Biomedical subjects

José V Castell

Publications and source records attributed to José V Castell.

At least 19 recordsLinked to original sources

PubMed2006

Underexpressed coactivators PGC1alpha and SRC1 impair hepatocyte nuclear factor 4 alpha function and promote dedifferentiation in human hepatoma cells.

Hepatocyte nuclear factor 4alpha (HNF4alpha) plays critical roles during liver development and in the transcriptional regulation of many hepatic genes in adult liver. Here we have demonstrated that in human hepatoma HepG2 cells, HNF4alpha is expressed at levels as high as in human liver but its activity on target genes is very low or absent. We have discovered that the low expression of key coactivators (PGC1alpha, SRC1, SRC2, and PCAF) might account for the lack of function of HNF4alpha in HepG2 cells. Among them, PGC1alpha and SRC1 are the two most important HNF4alpha coactivators as revealed by reporter assays with an Apo-CIII promoter construct. Moreover, the expression of these two coactivators was found to be down-regulated in all human hepatomas investigated. Overexpression of SRC1 and PGC1alpha by recombinant adenoviruses led to a significant up-regulation of well characterized HNF4alpha-dependent genes (ApoCIII, ApoAV, PEPCK, AldoB, OTC, and CYP7A1) and forced HepG2 cells toward a more differentiated phenotype as demonstrated by increased ureogenic rate. The positive effect of PGC1alpha was seen to be dependent on HNF4alpha. Finally, insulin treatment of human hepatocytes and HepG2 cells caused repression of PGC1alpha and a concomitant down-regulation of ApoCIII, PEPCK, AldoB, and OTC. Altogether, our results suggest that SRC1, and notably PGC1alpha, are key coactivators for the proper function of HNF4alpha in human liver and for an integrative control of multiple hepatic genes involved in metabolism and homeostasis. The down-regulation of key HNF4alpha coactivators could be a determinant factor for the dedifferentiation of human hepatomas.

Adult↗

PubMed2006

Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4alpha requires coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha and steroid receptor coactivator 1.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a key transcription factor for the constitutive expression of cytochromes P450 (P450s) in the liver. However, human hepatoma HepG2 cells show a high level of HNF4alpha but express only marginal P450 levels. We found that the HNF4alpha-mediated P450 transcription in HepG2 is impaired by the low level of coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha (PGC1alpha) and steroid receptor coactivator 1 (SRC1). Reporter assays with a chimeric CYP2C9-LUC construct demonstrated that the sole transfection of coactivators induced luciferase activity in HepG2 cells. In HeLa cells however, CYP2C9-LUC activity only significantly increased when coactivators were cotransfected with HNF4alpha. A deletion mutant lacking the two proximal HNF4alpha binding sites in the CYP2C9 promoter did not respond to PGC1alpha or SRC1, demonstrating that coactivators were acting through HNF4alpha response elements. Adenovirus-mediated transfection of PGC1alpha in human hepatoma cells caused a significant dose-dependent increase in CYP2C9, CYP1A1, and CYP1A2 and in the positive control CYP7A1. PGC1alpha also showed a moderate activating effect on CYP3A4, CYP3A5, and CYP2D6. Adenoviral transfection of SRC1 had a lessened effect on P450 genes. Chromatin immunoprecipitation assay demonstrated in vivo binding of HNF4alpha and PGC1alpha to HNF4alpha response sequences in the CYP2C9 promoter and to three new regulatory regions in the common 23.3 kilobase spacer sequence of the CYP1A1/2 cluster. Insulin treatment of HepG2 and human hepatocytes caused repression of PGC1alpha and a concomitant down-regulation of P450s. Our results establish the importance of coactivators PGC1alpha and SRC1 for the hepatic expression of human P450s and uncover a new HNF4alpha-dependent regulatory mechanism to constitutively control the CYP1A1/2 cluster.

Adenoviridae↗

PubMed2006

Potential impact of steatosis on cytochrome P450 enzymes of human hepatocytes isolated from fatty liver grafts.

Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of "cellular steatosis" by incubation of cultured hepatocytes with increasing concentrations (0.25-3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45-65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.

Adult↗

PubMed2006

Primary steps of the photochemical reactions of 2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine, the photoproduct of cyamemazine, a phototoxic neuroleptic: comparison with the sulfoxide.

