild type. Therefore, oxidative stress does not seem to be key actor in DCF toxicity in our model. Our hypothesis is that NAC potentiation effect is at least due to its ability to disrupt disulfide bridge in proteins required to overcome DCF toxicity in yeast.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetilcisteína/toxicidade , Anti-Inflamatórios não Esteroides/toxicidade , Antioxidantes/toxicidade , Diclofenaco/toxicidade , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Anti-Inflamatórios não Esteroides/metabolismo , Diclofenaco/metabolismo , Dissulfetos/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Genótipo , Mutação , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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
Hyperthermophilic Archaea colonizing unnatural habitats of extremes conditions such as volcanoes and deep-sea hydrothermal vents represent an unmeasurable bioresource for enzymes used in various industrial applications. Their enzymes show distinct structural and functional properties and are resistant to extreme conditions of temperature and pressure where their mesophilic homologs fail. In this review, we will outline carbohydrate-active enzymes (CAZymes) from hyperthermophilic Archaea with specific focus on the two largest families, glycoside hydrolases (GHs) and glycosyltransferases (GTs). We will present the latest advances on these enzymes particularly in the light of novel accumulating data from genomics and metagenomics sequencing technologies. We will discuss the contribution of these enzymes from hyperthermophilic Archaea to industrial applications and put the emphasis on newly identifed enzymes. We will highlight their common biochemical and distinct features. Finally, we will overview the areas that remain to be explored to identify novel promising hyperthermozymes.
Assuntos
Glicosídeo Hidrolases , Biotecnologia , TemperaturaRESUMO
Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.
Assuntos
DNA Helicases/genética , RNA Helicases/genética , Thermococcales/enzimologia , Adenosina Trifosfatases/genética , Proteínas Arqueais/química , DNA/química , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Filogenia , RNA/química , RNA Helicases/isolamento & purificação , RNA Helicases/metabolismo , Homologia de Sequência de Aminoácidos , Thermococcales/genética , Thermococcales/metabolismoRESUMO
Chlorpromazine (CPZ) is still a commonly prescribed antipsychotic which causes poorly understood idiosyncratic toxicity such as cholestasis, phospholipidosis and steatosis. CPZ has diverse cellular targets and exerts various toxicity mechanisms whose exploration is necessary to understand CPZ side effects. We report here that CPZ causes a decrease of total lipid content in Saccharomyces cerevisiae at the same dose range as that used on mammalian cells. The observed lipid decrease was obvious after 4 and 9 hours of treatment, and disappeared after 24 hours due to cells adaptation to the chemical stress. The inhibitory effect of CPZ was antagonized by the antioxidant N-acetyl L-cysteine and is likely caused by the parent compound. The obtained results demonstrate that yeast model is valid to investigate the involved CPZ toxicity mechanisms, particularly in terms of lipids alteration. This would contribute to understand CPZ side effects in simple model and reduce experimentation on animals.
RESUMO
Carcinogenic heterocyclic aromatic amines (HAAs) interact with some drug transporters, like the efflux pump BCRP and the organic anion transporters OAT1 and OAT3. The present study was designed to determine whether they can also target activities of the organic cation transporters (OCTs), using mainly OCT1-, OCT2- and OCT3-overexpressing HEK293 cells. Fifteen HAAs were demonstrated to differently alter OCT activities; with a cut-off of at least 50% reduction of transporter activity by 100⯵M HAAs, 5/15 HAAs, including Trp-P-1 and Trp-P-2, inhibited activities of OCT1, OCT2 and OCT3, whereas 7/15 HAAs, including PhIP and MeIQx, blocked those of OCT2 and OCT3, 1/15 HAAs reduced those of OCT1 and OCT2 and 2/15 HAAs, including AαC, only that of OCT2. IC50 values of Trp-P-1 and Trp-P-2 towards OCT activities were found to be in the 2-6⯵M range, likely not relevant for human exposure to HAAs through smoking or the diet. Trp-P-1 and Trp-P-2 additionally failed to trans-stimulate OCT1 and OCT2 activities and exhibited similar accumulation in OCT1/2-transduced HEK293 cells and control HEK293-MOCK cells. These data demonstrate that HAAs, notably Trp-P-1 and Trp-P-2, interact with OCT1/2, without however being transported, thus likely discarding a major role for OCT1/2 in HAA systemic toxicokinetics.
