M. José Gómez-Lechón

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.

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.

Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices: comparison with other hepatic cellular models

BACKGROUND/AIMS The pig is considered the best donor of hepatocytes for bioartificial liver devices, but little is known about the metabolic capability of pig hepatocytes. Therefore, we have evaluated drug metabolizing activities in pig hepatocytes and liver microsomes and compared the results with those of man and other animal hepatic cellular models that are potential sources of cells for bioreactors, such as rat, rabbit and dog hepatocytes and hepatoma cell lines. METHODS Total cytochrome P450 levels, six phase 1 activities representative of the most relevant cytochrome P450 enzymes (7-ethoxycoumarin O-deethylase, 7-ethoxy-, 7-methoxy- and 7-benzoxyresorufin O-dealkylases, coumarin 7-hydroxylase and p-nitrophenol hydroxylase), two phase 2 activities (glutathione S-transferase and UDP-glucuronyltransferase) and CYP-dependent regioselective testosterone metabolism were evaluated in in vitro models of different species. RESULTS The pattern of specific cytochrome P450 activities and the metabolic profile of testosterone in intact hepatocytes were essentially the same as those measured in liver microsomes. Relatively low ethoxy-, methoxy-, and benzoxyresorufin O-dealkylation rates were found in pig liver microsomes and hepatocytes as compared to hepatic in vitro human models. However, in contrast with the other species studied, stereoselective testosterone oxidation profiles were practically identical in human and pig models. Finally, the metabolic capability of hepatoma cell lines was very limited in comparison with that of hepatocytes. CONCLUSIONS Pig hepatocytes are able to maintain in culture the phase 1 and phase 2 activities found in liver microsomes. The high metabolic similarities found between pig and human hepatocytes lend support to the use of pig hepatocytes in bioartificial liver devices.

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

SummaryHigh yields of human hepatocytes (up to 23×106 viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10−8M insulin, and 10−8M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg−1·min−1) similar to that reported for human liver. Insulin at 10−8M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10−9M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α1-antitrypsin, α1-antichymotrypsin, α1-acid glycoprotein, haptoglobin, α2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10−9M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg−1 cell protein·min−1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg−1).The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.

Development of a Multiparametric Cell-based Protocol to Screen and Classify the Hepatotoxicity Potential of Drugs

Hepatotoxicity is a major reason for drug nonapprovals and withdrawals. The multiparametric analysis of xenobiotic toxicity at the single cells level using flow cytometry and cellular imaging-based approaches, such as high-content screening (HCS) technology, could play a key role in the detection of toxicity and the classification of compounds based on patterns of cellular injury. This study aimed to develop and validate a practical, reproducible, in vitro multiparametric cell-based protocol to assess those drugs that are potentially hepatotoxic to humans and to suggest their mechanisms of action. The assay was applied to HepG2 human cell line cultured in 96-well plates and exposed to 78 different compounds for 3 and 24 h at a range of concentrations (1-1000μM). After treatments, cells were simultaneously loaded with five fluorescent dyes showing optical compatibility and were then analyzed with the High-Content Screening Station Scan^R (Olympus). By using the new technology of HCS cell parameters associated with nuclear morphology, plasma membrane integrity, mitochondrial function, intracellular calcium concentration, and oxidative stress, indicative of prelethal cytotoxic effects and representative of different mechanisms of toxicity, were measured at the single cells level, which allows high-throughput screening. This strategy appears to identify early and late events in the hepatotoxic process and also suggests the mechanism(s) implicated in the toxicity of compounds to thereby classify them according to their degree of injury (no injury, low, moderate, and high injury).

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.

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.

Drug metabolizing enzymes in rat hepatocytes co-cultured with cell lines

SummaryWe have developed new co-cultures of continuous cell lines 3T3 (clone A31) and C3H/10T1/2 (colone 8) with hepatocytes as an alternative to co-cultures with nonconinuous epithelial cells. In this biological system we studied in detail the expression of the hepatic biotransformation system. After 7 d in culture, total cytochrome P-450 content and the monooxygenase activities aryl hydrocarbon hydroxylase and 7-ethoxycoumarino-deethylase still maintained about 30% of their initial value, whereas in pure cultured hepatocytes these activities were undetectable. A significant response to induction by methylcholanthrene and phenobarbital of monooxygenase activities was observed in co-cultures for 7 d. NADPH-cytochrome c reductase activity remained unchanged for at least 7 d in co-cultured hepatocytes, whereas in pure cultures this activity was reduced to about 75% of the initial value after only 24 h. Finally, the activity of the conjugating enzymes UDP-Gt and GSH-t was maintained at nearly the initial levels during the complete period of study. The easy handling of continuous cell lines and the maintenance of the biotransformation system of hepatocytes in co-culture make this approach simpler and easier to standardize.

Competency of different cell models to predict human hepatotoxic drugs

Introduction: The liver is the most important target for drug-induced toxicity. This vulnerability results from functional liver features and its role in the metabolic elimination of most drugs. Drug-induced liver injury is a significant leading cause of acute, chronic liver disease and an important safety issue when developing new drugs. Areas covered: This review describes the advantages and limitations of hepatic cell-based models for early safety risk assessment during drug development. These models include hepatocytes cultured as monolayer, collagen-sandwich; emerging complex 3D configuration; liver-derived cell lines; stem cell-derived hepatocytes. Expert opinion: In vitro toxicity assays performed in hepatocytes or hepatoma cell lines can potentially provide rapid and cost–effective early feedback to identify toxic candidates for compound prioritization. However, their capacity to predict hepatotoxicity depends critically on cells’ functional performance. In an attempt to improve and prolong functional properties of cultured cells, different strategies to recreate the in vivo hepatocyte environment have been explored. 3D cultures, co-cultures of hepatocytes with other cell types and microfluidic devices seem highly promising for toxicological studies. Moreover, hepatocytes derived from human pluripotent stem cells are emerging cell-based systems that may provide a stable source of hepatocytes to reliably screen metabolism and toxicity of candidate compounds.

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.