RESUMO
Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Metilação de DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Metabólica , Nicotina/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Fumaça/efeitos adversos , Animais , Animais Recém-Nascidos , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Regiões Promotoras GenéticasRESUMO
Drug-induced cholestasis (DIC) is one of the leading manifestations of drug-induced liver injury (DILI). As the underlying mechanisms for DIC are not fully known and specific and predictive biomarkers and pre-clinical models are lacking, the occurrence of DIC is often only reported when the drug has been approved for registration. Therefore, appropriate models that predict the cholestatic potential of drug candidates and/or provide insight into the mechanism of DIC are highly needed. We investigated the application of rat precision-cut liver slices (PCLS) to predict DIC, using several biomarkers of cholestasis: hepatocyte viability, intracellular accumulation of total as well as individual bile acids and changes in the expression of genes known to play a role in cholestasis. Rat PCLS exposed to the cholestatic drugs chlorpromazine, cyclosporine A and glibenclamide for 48 h in the presence of a 60 µM physiological bile acid (BA) mix reflected various changes associated with cholestasis, such as decrease in hepatocyte viability, accumulation and changes in the composition of BA and changes in the gene expression of Fxr, Bsep and Ntcp. The toxicity of the drugs was correlated with the accumulation of BA, and especially DCA and CDCA and their conjugates, but to a different extent for different drugs, indicating that BA toxicity is not the only cause for the toxicity of cholestatic drugs. Moreover, our study supports the use of several biomarkers to test drugs for DIC. In conclusion, our results indicate that PCLS may represent a physiological and valuable model to identify cholestatic drugs and provide insight into the mechanisms underlying DIC.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/efeitos dos fármacos , Técnicas de Cultura de Órgãos/métodos , Testes de Toxicidade/métodos , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Ácidos e Sais Biliares/metabolismo , Colestase/induzido quimicamente , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Ratos Wistar , Simportadores/genéticaRESUMO
Although intestinal P-glycoprotein (P-gp) has been extensively studied in vitro and in animals, its activity and the consequences of P-gp inhibition for drug disposition and toxicity in humans are still difficult to accurately extrapolate from these studies. Moreover, existing in vitro models do not take into consideration that the intestine is heterogeneous with respect to P-gp expression. Recently, we reported rat precision-cut intestinal slices (PCIS) as a physiological ex vivo model to study the regional gradient of P-gp activity and inhibition. Here we extended the application of PCIS to the human intestine. For this purpose rhodamine 123 (R123) accumulation in the presence or absence of the P-gp inhibitors verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 was measured in PCIS of human duodenum, jejunum, ileum and colon. R123 accumulation in the presence of the P-gp inhibitors appeared to be most enhanced in the ileum compared to the other regions. Moreover, the regional differences in accumulation are in line with published differences in abundance of P-gp. The rank order of the potency of the P-gp inhibitors, reflected by their IC50 , was comparable to that in rat PCIS. However, the increase in accumulation of the P-gp substrate R123 by the inhibitors was larger in human ileum PCIS than in rat PCIS, indicating species difference in P-gp abundance. These data show that human PCIS are an appropriate ex vivo model to study the activity of intestinal P-gp and predict the inhibitory effect of drugs and of transporter-mediated drug-drug interactions in the human intestine. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Colo/efeitos dos fármacos , Colo/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Transporte Biológico , Relação Dose-Resposta a Droga , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Feminino , Corantes Fluorescentes/metabolismo , Humanos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Técnicas In Vitro , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Cinética , Masculino , Pessoa de Meia-Idade , Rodamina 123/metabolismoRESUMO
P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas/fisiologia , Íleo/metabolismo , Jejuno/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Ciclosporinas/metabolismo , Absorção Intestinal , Cetoconazol/metabolismo , Masculino , Quinidina/metabolismo , Ratos , Ratos Wistar , Valina/metabolismo , Verapamil/metabolismoRESUMO