The UVA-absorbing photoproduct resulting from the oxidation of the sulfur atom and of the side chain nitrogen of the phototoxic drug cyamemazine (CMZ) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-phenothiazine) is a potent photodynamic photosensitizer. The photophysical and photochemical properties of this photoproduct (P) (2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine)) have been investigated in neutral buffered aqueous solutions and in ethanol and compared to those of the sulfoxide (S) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-5-oxide-phenothiazine), a CMZ oxidation product of cells. The fluorescence quantum yield (PhiF) of P is 0.25 and 0.21 in pH 7 phosphate buffer and ethanol, respectively. By contrast, S (PhiF = 0.14 in buffer) is practically unfluorescent in alcohol. In buffer, the fluorescence lifetimes of P and S are 10.5 and 11.8 ns, respectively. The transient absorbance of the first excited triplet state (3P1) with a characteristic absorption band peaking at 660 nm (epsilon = 5,300 M(-1) cm(-1)) has been observed by 355 nm laser flash spectroscopy of deaerated phosphate buffer or ethanol solutions. In buffer, the 3P1 lifetime is 0.5 micros. The energy transfer which occurs from the 3P1 to naproxen suggests that the 3P1 energy is greater than 62 kcal mol(-1). Triplet quenching by dioxygen occurs at rate 2.3 x 10(9) M(-1) s(-1). With the triplet benzophenone as actinometer, the 3P1 formation quantum yield is found to be 0. 40 in buffer. The 3P1 state is quenched by ethanol and 2-propanol with bimolecular reaction rate constants of 1.6 and 2.4 x 10(6) M(-1) s(-1), respectively. In buffer, P and S triplet states react with tryptophan, indole and cysteine at rate constants of the order of 10(9) M(-1) s(-1) for Trp and indole and 10(8) M(-1) s(-1) for Cys.

Antipsychotic Agents↗

PubMed2006

Allergic hepatitis induced by drugs.

PURPOSE OF REVIEW: To examine recent advances in our understanding of how drugs can trigger a hypersensitivity reaction in the liver, how tolerance is lost, the mechanisms of damage to hepatocytes and the strategies towards a better assessment of an idiosyncratic drug liver reaction. RECENT FINDINGS: Formation and presentation of drug-protein adducts, or a direct interaction with the major histocompatibility complex/T-cell receptor complex is a necessary but not sufficient stimulus to trigger a hypersensitivity reaction. Liver shows considerable tolerogenic potential towards drug adducts. Recent studies highlight allergic hepatitis as a loss of liver tolerance towards drug antigens, the mechanisms of which are beginning to be unravelled. Cell injury caused by the drug itself, a concomitant inflammatory process, or a coincidental stimulus probably represents the additional signal needed to initiate the allergic process. SUMMARY: Drug-induced liver injury is of concern due to its unpredictable nature and serious clinical implications. Clinically, both hepatocellular injury and cholestasis can occur and most episodes have good clinical prognoses upon drug discontinuation. In a few cases, damage to the liver cells may continue in the form of an autoimmune hepatitis. The available diagnostic tools to confirm an immune-mediated hepatic injury are still very limited, and rely on the lymphocyte transformation test.

Animals↗

PubMed2006

Hepatocyte cell lines: their use, scope and limitations in drug metabolism studies.

Gaining knowledge on the metabolism of a drug, the enzymes involved and its inhibition or induction potential is a necessary step in pharmaceutical development of new compounds. Primary human hepatocytes are considered a cellular model of reference, as they express the majority of drug-metabolising enzymes, respond to enzyme inducers and are capable of generating in vitro a metabolic profile similar to what is found in vivo. However, hepatocytes show phenotypic instability and have a restricted accessibility. Different alternatives have been explored in the past recent years to overcome the limitations of primary hepatocytes. These include immortalisation of adult or fetal human hepatic cells by means of transforming tumour virus genes, oncogenes, conditionally immortalised hepatocytes, and cell fusion. New strategies are currently being used to upregulate the expression of drug-metabolising enzymes in cell lines or to derive hepatocytes from progenitor cells. This paper reviews the features of liver-derived cell lines, their suitability for drug metabolism studies as well as the state-of-the-art of the strategies pursued in order to generate metabolically competent hepatic cell lines.

Animals↗

PubMed2005

Transcriptional regulation of the human hepatic CYP3A4: identification of a new distal enhancer region responsive to CCAAT/enhancer-binding protein beta isoforms (liver activating protein and liver inhibitory protein).

