Ramiro Jover

Human Hepatocytes in Primary Culture: The Choice to Investigate Drug Metabolism in Man

Different types of hepatic tissue, including whole or split livers from organ donors or waste liver from therapeutic liver resections, are used to prepare human hepatocyte cultures. Characteristics of liver samples from different origins (gender, age, healthy/pathological status, xenobiotic treatment) as sources of human hepatocytes are key factors which notably determine viability and functionality of hepatocytes. The characterisation of the CYP system can be assessed in terms of activity (using specific substrates/inhibitors), protein (antibody analysis) and molecular biology-based mRNA amplification techniques (PCR technology and DNA microarrays). It could reasonably be considered that human hepatocytes reflect the heterogeneity of CYP expression in human liver and is a suitable model for drug metabolism studies. Several key issues need to be addressed at the early stages of drug development to better select drug candidates (metabolic profile and rate, identification of CYPs involved, drug-drug interactions due to enzyme induction/inhibition). The metabolic stability and metabolite profile of new chemicals can be easily investigated by incubating the drugs with fully competent metabolic models like hepatocyte suspensions or 24 h-cultured hepatocytes. CYP inhibitory effects are usually screened in recombinant CYP enzymes or microsomes, however, the actual concentration of substrate and inhibitor available to the CYP enzyme depends on processes missing in subcellular models (transport mechanisms, cytosolic enzymes, binding to intracellular proteins). Since intact cells more closely reflect the environment to which drugs are exposed in the liver, cultured hepatocytes constitute a more predictive model for drug-drug interactions. Screening of CYP inducers cannot be done in microsomes as it requires a cellular system fully capable of expressing CYP genes. Primary hepatocytes are still the unique in vitro model for global examination of inductive potential of drugs (monitored as increases in mRNA content or activity).

Human Hepatocytes as a Tool for Studying Toxicity and Drug Metabolism

Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.

Down-regulation of human CYP3A4 by the inflammatory signal interleukin-6: molecular mechanism and transcription factors involved

The hepatic drug‐metabolizing cytochrome P‐450 (CYP) enzymes are down‐regulated during inflammation. In vitro studies with hepatocytes have shown that the cytokines released during inflammatory responses are largely responsible for this CYP repression. However, the signaling pathways and the cytokine‐activated factors involved remain to be properly identified. Our research has focused on the negative regulation of CYP3A4 (the major drug‐metabolizing human CYP) by interleukin 6 (IL‐6) (the principal regulator of the hepatic acute‐phase response). CYP3A4 down‐regulation by IL‐6 requires activation of the glycoprotein receptor gp130; however, it does not proceed through the JAK/STAT pathway, as demonstrated by the overexpression of a dominant‐negative STAT3 factor by means of an adenoviral vector. The involvement of IL‐6‐activated kinases such as extracellular signal‐regulated kinase ERK1/2 or p38 is also unlikely, as evidenced by the use of specific chemical inhibitors. It is noteworthy that IL‐6 caused a moderated induction in the mRNA of the transcription factor C/EBPβ (CCAAT‐ enhancer binding protein β) and a marked increase in the translation of C/EBPβ‐LIP, a 20‐kDa C/EBPβ isoform lacking a transactivation domain. Adenovirus‐mediated expression of C/EBPβ‐ LIP caused a dose‐dependent repression of CYP3A4 mRNA, whereas overexpression C/EBPα and C/EBPβ‐LAP (35 kDa) caused a significant induction. Our results support the idea that IL‐6 down‐regulates CYP3A4 through translational induction of C/EBPβ‐LIP, which competes with and antagonizes constitutive C/EBP transactivators. From a clinical point of view, these findings could be relevant in the development of therapeutic cytokines with a less repressive effect on hepatic drug‐metabolizing enzymes.