The use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is associated with a high prevalence of gastrointestinal side effects. In vivo studies in rodents suggested that reactive metabolites of DCF produced by the liver or the intestine might be responsible for this toxicity. In the present study, precision-cut intestinal slices (PCIS) prepared from the jejunum of 18 human donors were used as an ex vivo model to investigate whether DCF intestinal metabolites are responsible for its intestinal toxicity in man. PCIS were incubated with a concentration range of DCF (0-600 µM) up to 24 h. DCF (≥400 µM) caused direct toxicity to the intestine as demonstrated by ATP depletion, morphological damage, caspase 3 activation, and lactate dehydrogenase leakage. Three main metabolites produced by PCIS (4'-hydroxy DCF, 5-hydroxy DCF, and DCF acyl glucuronide) were detected by HPLC. Protein adducts were detected by immunohistochemical staining and showed correlation with the intestinal metabolites. DCF induced similar toxicity to each of the samples regardless of the variation in metabolism among them. Less metabolites were produced by slices incubated with 400 µM DCF than with 100 µM DCF. The addition of the metabolic inhibitors such as ketoconazole, cimetidine, or borneol decreased the metabolite formation but increased the toxicity. The results suggest that DCF can induce intestinal toxicity in human PCIS directly at therapeutically relevant concentrations, independent of the reactive metabolites 4'-OH DCF, 5-OH DCF, or diclofenac acylglucuronide produced by the liver or formed in the intestine.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Diclofenaco/análogos & derivados , Glucuronídeos/toxicidade , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Anti-Inflamatórios não Esteroides/metabolismo , Caspase 3/metabolismo , Diclofenaco/metabolismo , Diclofenaco/toxicidade , Feminino , Glucuronídeos/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Jejuno/patologia , L-Lactato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Amanita phalloides poisonings account for the majority of fatal mushroom poisonings. Recently, we identified hematotoxicity as a relevant aspect of Amanita poisonings. In this study, we investigated the effects of the main toxins of Amanita phalloides, α- and ß-amanitin, on hematopoietic cell viability in vitro. Hematopoietic cell lines were exposed to α-amanitin or ß-amanitin for up to 72 h with or without the pan-caspase inhibitor Z-VAD(OH)-FMK, antidotes N-acetylcysteine, silibinin, and benzylpenicillin, and organic anion-transporting polypeptide 1B3 (OATP1B3) inhibitors rifampicin and cyclosporin. Cell viability was established by trypan blue exclusion, annexin V staining, and a MTS assay. Caspase-3/7 activity was determined with Caspase-Glo assay, and cleaved caspase-3 was quantified by Western analysis. Cell number and colony-forming units were quantified after exposure to α-amanitin in primary CD34+ hematopoietic stem cells. In all cell lines, α-amanitin concentration-dependently decreased viability and mitochondrial activity. ß-Amanitin was less toxic, but still significantly reduced viability. α-Amanitin increased caspase-3/7 activity by 2.8-fold and cleaved caspase-3 by 2.3-fold. Z-VAD(OH)-FMK significantly reduced α-amanitin-induced toxicity. In CD34+ stem cells, α-amanitin decreased the number of colonies and cells. The antidotes and OATP1B3 inhibitors did not reverse α-amanitin-induced toxicity. In conclusion, α-amanitin induces apoptosis in hematopoietic cells via a caspase-dependent mechanism.
Assuntos
Alfa-Amanitina , Intoxicação Alimentar por Cogumelos , Humanos , Alfa-Amanitina/toxicidade , Caspase 3 , Antídotos/farmacologia , AmanitaRESUMO
The inclusion of nanoparticles dispersed in a hydrophilic matrix is one of the formulation strategies to improve the bioavailability of orally administered Biopharmaceutics Classification System (BCS) class II and IV drugs by increasing their dissolution rate in the intestine. To confirm that the increased dissolution rate results in increased bioavailability, in vitro and in vivo animal experiments are performed, however, translation to the human situation is hazardous. In this study, we used a range of in vitro and ex vivo methods, including methods applying human tissue, to predict the in vivo oral bioavailability of a model BCS class II CB-1 antagonist, formulated as a nanoparticle solid dispersion. The enhanced dissolution rate from the nanoparticle formulation resulted in an increased metabolite formation in both rat and human precision-cut intestinal slices, suggesting increased uptake and intracellular drug concentration in the enterocytes. In Ussing chamber experiments with human tissue, both the metabolite formation and apical efflux of the metabolite were increased for the nanoparticulate solid dispersion compared with a physical mixture, in line with the results in intestinal slices. The pharmacokinetics of the different formulations was studied in rats in vivo. The nanoparticle formulation indeed improved the absorption of the cannabinoid receptor 1 (CB-1) antagonist and the delivery into the brain compared with the physical mixture. In conclusion, the combined approach provides a valuable set of tools to investigate the effects of formulation on the absorption of poorly soluble compounds in human intestine and may provide relevant information on the oral bioavailability in humans early in the development process.