CCAAT/enhancer-binding proteins (C/EBPs) are key transcription factors involved in the constitutive expression of several cytochrome P450 genes in the liver. Their concentration and activity change in several pathophysiological conditions. For instance, during inflammation, released cytokines induce repressive C/EBPbeta-liver inhibitory protein (LIP), which antagonizes constitutive C/EBP transactivators [C/EBPalpha and C/EBPbeta-liver activating protein (LAP)], down-regulating genes such as CYP3A4. However, the mechanism by which hepatic C/EBP factors modulate transcription of the CYP3A4 gene is not known. To elucidate the mechanism of action, we cotransfected luciferase reporter vectors, containing 5'-flanking deletions of the CYP3A4 gene, along with expression vectors for C/EBPbeta-LAP, C/EBPbeta-LIP, and C/EBPalpha, in hepatic (HepG2) and nonhepatic (HeLa) cells. Analysis of the -3557 to -6954 base pair (bp) region demonstrated the existence of a 288-bp sequence at -5.95 kilobases (kb), which showed maximal response to C/EBPbeta-LAP ( approximately 30-fold increase in HepG2 cells). Coexpression of LAP with increasing amounts of LIP reduced the activating effect by approximately 70%. Site-directed mutagenesis of predicted C/EBPbeta binding sites demonstrated the presence of four functional C/EBPbeta-responsive motifs within this distal flanking region. Further experiments using chromatin immunoprecipitation proved the binding of endogenous C/EBPbeta to the -5.95-kilobase enhancer of the CYP3A4 gene in human hepatocytes. Expression of recombinant LAP and LIP by means of adenoviral vectors resulted in their binding to this region, which was followed by activation/repression of CYP3A4. Together, our results uncover a new distal enhancer site in the CYP3A4 gene where C/EBPbeta-LAP binds and activates transcription, whereas the truncated form, C/EBPbeta-LIP, antagonizes LAP activity and causes gene repression.

Animals↗

PubMed2005

Multiparametric characterization by flow cytometry of flow-sorted subpopulations of a human hepatoma cell line useful for drug research.

BACKGROUND: Primary cultured hepatocytes are the closest model to the liver for drug research. However, to overcome its limited availability, the search for hepatic cell lines as an alternative to primary cultures is a matter of current interest. In particular, highly differentiated hepatocellular carcinomas have been proposed as in vitro tools for routine experiments in hepatotoxicity and drug metabolism. METHODS: Cell populations were selected by fluorescence-activated cell sorting based on low and high relative expressions of P-glycoprotein. These cell lines were characterized after 21 days in culture by multiparametric analysis with flow cytometry providing direct information on key cellular functions (stability in culture, intracellular ionic homeostasis, plasmatic and mitochondrial membrane-related parameters, red-ox status, drug transport, and metabolism). RESULTS: Two subpopulations (ADV-1 and ADV-2) from the differentiated and well-characterized human hepatoma BC2 cell line showed increased activity of drug transport and drug biotransformation capability (cytochrome P450 [CYP] 1A2, CYP2B6, CYP3A4, and CYP2Cs). These subpopulations were characterized extensively by multiparametric flow cytometric analysis. CONCLUSION: ADV-1 subpopulation showed greater stability in culture, better efficiency regarding intracellular pH maintenance through the operation of Na+/H+ exchange antiporter, and significantly greater CYP-dependent biotransformation activity than the BC2 parental cells and ADV-2 cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Metabolism and bioactivation of toxicants in the lung. The in vitro cellular approach.