Assuntos
Aminas/farmacologia , Compostos Heterocíclicos/farmacologia , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Células HEK293 , Humanos , Proteínas de Transporte de Cátions Orgânicos/metabolismoRESUMO
In animals, cigarette smoke may alter pharmacokinetics by altering activity and expression of ABC drug transporters. We previously demonstrated that cigarette smoke condensate (CSC) impairs activity and expression of several hepatic ABC drug transporters which mediate toxicant efflux. However, CSC effects on efflux transporters are still unknown in Saccharomyces cerevisiae which resists diverse chemical stresses, by inducing pleiotropic drug resistance (PDR) genes among others. The yeast ABC transporters are functionally and structurally homologous to the mammalian ones. In this study, Saccharomyces cerevisiae exposure to CSC for 15 min caused a dose-dependent inhibition of rhodamine 123 efflux, whereas a longer exposure (3 h) induced mRNA expression of the ABC PDR efflux pumps Pdr5, Snq2, Pdr 10 and Pdr15, and of Tpo1, a member of the major facilitator superfamily (MFS). CSC also increased toxicity of caffeine, which is handled by two PDR transporters, Pdr5 and Snq2. Taken together, these data demonstrated that yeast efflux transporters are targets of cigarette smoke chemicals, and that Saccharomyces cerevisiae may cope with CSC-induced stress, including the initial efflux inhibition, by induction of the mRNA of several plasma membrane PDR and MFS efflux transporters. Saccharomyces cerevisiae is therefore a valid model to investigate pollutant effects on ABC and MFS transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Cafeína/toxicidade , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Rodaminas/metabolismo , Saccharomyces cerevisiae/fisiologiaRESUMO
BACKGROUND: Discovery of mesenchymal stem cells (MSCs) in various adult human tissues opened the way to new therapeutic strategies involving tissue engineering from these cells. More recently, vascular substitutes have opened the era of vascular engineering by making replacement vessels from purely biological material. The objective of our study was to create a vascular substitute from MSCs using a multilayer polyelectrolyte film based on natural polymers (Chitosan and Hyaluronic Acid). METHODS: Biocompatibility and cellular behavior were evaluated in this study using MSCs from the Wharton's jelly (WJ) of human umbilical cords. WJ-MSCs adherence was assessed and cells morphology was investigated by Scanning Electron Microscopy (SEM) and actin visualization (Phalloidin). RESULTS: The number of WJ-MSCs seeded on the (CHI/HA)10 films was greater than the number of cells seeded on the collagen, as the spectrophotometric measurement showed a large cell proliferation on (CHI/HA)10 in comparison with collagen. After adhesion, WJ-MSCs showed a fibroblastic morphology on CHI/HA as for control (collagen I). These results were confirmed by cytoskeleton staining. CONCLUSIONS: The biocompatibility of WJ-MSCs and (CHI/HA)10 showed the possibility to combine the use of WJ-MSCs and (CHI/HA)10 films in vascular tissue engineering.
RESUMO
Cigarette smoke condensate (CSC) has previously been shown to impair activity and expression of hepatic drug transporters. In the present study, we provided evidence that CSC also hinders activity of organic anion transporters (OATs), notably expressed at the kidney level. CSC thus cis-inhibited OAT substrate uptake in OAT1- and OAT3-transfected HEK293 cells, in a concentration-dependent manner (IC50=72.1µg/mL for OAT1 inhibition and IC50=27.3µg/mL for OAT3 inhibition). By contrast, OAT4 as well as the renal organic cation transporter (OCT) 2 were less sensitive to the inhibitory effect of CSC (IC50=351.5µg/mL and IC50=226.2µg/mL, for inhibition of OAT4 and OCT2, respectively). OAT3 activity was further demonstrated to be blocked by some single chemicals present in cigarette smoke such as the heterocyclic amines AαC (IC50=11.3µM) and PhIP (IC50=1.9µM), whereas other major cigarette smoke components used at 100µM, like nicotine, the nitrosamine NNK and the polycyclic aromatic hydrocarbons benzo(a)pyrene and phenanthrene, were without effect. AαC and PhIP however failed to trans-stimulate activity of OAT3, suggesting that they were not substrates for this transporter. Taken together, these data establish OAT1 and OAT3 transporters as targets of cigarette smoke chemicals, which may contribute to smoking-associated pharmacokinetics alterations.