Assuntos
Antagonistas de Receptores de Canabinoides/administração & dosagem , Absorção Intestinal , Nanopartículas/administração & dosagem , Receptor CB1 de Canabinoide/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/química , Antagonistas de Receptores de Canabinoides/farmacocinética , Química Farmacêutica , Humanos , Masculino , Ratos , Ratos Wistar , SolubilidadeRESUMO
1.The precision-cut intestinal slice (PCIS) technology is a relatively new addition to the battery of in vitro assays for evaluation of xenobiotic toxicity, metabolism, and transport. 2.The intestine is an important target for drug-induced toxicity due to its high exposure after oral administration. Therefore, the prediction of drug-induced intestinal side effects remains a significant safety issue in pharmaceutical development. Although animal experiments have been proven useful, species differences and the requirement for reduction of animal use warrant the development of in vitro methods which can apply human tissue. 3.The enterocytes lining the villi express high activities of enzymes and transporters involved in drug disposition. They vary highly in activities: along the length of the intestine and along the villi, gradients of expression levels of the enzymes and proteins exist, which necessitates an in vitro model that can reflect the different regions of the intestine. 4.In this chapter, the application of PCIS in studies on transport and toxicity of xenobiotics is reviewed. PCIS can be prepared from each region of the intestine and from various species in a similar manner, and the results published so far indicate that they represent a promising model to evaluate intestinal toxicity and transport.
Assuntos
Proteínas de Transporte/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Modelos Biológicos , Xenobióticos , Animais , Transporte Biológico/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Intestinos/patologia , Microdissecção/métodos , Técnicas de Cultura de Órgãos/métodos , Xenobióticos/efeitos adversos , Xenobióticos/farmacocinética , Xenobióticos/farmacologiaRESUMO
1.Cryopreservation of precision-cut tissue slices (PCTS) would have many advantages for drug development and would encourage more extensive use of the PCTS preparation. 2.Three methods have been studied to date: slow freezing, fast freezing, and vitrification. 3.Slow freezing can be very effective for some PCTS but is devastating to rat liver PCTS. Fast freezing can be successful for rat liver PCTS but is devastating to renal PCTS and has given inconsistent results even for rat liver PCTS. Vitrification has been effective for some slice systems but less effective for rat liver PCTS. Rat liver PCTS appear to be particularly difficult to cryopreserve well. 4.The general cryobiological principles of slow freezing, rapid freezing, and vitrification are reviewed. The empirical literature on the cryopreservation of PCTS has not taken sufficient account of these principles, and may, for example, include the effects of easily preventable osmotic injury. 5.More attention is needed to the effects of cryopreservation on specific cell types within PCTS and to the general integrity and viability of cryopreserved PCTS. Drug metabolism as a sole endpoint of study can be highly misleading. 6.Better application of cryobiological principles may enable improved results in the future.
Assuntos
Criopreservação/métodos , Congelamento , Fígado , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , RatosRESUMO
Coumarin (1,2-benzopyrone) is a natural compound that has been used as a fragrance in the food and perfume industry and could have therapeutic usefulness in the treatment of lymphedema and different types of cancer. Several previous pharmacokinetic studies of coumarin have been performed in humans, which revealed extensive first-pass metabolism of the compound. 7-Hydroxycoumarin (7-HC) and its glucuronide (7-HC-G) are the main metabolites formed in humans, and via this route, 80 to 90% of the absorbed coumarin is excreted into urine, mainly as 7-HC-G. Active transport processes play a role in the urinary excretion of 7-HC-G; however, until now, the transporters involved remained to be elucidated. In this study, we investigated whether the efflux transporters multidrug resistance-associated proteins (MRP)1-4, breast cancer resistance protein, or P-glycoprotein play a role in 7-HC and 7-HC-G transport. For this purpose, we measured uptake of the metabolites into membrane vesicles overexpressing these transporters. Our results showed that 7-HC is not transported by any of the efflux transporters tested, whereas 7-HC-G was a substrate of MRP3 and MRP4. These results are in line with the pharmacokinetic profile of coumarin and suggest that MRP3 and MRP4 are the main transporters involved in the excretion of the coumarin metabolite 7-HC-G from liver and kidney.