Lung is a target organ for the toxicity of inhalated compounds. The respiratory tract is frequently exposed to elevated concentrations of these compounds and become the primary target site for toxicity. Occupational, accidental or prolonged exposure to a great variety of chemicals may result in acute or delayed injury to cells of the respiratory tract. Nevertheless, lung has a significant capability of biotransforming such compounds with the aim of reducing its potential toxicity. In some instances, the biotransformation of a given compound can result in the generation of more reactive, and frequently more toxic, metabolites. Indeed, lung tissue is known to activate pro-carcinogens (i.e. polycyclic aromatic hydrocarbons or N-nitrosamines) into more reactive intermediates that easily form DNA adducts. Lungs express several enzymes involved in the metabolising of xenobiotics. Among them, cytochrome P450 enzymes are major players in the oxidative metabolism as well metabolic bioactivation of many organic toxicants, including pro-carcinogens. Xenobiotic-metabolising P450 enzymes are expressed in bronchial and bronchiolar epithelium, Clara cells, type II pneumocytes, and alveolar macrophages Individual CYP isoforms have different patterns of localisation within pulmonary tissue. With the aid of sensitive techniques (i.e. reverse transcriptase-polymerase chain reaction, RT-PCR) it has become possible to detect CYP1A1, CYP1B1, CYP2A6, CYP2B6, CYP2E1 and CYP3A5 mRNAs in lung cells. Less conclusive results have been obtained concerning CYP2Cs, CYP2D6 and CYP3A4. CYP3A5 protein appears to be widely present in all lung samples and is localised in the ciliated and mucous cells of the bronchial wall, bronchial glands, bronchiolar ciliated epithelium and in type I and type II alveolar epithelium. Lung cells also express Phase II enzymes such as epoxide hydrolase, UGT1A (glucuronyl transferase) and GST-P1 (glutathione S-transferase), which largely act as detoxifying enzymes. A key question concerning organ-specific chemical toxicity is whether the actual target has the capacity to activate (or efficiently inactivate) chemicals. Results of several studies indicate that the different xenobiotic-metabolising CYPs, present in the human lung and lung-derived cell lines, likely contribute to in situ activation of pulmonary toxins, among them, pro-carcinogens. Some CYPs, in particular CYP1A, are polymorphic and inducible. Interindividual differences in the expression of these CYPs may explain the different risk of developing lung toxicity (possibly cancer), by agents that require metabolic activation. Few cell lines, principally A549, have been used with variable success as an experimental model for investigating the mechanisms of toxicity. Although RT-PCR analysis has evidenced the presence of the major human pulmonary CYP mRNAs, the measurable P450 specific activities are, however, far below those present in human lungs. Detection of the toxicity elicited by reactive metabolites requires the use of metabolically competent cells; consequently, better performing cells are needed to ensure realistic in vitro prediction of toxicity. Genetic manipulation of lung-derived cells allowing them to re-express key biotransformation enzymes appear to be a promising strategy to improve their functionality and metabolic performance.

Air Pollutants↗

PubMed2005

Antitumour activity of fatty acid maltotriose esters obtained by enzymatic synthesis.

The antitumour properties of two fatty acid esters of maltotriose, 6''-O-dodecanoylmaltotriose and 6''-O-palmitoylmaltotriose, in which the 6-OH at the non-reducing end of the maltotriose has been regioselectively acylated, were studied. Both compounds were synthesized by transesterification of vinyl laurate or vinyl palmitate with maltotriose, in the presence of immobilized lipase from Thermomyces lanuginosus. A mixture of 2-methylbutan-2-ol and DMSO (80:20, v/v) was the reaction medium. Their cytotoxic activities against two human cancer cell lines, Hep-G2 and HeLa, were studied. 6''-O-palmitoylmaltotriose showed IC50 values of 2.3 microM (1.7 microg/ml) for Hep-G2 and 3.6 microM (2.7 microg/ml) for HeLa cells, whereas 6''-O-dodecanoylmaltotriose displayed a lower inhibitory effect. 6''-O-palmitoylmaltotriose showed a marginal cytotoxicity to rat hepatocytes, confirming its potential as a new antitumour agent.

Acetylation↗

PubMed2004

Liver grafts preserved in Celsior solution as source of hepatocytes for drug metabolism studies: comparison with surgical liver biopsies.

Suitability of human liver grafts preserved in Celsior solution (CS) for preparing metabolically competent hepatocyte cultures has been examined. To this end, basal and induced activity and mRNA levels of major hepatic cytochrome P450 (P450) enzymes have been measured. By 24 h in culture, measurable levels of the 10 P450 mRNAs studied were found in all hepatocyte preparations examined, with CYP2E1, CYP2C9, and CYP3A4 mRNAs being the most abundant. Compared with hepatocytes obtained from surgical liver resections (SLRs), lower content of each P450 mRNA was found in hepatocytes from the CS group; however, the relative distribution of individual P450 mRNAs was similar. Similar results were observed after measuring P450 activities. CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 activities in hepatocytes from CS-flushed grafts were lower than but comparable with those of cultures prepared from SLRs. No differences in the metabolite profile of testosterone were found. Treatment of hepatocytes from CS-preserved grafts with model P450 inducers shows that 2 microM methylcholanthrene only increased CYP1A1 and CYP1A2 mRNAs (>100-fold over control), 1 mM phenobarbital markedly increased CYP2A6, CYP2B6, and CYP3A4 mRNA content (>7-fold), and 50 microM rifampicin highly increased CYP3A4 mRNA levels (>10-fold), whereas minor effects (<3-fold) were observed in CYP2A6, CYP2B6, and CYP2C9 mRNAs. This induction pattern of P450s was similar, in terms of magnitude, reproducibility, and specificity, to that shown in primary hepatocytes from surgical biopsies. Overall, our results indicate that, cold-preserved in CS, liver grafts constitute a valuable source of human hepatocytes for drug metabolism studies.