Assuntos
Transportadores de Ânions Orgânicos/antagonistas & inibidores , Fumaça/efeitos adversos , Produtos do Tabaco , Células HEK293 , Humanos , Transportadores de Ânions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/genéticaRESUMO
PURPOSE: Superfamily 1 and Superfamily 2 helicases, two of the largest helicase protein families, play vital roles in many biological processes including replication, transcription and translation. Study of helicase proteins in the model microorganisms of archaea have largely contributed to the understanding of their function, architecture and assembly. Based on a large phylogenomics approach, we have identified and classified all SF1 and SF2 protein families in ninety five sequenced archaea genomes. Here we developed an online webserver linked to a specialized protein database named ARCPHdb to provide access for SF1 and SF2 helicase families from archaea. METHODS: ARCPHdb was implemented using MySQL relational database. Web interfaces were developed using Netbeans. Data were stored according to UniProt accession numbers, NCBI Ref Seq ID, PDB IDs and Entrez Databases. RESULTS: A user-friendly interactive web interface has been developed to browse, search and download archaeal helicase protein sequences, their available 3D structure models, and related documentation available in the literature provided by ARCPHdb. The database provides direct links to matching external databases. CONCLUSIONS: The ARCPHdb is the first online database to compile all protein information on SF1 and SF2 helicase from archaea in one platform. This database provides essential resource information for all researchers interested in the field.
Assuntos
Proteínas Arqueais , Biologia Computacional , Bases de Dados de Proteínas , RNA Helicases , Archaea , Sistemas de Gerenciamento de Base de Dados , Interface Usuário-ComputadorRESUMO
The role of reactive metabolites and inflammatory stress has been largely evoked in idiosyncratic hepatotoxicity of diclofenac (DCF); however mechanisms remain poorly understood. We aimed to evaluate the influence of liver cell phenotype on the hepatotoxicity of DCF combined or not with TNF-α using differentiated and undifferentiated HepaRG cells, and for comparison, HepG2 cells. Our results demonstrate that after a 24h-treatment metabolizing HepaRG cells were less sensitive to DCF than their undifferentiated non-metabolizing counterparts as shown by lower oxidative and endoplasmic reticulum stress responses and lower activation of caspase 9. Differentiated HepaRG cells were also less sensitive than HepG2 cells. Their lower sensitivity to DCF was related to their high content in glutathione transferases. DCF-induced apoptotic effects were potentiated by TNF-α only in death receptor-expressing differentiated HepaRG and HepG2 cells and were associated with marked activation of caspase 8. TNF-α co-treatment did not aggravate DCF-induced cholestatic features. Altogether, our results demonstrate that (i) lower sensitivity to DCF of differentiated HepaRG cells compared to their non-metabolically active counterparts was related to their high detoxifying capacity, giving support to the higher sensitivity of nonhepatic tissues than liver to this drug; (ii) TNF-α-potentiation of DCF cytotoxicity occurred only in death receptor-expressing cells.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Diclofenaco/farmacologia , Resistência a Medicamentos , Hepatócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/agonistas , Anti-Inflamatórios não Esteroides/agonistas , Anti-Inflamatórios não Esteroides/metabolismo , Biotransformação/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Diclofenaco/agonistas , Diclofenaco/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/metabolismo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Microscopia de Contraste de Fase , Estresse Oxidativo/efeitos dos fármacos , Receptor fas/metabolismoRESUMO
Smoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRG cells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B1, OATP2B1, OAT2, NTCP, OCT1 and BSEP, and enhanced that of MRP4. Such changes in transporter gene expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reference activator of the aryl hydrocarbon receptor (AhR) pathway, and were counteracted, for some of them, by siRNA-mediated AhR silencing. This suggests that CSC alters hepatic drug transporter levels via activation of the AhR cascade. Importantly, drug transporter expression regulations as well as some transporter activity inhibitions occurred for a range of CSC concentrations similar to those required for inducing drug metabolizing enzymes and may therefore be hypothesized to be relevant for smokers. Taken together, these data established human hepatic transporters as targets of cigarette smoke, which could contribute to known alteration of pharmacokinetics and some liver adverse effects caused by smoking.