Assuntos
Cumarínicos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Umbeliferonas/metabolismo , Transporte Biológico Ativo/fisiologia , Células HEK293 , HumanosRESUMO
Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time. With this method, CPA concentration versus time profiles were varied using various commercially available CPA mixtures [VMP, VM3, M22, and modified M22 (mM22)]. The viability of PCLS was determined after CPA loading and unloading and subsequent incubation during 3h at 37°C. Despite the reduction of osmotic stress, the viability of slices did not improve with gradual loading and unloading and remained considerably lower than that of untreated slices. The toxicity of the three CPA solutions did not correlate with either their potential osmotic effects or their total concentrations, and did not change strongly with exposure time in 100% CPA. The most likely explanation for these observations is that PCLS are not very sensitive to osmotic changes of the magnitude imposed in our study, and chemical toxicity of the CPA solutions is the main barrier to be overcome. The chemical toxicity of the CPAs used in this study probably originates from a source other than the total concentration of the solutions. The presented gradient method using the specially designed chamber is more time and cost effective than the step-wise method and can be universally applied to efficiently evaluate different CPA solutions.
Assuntos
Crioprotetores/metabolismo , Fígado/metabolismo , Soluções para Preservação de Órgãos/metabolismo , Preservação de Órgãos/instrumentação , Trifosfato de Adenosina/metabolismo , Animais , Desenho de Equipamento , Fígado/ultraestrutura , Masculino , Preservação de Órgãos/métodos , Osmose , Pressão Osmótica , Permeabilidade , Ratos , Ratos Wistar , VitrificaçãoRESUMO
A novel approach for on-line monitoring of drug metabolism in continuously perifused, precision-cut liver slices (PCLS) in a microfluidic system has been developed using high-performance liquid chromatography with UV detection (HPLC-UV). In this approach, PCLS are incubated in a microfluidic device made of poly(dimethylsiloxane) (PDMS) by continuous, single-pass perifusion with fresh medium. Two syringe pumps are incorporated into the system to infuse substrates or inhibitors at varying concentrations into the perfusion medium just before the chip entrance. The medium containing the metabolites produced by the PCLS is directed toward an injection loop. Once filled, the content of this injection loop is automatically injected onto an HPLC for analysis. The on-line analysis of metabolites was tested by using the substrate, 7-hydroxycoumarin (7-HC). Rapid switching between substrate and solvent control was possible, and a direct metabolic response of the liver slice to perifusion with substrate was detected. Very stable phase II metabolism over a period of 24 h was observed. The inhibitory effect of phloxine B on the formation of 7-hydroxycoumarin glucuronide (phase II product of 7-HC) was also investigated. Phloxine B was injected into the incubation medium in increasing concentrations varying from 0 to 200 µM. The results showed a concentration-dependent inhibition of 7-HC glucuronide formation and allowed the calculation of an IC50 value (concentration in which 50% of the enzyme is inhibited) of â¼85 µM using one single liver slice. On-line detection was also shown to be advantageous for the detection of unstable metabolites. This was demonstrated by determination of the metabolites of the drug diclofenac. The reactive metabolite, acyl glucuronide, was detected at relatively high concentrations which remained very constant over a period of 4 h. In contrast, only low and decreasing amounts of diclofenac acyl glucuronide could be measured in the conventional well-plate incubation system. The advantages of this novel on-line analysis system for PCLS include the capability to obtain direct information about tissue function, assess the concentration dependence of drug-drug interactions in one single slice, and detect unstable metabolites. The system also enables fast analysis without the need to store samples, thus eliminating the associated freeze-thaw problems, and allows the simultaneous analysis of multiple metabolites.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Sistemas On-Line , Preparações Farmacêuticas/metabolismo , Animais , Diclofenaco/metabolismo , Azul de Eosina I/farmacologia , Técnicas In Vitro , Inativação Metabólica , Fígado/citologia , Masculino , Microtomia , Ratos , Ratos Wistar , Umbeliferonas/metabolismoRESUMO
P-glycoprotein (ABCB1), an ATP-binding cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions. Drug-mediated induction of intestinal ABCB1 is a clinically relevant phenomenon associated with significantly decreased drug bioavailability. Currently, there are no well-established human models for evaluating its induction, so drug regulatory authorities provide no recommendations for in vitro/ex vivo testing drugs' ABCB1-inducing activity. Human precision-cut intestinal slices (hPCISs) contain cells in their natural environment and express physiological levels of nuclear factors required for ABCB1 induction. We found that hPCISs incubated in William's Medium E for 48 h maintained intact morphology, ATP content, and ABCB1 efflux activity. Here, we asked whether rifampicin (a model ligand of pregnane X receptor, PXR), at 30 µM, induces functional expression of ABCB1 in hPCISs over 24- and 48-h incubation (the time to allow complete induction to occur). Rifampicin significantly increased gene expression, protein levels, and efflux activity of ABCB1. Moreover, we described dynamic changes in ABCB1 transcript levels in hPCISs over 48 h incubation. We also observed that peaks of induction are achieved among donors at different times, and the extent of ABCB1 gene induction is proportional to PXR mRNA levels in the intestine. In conclusion, we showed that hPCISs incubated in conditions comparable to those used for inhibition studies can be used to evaluate drugs' ABCB1-inducing potency in the human intestine. Thus, hPCISs may be valuable experimental tools that can be prospectively used in complex experimental evaluation of drug-drug interactions.