Biopsy↗

PubMed2004

Role of hepatocyte nuclear factor 3 gamma in the expression of human CYP2C genes.

Hepatocyte nuclear factor 3 gamma (HNF-3 gamma) is an important transcription factor for the maintenance of specific liver functions. However, its relevance in the expression of human cytochrome P450 (CYP) genes has not yet been explored. Several HNF3 putative binding sites can be identified in human CYP2C 5'-flanking regions. Gene reporter experiments with proximal promoters revealed that HNF-3 gamma transactivated CYP2C8, CYP2C9, and CYP2C19 (25-, 4-, and 4-fold, respectively), but it did not transactivate CYP2C18. However, overexpression of HNF-3 gamma in hepatoma cells by means of a recombinant adenovirus induced CYP2C9, CYP2C18, and CYP2C19 mRNA (4.5-, 20-, and 50-fold, respectively) but did not activate endogenous CYP2C8. The lack of effect of HNF-3 gamma on endogenous CYP2C8 could be reversed by treating cells with the deacetylase inhibitor, trichostatin A, suggesting the existence of chromatin condensation around functional HNF3 elements in this gene. We conclude that HNF3 gamma is an important transcription factor for the hepatic-specific expression of human CYP2C genes. Our results also evidence that efficient transfection tools, such as adenoviral vectors, may be decisive for assessing the role of transcription factor on chromatin organized genes.

Adenoviridae↗

PubMed2004

Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes.

In this study we describe a battery of fluorescence assays for rapid measurement in intact cells of the activity of nine cytochromes P450 (P450s) involved in drug metabolism. The assays are based on the direct incubation of monolayers of cells expressing individual P450 enzymes with a fluorogenic substrate followed by fluorimetric quantification of the product formed and released into incubation medium. For each individual P450 activity, different fluorescence probes were examined, and the one showing the best properties (highest metabolic rates, lowest background fluorescence) was selected: 3-cyano-7-ethoxycoumarin for CYP1A2 and CYP2C19, coumarin for CYP2A6, 7-ethoxy-4-trifluoromethylcoumarin for CYP2B6, dibenzylfluorescein for CYP2C8, 7-methoxy-4-trifluoromethylcoumarin (MFC) for CYP2C9 and CYP2E1, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin for CYP2D6, and 7-benzyloxy-4-trifluoromethylcoumarin for CYP3A4. Fluorescence-based assays are highly sensitive and allow the simultaneous measurement of a large number of samples using plate readers, thus enhancing sample throughput. Major advantages over high-throughput assays in subcellular fractions are that, as living cells are used, manual handling and enzyme damage are minimized, the endoplasmic reticulum of the cells remains intact, exogenous cofactors or NADPH-regenerating systems are not required, and transport processes are maintained. These assays can be applied to preliminary screening of inhibitory effects of new drugs on individual P450 enzymes. After comparison of the results obtained using the fluorescent probes in intact P450-expressing cells and those obtained using the high-performance liquid chromatography-based selective assays in the same cells, in primary human hepatocytes or in human liver microsomes, a fairly good agreement was found.

Cells, Cultured↗

PubMed2004

Drug metabolism by cultured human hepatocytes: how far are we from the in vivo reality?

