Hepatocyte transplantation for inherited metabolic diseases of the liver.

Inherited metabolic diseases of the liver are characterized by deficiency of a hepatic enzyme or protein often resulting in life-threatening disease. The remaining liver function is usually normal. For most patients, treatment consists of supportive therapy, and the only curative option is liver transplantation. Hepatocyte transplantation is a promising therapy for patients with inherited metabolic liver diseases, which offers a less invasive and fully reversible approach. Procedure-related complications are rare. Here, we review the experience of hepatocyte transplantation for metabolic liver diseases and discuss the major obstacles that need to be overcome to establish hepatocyte transplantation as a reliable treatment option in the clinic.

Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in nontransformed, normal cells. Preclinical studies in mice and nonhuman primates have shown that administration of TRAIL can induce apoptosis in human tumors, but that no cytotoxicity to normal organs or tissues is found. The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells has lead to a proposal to use TRAIL in cancer therapy. Here, we assessed the sensitivity of hepatocytes from rat, mouse, rhesus monkey and human livers to TRAIL-induced apoptosis. TRAIL induced apoptosis in normal human hepatocytes in culture but not in hepatocytes isolated from the other species. Human hepatocytes showed characteristic features of apoptosis, including cytoplasmic shrinkage, the activation of caspases and DNA fragmentation. Apoptosis and cell death in human hepatocytes was massive and rapid, occurring in more than 60% of the cells exposed to TRAIL within 10 hours. These results indicate that there are species differences in sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used in human cancer therapy.

Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium.

Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFalpha and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPalpha and C/EBPbeta, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFkappaB. The liver associated transcription factors HNFI, HNF3, and HNF4 are preserved throughout this process. After population expansion and clonal growth, the proliferating hepatocytes can return to mature hepatocyte phenotype in the presence of EHS gel (Matrigel). This includes complete restoration of electron microscopic structure and albumin expression. The hepatocyte cultures however can instead be induced to form acinar/ductular structures akin to bile ductules (in the presence of HGF/SF and type I collagen). These transformations affect the entire population of the hepatocytes and occur even when DNA synthesis is inhibited. Similar acinar/ductular structures are seen in embryonic liver when HGF/SF and its receptor are expressed at high levels. These findings strongly support the hypothesis that mature hepatocytes can function as or be a source of bipotential facultative hepatic stem cells (hepatoblasts). These studies also provide evidence for the growth factor and matrix signals that govern these complex phenotypic transitions of facultative stem cells which are crucial for recovery from acute and chronic liver injury.

In vitro methods in human drug biotransformation research: implications for cancer chemotherapy.

Anticancer drugs have a complex pharmacological and toxicological profile with a narrow therapeutic index. It is therefore critical to understand the factors that contribute to the marked intersubject variability in the pharmacokinetics and pharmacodynamics often observed with anticancer compounds. Since hepatic and extra-hepatic drug metabolism represents a major drug disposition pathway, extensive efforts are made to thoroughly investigate metabolism of anticancer compounds during the pre-clinical and clinical development phases as well as to address issues encountered during the clinical use of an approved drug. In recent years there has been a significant paradigm shift in pre-clinical/non-clinical drug metabolism studies. Most importantly, this has included a reduced reliance on animal models and increased use of human tissues (i.e. human liver microsomes and other cellular fractions, primary culture of human hepatocytes, cDNA expressed human-specific enzymes and cell-based reporter assays). Typically, experiments are performed using these tools to identify the phase I and/or phase II enzymes involved in metabolism of the drug/investigational agent and for metabolic fingerprinting. Additionally, issues pertaining to the rate, extent and mechanism(s) of the inhibition or induction of the metabolic pathways are also investigated. These studies provide important clues about various aspects of the disposition of a therapeutic agent including first-pass metabolism, elimination half-life, overall bioavailability and the potential for drug-drug interactions. The methodologies used for in vitro assessment of drug metabolism and their applications to drug development and clinical therapeutics with special emphasis on anticancer drugs are reviewed in this manuscript.

Historical aspects of hepatocyte transplantation.

Hepatocyte transplantation has been used for temporary metabolic support of patients in end-stage liver failure awaiting whole organ transplantation as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure and as a "cellular therapy" for patients with genetic defects in vital liver functions. The aim of this paper was to discuss the basic research that led to clinical hepatocyte transplantation, the published clinical experience with this experimental technique, and some possible future uses of hepatocyte transplantation.

Isolation, culture, and transplantation of human hepatocytes.

The in situ two-step collagenase perfusion technique used for the isolation of hepatocytes from rat liver was adapted into a procedure applicable to pieces of human liver obtainable from surgical procedures. Human hepatocytes obtained by this method were maintained in primary culture for 10 days. The cellular changes observed at the light microscopic and electron microscopic levels are described. The changes in microsomal enzymes as a function of the age of the cultures were also measured. Exposure of the human hepatocytes to procarcinogens known to be metabolized by rodent liver resulted in unscheduled DNA synthesis. The isolated hepatocytes were also transplanted into two-thirds partially hepatectomized athymic nude mice. The transplanted cells formed nodules with characteristic hepatic architecture. These studies demonstrate that hepatocytes obtained from human liver by the described modified collagenase technique can be used for in vitro studies in chemical carcinogenesis.