RESUMO
The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as the master regulator of antioxidant and cytoprotective gene expressions. Moreover, it plays a pivotal role in cancer progression. Nrf2 mediates the adaptive response which contributes to the resistance to chemotherapeutic pro-oxidant drugs, such as cisplatin (CDDP), in various tumors, including bladder cancers. For this reason, Nrf2 could be a promising target to overcome chemoresistance. There are several known Nrf2 pharmacological inhibitors; however, most of them are not specific. The use of a specific small interfering RNA (siRNA) targeting the Nrf2 gene (siNrf2) loaded into nanovehicles is an attractive alternative, since it can increase specificity. This study aimed to evaluate the biological activity of siNrf2 loaded on guanidine-terminated carbosilane dendrimers (GCDs) in overcoming CDDP resistance in bladder cancer cells with a high level of Nrf2. Parameters such as viability, proliferation, apoptosis, migration, and oxidative stress level were taken into account. Results demonstrated that siNrf2-GCD treatment sensitized CDDP-resistant cells to CDDP treatment. Moreover, data obtained by treating the non-cancerous human kidney HK-2 cell line strongly suggest a good safety profile of the carbosilane dendrimers loaded with siNrf2. In conclusion, we suggest that siNrf2-GCD is a promising drug delivery system for gene therapy to be used in vivo; and it may represent an important tool in the therapy of CDDP-resistant cancer.
RESUMO
Prenatal smoke exposure (PSE) is a risk factor for nicotine dependence. One susceptibility gene for nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine and nicotine clearance in the liver. Higher activity of the CYP2A6 enzyme is associated with nicotine dependence, but no research has addressed the PSE effects on the CYP2A6 gene or its mouse homologue Cyp2a5. We hypothesized that PSE affects Cyp2a5 promoter methylation, Cyp2a5 mRNA levels, and nicotine metabolism in offspring. We used a smoke-exposed pregnant mouse model. RNA, DNA, and microsomal protein were isolated from liver tissue of foetal, neonatal, and adult offspring. Enzyme activity, Cyp2a5 mRNA levels, and Cyp2a5 methylation status of six CpG sites within the promoter region were analysed via HPLC, RT-PCR, and bisulphite pyrosequencing. Our data show that PSE induced higher cotinine levels in livers of male neonatal and adult offspring compared to controls. PSE-induced cotinine levels in neonates correlated with Cyp2a5 mRNA expression and promoter methylation at CpG-7 and CpG+45. PSE increased methylation in almost all CpG sites in foetal offspring, and this effect persisted at CpG-74 in male neonatal and adult offspring. Our results indicate that male offspring of mothers which were exposed to cigarette smoke during pregnancy have a higher hepatic nicotine metabolism, which could be regulated by DNA methylation. Given the detected persistence into adulthood, extrapolation to the human situation suggests that sons born from smoking mothers could be more susceptible to nicotine dependence later in life.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/genética , Metilação de DNA , Fígado/metabolismo , Nicotina/metabolismo , Efeitos Tardios da Exposição Pré-Natal/genética , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Ilhas de CpG , Feminino , Fígado/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The potential mammalian hepatotoxicity of a new class of GSH-responsive cyclodextrin-based nanosponges loaded with the anticancer drug doxorubicin (Dox-GSH-NS) was investigated. Previous studies showed that these nanosponges can release medicaments preferentially in cells having high GSH content, a common feature of chemoresistant cells, and showed enhanced anti-tumoral activity compared to free Dox in vitro and in vivo in cells with high GSH content. Following these promising results, we investigated here the Dox-GSH-NS hepatotoxicity in human HepG2 cells (in vitro) and in the organotypic cultures of rat precision-cut liver slices (PCLS, ex vivo), while their accumulation in rat liver was assessed in vivo. Moreover, the transport in Dox uptake, as well as its efflux, was studied in vitro. Overall, benefiting of the integration of different investigational models, a good safety profile of Dox-GSH-NSs was evidenced, and their hepatotoxicity resulted to be comparable with respect to free Dox both in vitro and ex vivo. Furthermore, in vivo studies showed that the hepatic accumulation of the Dox loaded in the NS is comparable with respect to the free drug. In addition, Dox-GSH-NSs are taken up by active mechanisms, and can escape the efflux drug pump, thus, contributing to overcoming drug resistance.