The investigation of metabolism is an important milestone in the course of drug development. Drug metabolism is a determinant of drug pharmacokinetics variability in human beings. Fundamental to this are phenotypic differences, as well as genotypic differences, in the expression of the enzymes involved in drug metabolism. Genotypic variability is easy to identify by means of polymerase chain reaction-based or DNA chip-based methods, whereas phenotypic variability requires direct measurement of enzyme activities in liver, or, indirectly, measurement of the rate of metabolism of a given compound in vivo. There is a great deal of phenotypic variability in human beings, only a minor part being attributable to gene polymorphisms. Thus, enzyme activity measurements in a series of human livers, as well as in vivo studies with human volunteers, show that phenotypic variability is, by far, much greater than genotypic variability. In vitro models are currently used to investigate the hepatic metabolism of new compounds. Cultured human hepatocytes are considered to be the closest model to the human liver. However, the fact that hepatocytes are placed in a microenvironment that differs from that of the cells in the liver raises the question of to what extent drug metabolism variability observed in vitro actually reflects that in the liver in vivo. This issue has been examined by investigating the metabolism of the model compound, aceclofenac (an approved analgesic/anti-inflammatory drug), both in vitro and in vivo. Hepatocytes isolated from programmed liver biopsies were incubated with aceclofenac, and the metabolites formed were investigated by HPLC. The patients were given the drug during the course of clinical recovery, and the metabolites, largely present in urine, were analysed. In vitro and in vivo data from the same individual were compared. There was a good correlation between the in vitro and in vivo relative abundance of oxidised metabolites (4'-OH-aceclofenac + 4'-OH-diclofenac; Spearman's rho = 0.855), and the hydrolysis of aceclofenac (diclofenac + 4'-OH-aceclofenac + 4'-OH-diclofenac; rho = 0.691), while the conjugation of the drug in vitro was somewhat lower than in vivo. Globally, the metabolism of aceclofenac in vitro correlated with the amount of metabolites excreted in urine after 16 hours (rho = 0.95). Overall, although differing among assays, the in vitro/in vivo metabolism data for each patient were surprisingly similar. Thus, the variability observed in vitro appears to reflect genuine phenotypic variability among the donors.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2003

Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS.

Diclofenac is a non-steroidal anti-inflammatory drug that is widely used clinically but side effects associated with the administration of the drug have been reported. The apoptotic effect of the drug has been evaluated in human and rat hepatocytes. Apoptosis was observed after exposure to sub-cytotoxic concentrations of the drug, without overlapping with cell necrosis. Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 8 and 9 mediate the cell-receptor and the mitochondria-initiated apoptotic pathways, respectively. Inhibition of both caspases prevented activation of downstream caspases, thus indicating that diclofenac at least activates caspase 3 and both effector caspases 8 and 9. The hierarchy of caspase activation by diclofenac was investigated. Analysis of kinetics revealed a simultaneous activation of these caspases that was maximal after 12 hr of exposure to the drug. Inhibitors of MPT, prevented the downstream activation of the caspase cascade, thus showing that diclofenac opened the mitochondrial pore. On the other hand, antioxidants were able to prevent caspase activation by diclofenac, revealing that oxidative stress at the mitochondrial level is in the root of MPT induction and caspase cascade activation. Caspase activation is not mediated by Bid cleavage, suggesting that the cell-receptor pathway seems not to be involved. However, a dose-dependent release of caspase 8 from the mitochondria was observed, indicating that caspase 8 can be processed independently of cell death receptors. Caspases 8 and 9 are very likely the apical caspases in diclofenac-induced apoptosis. In addition, an early dose-dependent increase of bclX(L) expression parallel to the generation of reactive oxygen species in the mitochondria was found. In conclusion, the mitochondrial pathway is very likely the only pathway involved in diclofenac-induced apoptosis, which was related to CYP-mediated metabolism of diclofenac, with the highest apoptotic effect produced by the metabolite 5OH-diclofenac.

Animals↗

PubMed2003

Induction of hepatic heme oxygenase-1 by diclofenac in rodents: role of oxidative stress and cytochrome P-450 activity.

BACKGROUND/AIMS: The role of oxidative stress in diclofenac hepatotoxicity is still not clear. This study examined whether the drug induced heme oxygenase-1 (HO-1), a stress protein. METHODS: HO-1 mRNA and HO activity were measured in mouse liver and in rat hepatocytes after treatment with diclofenac parallel to release of serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) as a marker of hepatic damage. RESULTS: HO-1 was transcriptionally and dose-dependently induced by diclofenac in mouse liver and rat hepatocytes. HO-1 mRNA, ALT and SDH peaked at the same time. Mechanistic studies revealed that the drug synergized with buthionine sulfoximine (BSO) in lowering hepatic glutathione, increased the formation of reactive oxygen intermediates and activated NF-kappaB and AP-1 in rat hepatocytes. HO-1 induction and hepatic damage were increased by BSO and only HO-1 induction was attenuated by the antioxidant N-acetylcysteine. HO-1 induction was also reduced by the cytochrome P-450 inhibitors ketoconazole and tranylcypromine, concomitantly with a significant decrease in the formation of diclofenac oxidative metabolites, which may give rise to reactive compounds. CONCLUSIONS: Acute treatment with diclofenac induces HO-1 in rodent hepatocytes. Induction is influenced by changes in the cellular redox states and by cytochrome P-450 activity and gives a new insight into the response of the liver to diclofenac.