Mutagenesis induced by procarcinogens at the hypoxanthine-guanine phosphoribosyl transferase locus of human fibroblasts cocultured with rat hepatocytes.

The addition of diethylnitrosamine or cyclophosphamide in cultures of hepatocytes overlaid on confluent diploid human fibroblasts resulted in a dose-dependent increase in the frequency of hypoxanthine-guanine phosphoribosyl transferase human fibroblast mutants with both chemicals. Different toxicity patterns for the two cell types were seen. Diethylnitrosamine was more toxic to the hepatocytes, whereas cyclophosphamide was more toxic to the fibroblasts. These data open the possibility of using strains of human fibroblasts as in vitro screens for mutagenicity of procarcinogens.

Liver regeneration studies with rat hepatocytes in primary culture.

Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.

Introduction of a Ha-ras oncogene into rat liver epithelial cells and parenchymal hepatocytes confers resistance to the growth inhibitory effects of TGF-beta.

Growth of rat liver epithelial cells (RLEC) and primary cultures of parenchymal hepatocytes is potently inhibited by TGF-beta. Transfection of a mutated Ha-ras oncogene, but not a human c-myc oncogene, into RLEC resulted in cell lines resistant to growth inhibition by TGF-beta under anchorage-dependent conditions. Infection of primary rat hepatocyte cultures with v-Ha-ras yielded a cell line likewise insensitive to inhibition by TGF-beta. Binding of [125I]TGF-beta to Ha-ras-transfected RLEC was reduced relative to control or c-myc-transfected cells. These data suggest that activation of a Ha-ras oncogene in epithelial cells may result in escape from negative growth control and hence be a critical step during carcinogenesis. However, although Ha-ras induced resistance to growth inhibition by TGF-beta under anchorage-dependent conditions, TGF-beta inhibited the spontaneous growth in soft agar of all cell lines containing the Ha-ras oncogene. This may reflect an alteration in regulation of extracellular matrix proteins and related enzymes responsible for anchorage-independent growth.

Human cytochrome P450 3A (CYP3A) mediated midazolam metabolism: the effect of assay conditions and regioselective stimulation by alpha-naphthoflavone, terfenadine and testosterone.

The effect of ionic strength, assay constituents, alpha-naphthoflavone (aNF), terfenadine and testosterone on human CYP3A mediated midazolam (MDZ) 1'-hydroxylation (MDZ 1'-OH) and 4-hydroxylation (MDZ 4-OH) in vitro was examined. Increasing concentration of Tris-HCl (Tris) and sodium phosphate (PO4) buffers differentially affected MDZ 1'-OH and MDZ 4-OH formation rates and had a different effect on MDZ metabolism mediated by microsomes containing CYP3A4 versus CYP3A4 and CYP3A5. MDZ metabolism was not affected by PO4 buffer concentration when cumene hydroperoxide (CUOOH) was used as the source of reactive oxygen. Interestingly, the ammonium ion present in the solution of glucose 6-phosphate dehydrogenase was found to inhibit MDZ metabolism. The addition of MgCl2 up to 50 mM and CaCl2 (5-30 mM) had no affect or inhibited MDZ metabolism, respectively. Formation of MDZ 1'-OH by microsomes from adult and fetal liver and expressed CYP3A4 was regioselectively stimulated by aNF (10 microM). In human hepatocytes, aNF stimulated MDZ 1'-OH formation (up to 100%). Terfenadine (20 microM) regioselectively stimulated MDZ 1'-OH formation in Tris (1-200 mM) and PO4 (1-10 mM) buffers by up to 159%. Surprisingly, with expressed CYP3A4, terfenadine (20 microM) inhibited MDZ 1'-OH formation. Terfenadine (20 microM) had little effect on MDZ 1'-OH formation by fetal liver microsomes. Testosterone (10 and 100 microM) regioselectively stimulated (up to 269%) MDZ 4-OH formation by adult liver microsomes and expressed CYP3A4. Testosterone (100 microM) inhibited (> 40%) MDZ 1'-OH and MDZ 4-OH formation by fetal liver microsomes. With adult liver microsomes, aNF and terfenadine had little effect on the Km for MDZ 1'-OH formation. However, the Km for MDZ 4-OH formation was decreased (up to 94%) by 100 microM testosterone. In the presence of CUOOH, no stimulation of MDZ metabolism was observed by aNF, terfenadine or testosterone in adult liver microsomes. These studies indicate that because assay conditions can substantially alter the catalytic activity of CYP3A, caution should be exerted when extrapolating results between in vitro and in vivo, and when results from different laboratories are compared. Further, these results suggest that the stimulation of CYP3A4 may also occur in vivo and, consequently, may have clinical importance.