Assuntos
Antineoplásicos/administração & dosagem , Ciclodextrinas/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Glutationa/administração & dosagem , Nanoestruturas/administração & dosagem , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/administração & dosagem , Cumarínicos/química , Cumarínicos/toxicidade , Ciclodextrinas/química , Ciclodextrinas/toxicidade , Doxorrubicina/química , Doxorrubicina/toxicidade , Glutationa/química , Glutationa/toxicidade , Células Hep G2 , Humanos , Fígado/metabolismo , Masculino , Nanoestruturas/química , Nanoestruturas/toxicidade , Ratos WistarRESUMO
Precision-cut intestinal slices (PCIS) is an ex vivo culture technique that found its applications in toxicology, drug transport and drug metabolism testing, as well as in fibrosis research. The main limiting factor of PCIS as experimental model is the relatively short viability of tissue slices. Here, we describe a strategy for extending the life-span of PCIS during culture using medium that is routinely used for growing intestinal organoids. Mouse and rat PCIS cultured in standard medium progressively showed low ATP/protein content and severe tissue degradation, indicating loss of tissue viability. In turn, organoid medium, containing epithelial growth factor (EGF), Noggin and R-spondin, maintained significantly higher ATP/protein levels and better preserved intestinal architecture of mouse PCIS at 96â¯h. In contrast, organoid medium that additionally contained Wnt, had a clear positive effect on the ATP content of rat PCIS during 24â¯h of culture, but not on slice histomorphology. Our proof-of-concept study provides early evidence that employing organoid medium for PCIS culture improved tissue viability during extended incubation. Enabling lasting PCIS cultures will greatly widen their range of applications in predicting long-term intestinal toxicity of xenobiotics and elucidating their mechanism of action, among others.
Assuntos
Intestinos , Técnicas de Cultura de Tecidos , Trifosfato de Adenosina , Animais , Proteínas de Transporte/farmacologia , Meios de Cultivo Condicionados/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Ratos Wistar , Nicho de Células-Tronco , Trombospondinas/genética , Proteína Wnt3A/farmacologiaRESUMO
Gold-based compounds are of great interest in the field of medicinal chemistry as novel therapeutic (anticancer) agents due to their peculiar reactivity and mechanisms of action with respect to organic drugs. Despite their promising pharmacological properties, the possible toxic effects of gold compounds need to be carefully evaluated in order to optimize their design and applicability. This study reports on the potential toxicity of three experimental gold-based anticancer compounds featuring lansoprazole ligands (1-3) studied in an ex vivo model, using rat precision cut kidney and liver slices (PCKS and PCLS, respectively). The results showed a different toxicity profile for the tested compounds, with the neutral complex 2 being the least toxic, even less toxic than cisplatin, followed by the cationic complex 1. The dinuclear cationic gold complex 3 was the most toxic in both liver and kidney slices. This result correlated with the metal uptake of the different compounds assessed by ICP-MS, where complex 3 showed the highest accumulation of gold in liver and kidney slices. Interestingly compound 1 showed the highest selectivity towards cancer cells compared to the healthy tissues. Histomorphology evaluation showed a similar pattern for all three Au(i) complexes, where the distal tubular cells suffered the most extensive damage, in contrast to the damage in the proximal tubules induced by cisplatin. The binding of representative gold compounds with the model ubiquitin was also studied by ESI-MS, showing that after 24 h incubation only 'naked' Au ions were bound to the protein following ligands' loss. The mRNA expression of stress response genes appeared to be similar for both evaluated organs, suggesting oxidative stress as the possible mechanism of toxicity. The obtained results open new perspectives towards the design and testing of bifunctional gold complexes with chemotherapeutic applications.