Acetylcysteine↗

PubMed2003

Comparative studies on the cytochrome p450-associated metabolism and interaction potential of selegiline between human liver-derived in vitro systems.

Selegiline was used as a model compound in a project aimed at comparing, evaluating, and integrating different in vitro approaches for the prediction of cytochrome p450 (p450)-catalyzed hepatic drug metabolism in humans (EUROCYP). Metabolic predictions were generated using homology modeling, cDNA-expressed p450 enzymes, human liver microsomes, primary cultured human hepatocytes, and precision-cut human liver slices. All of the in vitro systems correctly indicated the formation of two dealkylated metabolites, desmethylselegiline and methamphetamine. The metabolic instability of selegiline was demonstrated by all of the in vitro systems studied. Estimates of clearance varied from 16 l/h to 223 l/h. With the exception of one approach, all systems underpredicted the in vivo clearance in humans (236 l/h). Despite this, all approaches successfully classified selegiline as a high clearance compound. Homology modeling suggested the participation of CYP2B6 in the demethylation of selegiline and of CYP2D6 in the depropargylation of the drug. Studies with recombinant expressed enzymes and with human hepatic microsomal fraction supported the involvement of CYP2B6 but not of CYP2D6. These techniques also suggested the involvement of CYP1A2, CYP2C8, and CYP2C19 in the biotransformation of selegiline. In vitro, CYP2B6 was the most active form of p450 involved in selegiline metabolism. Metabolism by several enzymes operating in parallel implies a low interaction potential for the drug. None of the techniques alone was able to predict all aspects of the metabolic and kinetic behavior of selegiline in vivo. However, when used as an integrated package, all significant characteristics were predictable.

Cells, Cultured↗

PubMed2003

Strategies and molecular probes to investigate the role of cytochrome P450 in drug metabolism: focus on in vitro studies.

Drug metabolism is the major determinant of drug clearance and, because of polymorphic or inducible expression of drug-metabolising cytochrome P450s (CYPs), is the factor most frequently responsible for interindividual differences in pharmacokinetics. A number of well characterised CYP substrates and inhibitors have been identified that allow precise measurements of individual CYP isoforms. Their use, alone or in combination, facilitates the phenotype characterisation of hepatocytes in vitro and in vivo. Two procedures are used for in vitro investigation of the metabolic profile of a drug: incubation with microsomes and incubation with metabolically competent cells. The major limitation of microsomes is that they express phase I activities, but only part of phase II activities, and can only be used for short incubation times. When intact cells are used, gene expression, metabolic pathways, cofactors/enzymes and plasma membrane are largely preserved, but fully differentiated cells such as primary cultured hepatocytes need to be used, since hepatoma cell lines have only very low and partial CYP expression. CYP-engineered cells or their microsomes ('supersomes') have made the identification of the CYPs involved in the metabolism of a drug candidate straightforward and easier. Inhibition of CYP is an undesirable feature for a drug candidate, and needs to be addressed by examining whether the drug candidate inhibits the metabolism of other compounds or whether other compounds inhibit the metabolism of the drug candidate. Such experiments can be conducted both with microsomes and in cells. The major limitation of microsomes is that inhibition parameters may not accurately reflect the situation in vivo, since the contribution of drug transport is not considered. The best picture of a potential drug-drug interaction can be obtained in metabolically competent hepatocytes. Screening of CYP inducers cannot be done in microsomes. It requires the use of a cellular system fully capable of transcribing and translating CYP genes, and can be monitored in vitro as an increase in enzyme mRNA or activity. Human hepatocytes in primary culture respond well to enzyme inducers during the first few days; this ability is lost thereafter. Rat hepatocytes are much less stable and soon become unresponsive to inducers. Hepatoma cell lines respond poorly to inducers, although the induction of a few isoenzymes has been reported. Primary cultured hepatocytes are still the unique in vitro model that allows global examination of the inductive potential of a drug.

Animals↗