Long‐term expression of differentiated functions in hepatocytes cultured in three‐dimensional collagen matrix

Hepatocytes entrapped in collagen gel and cultured in serum‐free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c‐fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin‐stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 ± 0.37 and 9 ± 2.7 nmol glucose/h/μg DNA). Collagen‐cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 ± 4 nmol urea/h/μg DNA). The rate of albumin synthesis in collagen‐entrapped cells was maintained above the day‐1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 ± 152 pmol/μg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen‐cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and gluthatione‐transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione‐transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP‐α and ‐β, and HNF‐1 and ‐4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate‐synthetase I). J Cell Physiol 177:553–562, 1998.

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 invitro 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.

Re-expression of C/EBPα induces CYP2B6, CYP2C9 and CYP2D6 genes in HepG2 cells

Cytochrome P450 (CYP) activity is very low or even absent in human hepatomas, a phenomenon that is accompanied by low levels of some liver transcription factors, notably C/EBPα. To investigate a possible link between this transcription factor and hepatic CYP expression, we have stably transfected HepG2 cells with a C/EBPα vector containing a Zn‐inducible metallothionein promoter. Expression of functional C/EBPα up to liver levels concomitantly increased the mRNAs of several members of the CYP2 family (2B6, 2C9 and 2D6), suggesting that this transcription factor may play a relevant role in controlling the hepatic expression of CYP enzymes.

Enhanced steatosis by nuclear receptor ligands: A study in cultured human hepatocytes and hepatoma cells with a characterized nuclear receptor expression profile

Steatosis is the first step in the development of non-alcoholic fatty liver disease (NAFLD). However, the mechanisms involved in its pathogenesis are not fully understood. Many nuclear receptors (NRs) involved in energy homeostasis and biotransformation constitute a network connecting fatty acids, cholesterol and xenobiotic metabolisms; therefore, multiple NRs and their ligands may play a prominent role in liver fat metabolism and accumulation. In this study we have attempted to gain insight into the relevance of the NR superfamily in NAFLD by investigating the steatogenic potential of 76 different NR ligands in fatty acid overloaded human hepatocytes and hepatoma cells. Moreover, we have determined the mRNA expression level of 24 NRs to correlate the steatogenic potential of the ligands with the expression of their associated NRs in the cultured cells. Our results demonstrate that 18% of the examined NR ligands enhanced lipid accumulation in human hepatocytes and/or hepatoma cells. Among them, ligands of PPARgamma (e.g., thiazolidinediones), LXR (paxilline and 24(S),25-epoxycholesterol), PXR (hyperforin), CAR (3alpha,5alpha-androstenol), ERalpha (tamoxifen), FXR (Z-guggulsterone), VDR (25-hydroxyvitamin D3) and particular retinoids and farnesoids showed a significant pro-steatotic effect. The mRNA level of most of the NRs examined was well preserved in human hepatocytes, but HepG2 showed a deranged profile, where many of the receptors had a marginal or negligible level of expression in comparison with the human liver. By comparing the steatogenic effect of NR ligands with the NR expression levels, we conclude that LXR, PXR, RAR and PPARgamma ligands likely induce fat accumulation by a NR-dependent mechanism. Indeed, over-expression of PXR in HepG2 cells enhanced the steatogenic effect of hyperforin and rifampicin. However, the accumulation of fat induced by other ligands did not correlate with the expression of their associated NR. Our results also suggest that human hepatocytes cultured with free fatty acids offer a highly valuable in vitro system to investigate the pathogenesis and therapeutics of the human fatty liver.

Evaluation of the cytotoxicity of ten chemicals on human cultured hepatocytes: Predictability of human toxicity and comparison with rodent cell culture systems