RESUMO
Induction of drug enzyme activity in the intestine can strongly determine plasma levels of drugs. It is therefore important to predict drug-drug interactions in human intestine in vitro. We evaluated the applicability of human intestinal precision-cut slices for induction studies in vitro. Morphological examination and intracellular ATP levels indicated tissue integrity up to 24 h of incubation, whereas in proximal jejunum slices, the metabolic rate toward most substrates remained at 40 to 50% of initial values. In colon slices, the cytochrome P450 conversions were below the detection limit, but conjugation rates remained relatively constant during incubation. The inducibility of drug-metabolizing enzymes and P-glycoprotein was evaluated using prototypical inducers for five induction pathways. beta-Naphthoflavone (aryl hydrocarbon receptor ligand) induced CYP1A1 (132-fold in colon and 362-fold in proximal jejunum) and UDP glucuronosyltransferase (UGT) 1A6 mRNA (9.8-fold in colon and 3.2-fold in proximal jejunum). In proximal jejunum, rifampicin (RIF) [pregnane X receptor (PXR) ligand] induced CYP3A4 (5.2-fold), CYP2B6 (2-fold), UGT1A6 (2.2-fold), and multidrug resistance-1 (MDR1)/ABCB1 mRNA (2.7-fold), whereas 6beta-hydroxytestosterone formation (CYP3A4) increased 2-fold. In colon, RIF induced UGT1A6 32-fold and MDR1 2.2-fold. Dexamethasone (glucocorticoid receptor and PXR ligand) induced CYP3A4 mRNA (3.5-fold) and activity (5-fold) in proximal jejunum. Phenobarbital (constitutive androstane receptor activator) induced CYP3A4 (4.1-fold, only in jejunum), CYP2B6 (4.9-fold in colon and 2.3-fold in proximal jejunum), and MDR1/ABCB1 mRNA and CYP3A4 activity (2-fold only proximal jejunum). Quercetin (nuclear factor-E2-related factor 2 activator) induced UGT1A6 mRNA (6.7-fold in colon and 2.2-fold in proximal jejunum). In conclusion, this study shows that human intestinal precision-cut slices are useful to study induction of drug-metabolizing enzymes and transporters in the human intestine.
Assuntos
Colo/metabolismo , Jejuno/metabolismo , Preparações Farmacêuticas/metabolismo , Trifosfato de Adenosina/metabolismo , Colo/enzimologia , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Humanos , Técnicas In Vitro , Jejuno/enzimologia , Microtomia/métodos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reprodutibilidade dos TestesRESUMO
Although the liver has long been thought to play the major role in drug metabolism, also the metabolic capacity of the intestine is more and more recognized. In vivo studies eventually pointed out not only the significance of first-pass metabolism by the intestinal wall for the bioavailability of several compounds, but also the relevance of transporters in this process. Only a few methods are available to study drug metabolism in vivo or in situ and with most of these methods it remains difficult to discriminate between the contribution of liver and extrahepatic tissues. To study intestinal drug metabolism in vitro, apart from subcellular fractions, several intact cell systems are nowadays available. This review discusses the available intestinal in vitro methods to study drug metabolism. The advantages and limitations of intact cell systems (isolated intestinal perfusion, everted sac, Ussing chamber preparations, biopsies, precision-cut slices, primary cells), subcellular fractions (S9 fractions, microsomes) and intestinal cell lines (caco-2, LS180 cells amongst others) are discussed. Their applicability to different species and to study phase I and II metabolism/transport and drug-drug interactions are summarized. Furthermore, causes of variation within and between methods are discussed and metabolic rates obtained with different methods are compared. Whereas subcellular fractions and cell lines are efficient methods to study mechanistic aspects of drug metabolism at the enzyme level, the isolated intestinal perfusion, everted sac and Ussing chamber appear particularly useful for studying drug metabolism of rapidly metabolised drugs and interactions with transporters. Biopsies, precision-cut slices and primary cells seem all appropriate to study induction and metabolism of slowly metabolised drugs.