Species differences in the response of liver drug-metabolizing enzymes to (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949) in vivo and in vitro.

Induction of drug-metabolizing enzymes (DMEs) is highly species-specific and can lead to drug-drug interaction and toxicities. In this series of studies we tested the species specificity of the antidiabetic drug development candidate and mixed peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949, EMD) with regard to the induction of gene expression and activities of DMEs, their regulators, and typical PPAR target genes. EMD clearly induced PPARalpha target genes in rats in vivo and in rat hepatocytes but lacked significant induction of DMEs, except for cytochrome P450 (P450) 4A. CYP2C and CYP3A were consistently induced in livers of EMD-treated monkeys. Interestingly, classic rodent peroxisomal proliferation markers were induced in monkeys after 17 weeks but not after a 4-week treatment, a fact also observed in human hepatocytes after 72 h but not 24 h of EMD treatment. In human hepatocyte cultures, EMD showed similar gene expression profiles and induction of P450 activities as in monkeys, indicating that the monkey is predictive for human P450 induction by EMD. In addition, EMD induced a similar gene expression pattern as the PPARalpha agonist fenofibrate in primary rat and human hepatocyte cultures. In conclusion, these data showed an excellent correlation of in vivo data on DME gene expression and activity levels with results generated in hepatocyte monolayer cultures, enabling a solid estimation of human P450 induction. This study also clearly highlighted major differences between primates and rodents in the regulation of major inducible P450s, with evidence of CYP3A and CYP2C inducibility by PPARalpha agonists in monkeys and humans.

Isolation and culture of primary human hepatocytes.

As our knowledge of the species differences in drug metabolism and drug-induced hepatotoxicity has expanded significantly, the need for human-relevant in vitro hepatic model systems has become more apparent than ever before. Human hepatocytes have become the "gold standard" for evaluating hepatic metabolism and toxicity of drugs and other xenobiotics in vitro. In addition, they are becoming utilized more extensively for many kinds of biomedical research, including a variety of biological, pharmacological, and toxicological studies. This chapter describes methods for the isolation of primary human hepatocytes from liver tissue obtained from an encapsulated end wedge removed from patients undergoing resection for removal of liver tumors or resected segments from whole livers obtained from multiorgan donors. The maintenance of normal cellular physiology and intercellular contacts in vitro is of particular importance for optimal phenotypic gene expression and response to drugs and other xenobiotics. As such, methods are described for culturing primary hepatocytes under various matrix compositions and geometries. Differential expression of liver-selective properties occurs over time in primary hepatocytes dependent on the culture and study conditions. Overall, improved isolation and cultivation methods have allowed for exciting advances in our understanding of the pathology, biochemistry, and cellular and molecular biology of human hepatocytes.

Effect of chrysin and natural coumarins on UGT1A1 and 1A6 activities in rat and human hepatocytes in primary culture.

Flavonoids and coumarins are naturally occurring compounds that are widely distributed in vegetables and have a broad pharmacological activity. Inducibility of UDP-glucuronosyltransferases (UGTs) by xenobiotics is well documented and can be considered beneficial for health. In particular, UGT1A1-dependent bilirubin conjugation plays a critical role in the detoxification of neurotoxic bilirubin and phenobarbital-mediated UGT1A1 induction therapy is commonly used in the treatment of unconjugated hyperbilirubinemic diseases such as Crigler-Najjar type II disease. In the present study, the effects of the flavone chrysin and six natural coumarins isolated from various Rutaceous plants on UGT1A6-dependent P-nitrophenol and/or UGT1A1-dependent bilirubin glucuronoconjugation activities were evaluated in cultured rat and human hepatocytes and compared to those of the prototypical UGT1A inducers beta-naphthoflavone, phenobarbital and clofibric acid. After 3 days of treatment at a concentration of 25 microM, the pyranocoumarins avicennin and CIS-avicennol, and the furocoumarins bergapten and imperatorin, increased by 2-fold UGT1A1-dependent activity, equivalent to the increases obtained with chrysin at 25 microM, whereas in the presence of the simple coumarins such as coumarin or umbelliferone, UGT1A1-dependent activity was not modified. In terms of structural requirements for UGT1A1 induction, the present study suggests that the B-ring (phenyl) for chrysin and the furan or pyran rings for coumarins are essential for the biological activity.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study.

BACKGROUND: The European Center for Validation of Alternative Methods (ECVAM) has funded a prevalidation study in three laboratories (France, USA and UK) on the use of human hepatocyte cultures to predict cytochrome P-450 induction. AIMS AND METHODS: As first stage of this prevalidation study, the purpose of the present work was to set criteria for optimization and harmonization of hepatocyte isolation from human tissue among laboratories to establish a routine procedure. This was achieved by combining and/or comparing the data generated by the two independent European laboratories (France and UK). RESULTS: The results confirmed that surgical waste material is a valuable source for obtaining high quality hepatocytes under certain pre-, intra- and post-operative conditions: cell yield of viable hepatocytes was not significantly affected by age and sex of patients, nor indications for resection, steatosis or cholestasis. Cold ischeamia up to 5 hours did not influence viable cell yield allowing transport of material. CONCLUSION: The use of biopsy sizes between 50-100 g, cannulation with 2-4 cannulae, digestion with collagenase-containing digestion medium at a flow rate of 25 ml/cannula for 20 minutes, with cut surface being glued in order to reform Glisson's capsule, should optimize the total yield of viable human hepatocytes obtained per preparation of waste liver surgical resections.