The cytotoxic effect of the first 10 chemicals on the MEIC list (evaluated in the Multicentre Evaluation of In Vitro Cytotoxicity organized by the Scandinavian Society of Cell Toxicology) was evaluated on human and rat cultured hepatocytes and in the non-hepatic murine 3T3 cell line. The MTT test was used as an endpoint to evaluate cytotoxicity after 24 hr of exposure to the chemicals. The predictability of human toxicity using human hepatocytes was analysed and compared with the results using rodent cell culture systems and rat and mouse LD(50) tests. Ferrous sulphate, diazepam and isopropyl alcohol produced about the same toxicity in all three cell culture models; paracetamol and acetylsalicylic acid were more toxic to human and rat hepatocytes than to mouse 3T3 cells; amitriptyline, ethylene glycol, methanol and ethanol were more toxic to human hepatocytes than to rodent cells. Digoxin was the most cytotoxic chemical to human hepatocytes (IC(50), 4.9 nm), the alcoholic compounds (isopropanol, ethylene glycol, ethanol and methanol) were the least toxic (IC(50), 125-819 mm) and paracetamol, acetylsalicylic acid, ferrous sulphate, diazepam and amitriptyline showed intermediate cytotoxicities (IC(50), 0.05-6 mm). The data suggest that for these 10 chemicals, acute toxicity in humans was more accurately predicted using human hepatocytes than using rat hepatocytes or mouse non-hepatic 3T3 cells.

Transcriptional Regulation of Cytochrome P450 Genes by the Nuclear Receptor Hepatocyte Nuclear Factor 4-Alpha

Hepatocyte nuclear factor 4-alpha (HNF4alpha, NR2A1) is a nuclear receptor (NR) required for liver development and for controlling the expression of many hepatic-specific genes associated with important metabolic pathways. Many studies have also identified HNF4alpha as a direct transactivator of numerous xenobiotic-metabolizing cytochrome P450 (CYP) genes, suggesting that this factor is a global regulator which supports CYP transcription in the liver. Moreover, HNF4alpha expression displays a significant variability in human liver which may account for a proportion of the inter-individual variability in the expression of drug-metabolism genes and the clearance rate of a wide variety of prescribed drugs. In the last few years, a number of complex interactions and cross-talks between HNF4alpha and other transcription factors and coregulators have also surfaced, and the impact on CYP gene expression has been demonstrated. Thus, it is now clear that HNF4alpha modulates CYP expression in the liver by interacting with the xenosensor receptors (PXR and CAR), the glucocorticoid receptor (GR), the feeding-fasting cycle target PGC-1alpha, the sexual-dimorphism factor Stat5b, and other liver-enriched factors, such as C/EBPs. In addition to regulating drug elimination pathways, HNF4alpha also triggers pleiotropic effects on cholesterol and fatty acid metabolism, glucose homeostasis and inflammation. As a whole, current evidence indicates that HNF4alpha is a central regulator in the network of NRs that integrates drug-metabolism not only with the liver intermediate metabolism, but also with a number of patho-physiological conditions where the CYP expression is altered. The purpose of this review is to summarize and discuss these studies and their conclusions, with particular emphasis on the role of HNF4alpha in the regulation of drug-metabolizing CYP genes in the human liver.

Transcriptional Activation of CYP2C9, CYP1A1, and CYP1A2 by Hepatocyte Nuclear Factor 4α Requires Coactivators Peroxisomal Proliferator Activated Receptor-γ Coactivator 1α and Steroid Receptor Coactivator 1

Hepatocyte nuclear factor 4α (HNF4α) 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 HNF4α but express only marginal P450 levels. We found that the HNF4α-mediated P450 transcription in HepG2 is impaired by the low level of coactivators peroxisomal proliferator activated receptor-γ coactivator 1α (PGC1α) 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 HNF4α. A deletion mutant lacking the two proximal HNF4α binding sites in the CYP2C9 promoter did not respond to PGC1α or SRC1, demonstrating that coactivators were acting through HNF4α response elements. Adenovirus-mediated transfection of PGC1α in human hepatoma cells caused a significant dose-dependent increase in CYP2C9, CYP1A1, and CYP1A2 and in the positive control CYP7A1. PGC1α 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 HNF4α and PGC1α to HNF4α 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 PGC1α and a concomitant down-regulation of P450s. Our results establish the importance of coactivators PGC1α and SRC1 for the hepatic expression of human P450s and uncover a new HNF4α-dependent regulatory mechanism to constitutively control the CYP1A1/2 cluster.