Assuntos
Fígado/efeitos dos fármacos , Fumar/efeitos adversos , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Interações Medicamentosas , Humanos , Fígado/citologia , Fígado/metabolismo , Interferência de RNA/efeitos dos fármacos , Proteínas Carreadoras de Solutos/efeitos dos fármacos , Proteínas Carreadoras de Solutos/metabolismoRESUMO
The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na(+)-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.
Assuntos
Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Linhagem Celular , HumanosRESUMO
Cytochrome P450 2E1 (CYP2E1) gene expression is known to be induced by interleukin-4 (IL4) and repressed by inflammatory cytokines, such as interleukin-1beta3 (IL1beta3) in human hepatocytes. The mechanisms involved in these transcriptional regulations remain elusive. In order to study these mechanisms, various constructs of the human CYP2E1 promoter were prepared and transfected into the human HepG2 hepatoma cell line. Our findings revealed that an IL4-responsive region of 128bp (-671/-544) was required to mediate induction by IL4. IL1beta caused moderate but significant decrease of the promoter activity, which was abolished when the two cytokines were combined. The IL1beta inhibitory effect is mediated through a regulatory sequence independent of that of IL4. Furthermore, by using specific signaling pathway inhibitors, we demonstrated that IL4 activation required protein kinase C (PKC) activation. In addition, our results suggest that induction by IL4 was not dependent on a single binding site but rather on a complex region which includes putative binding sites for signal transducer and activator of transcription (STAT)6, activator protein (AP)-1, nuclear factor kappa-B (NFkappaB), nuclear factor of activated T cells (NFAT) and CCAAT enhancer binding protein (C/EBP). Electrophoretic mobility shift assays suggest that AP1 and NFAT transcription factors are able to bind to three sites in the IL4-responsive region.
Assuntos
Citocromo P-450 CYP2E1/genética , Interleucina-4/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Elementos de Resposta/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Citocromo P-450 CYP2E1/metabolismo , DNA/análise , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1/farmacologia , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição , Células Tumorais CultivadasRESUMO
AIM: This study investigated the effect of smoking, mother's age, body mass index (BMI), and parity number on density, lipids, proteins, and secreted immunoglobulin A (SIgA) of human milk. METHODS: Transitional and mature milk samples were collected from 23 nursing smoker mothers and 43 nursing nonsmoker mothers. Proteins, lipids, and SIgA concentrations were determined as well as the milk density and the general protein profile. RESULTS: Our investigation showed that the milk of smokers contained less lipids and proteins (statistically significant 26% and 12% decrease, respectively), whereas milk density was unchanged. SIgA concentration was 27% lower in milk from smokers, but the decrease was not statistically significant. The general protein profile showed no significant smoking-associated changes in the four identified proteins (ß-casein, immunoglobulin A heavy chain, serum albumin, and lactoferrin). Mothers' age and residential area showed noticeable but statistically nonsignificant differences in some of the measured parameters. However, parity number, lactation stage, and BMI were associated with a significant modification of milk composition. Mature milk contained more lipids and less protein, whereas the increase of parity number was associated with an increase in lipid concentration. The group of overweight mothers showed lower milk protein concentration in comparison with the normal group. Multivariate analysis showed a statistically significant interaction effect of the variables (smoking, parity number, lactation stage, age, and BMI) on lipids and between some of them on proteins and SIgA. CONCLUSION: Our study showed that smoking was associated with lower milk lipid and protein concentrations and that the parity number and BMI were associated with a change in milk lipids and proteins content, respectively.