Cultured human primary hepatocytes

The application of this technology for determining CYP induction in primary hepatocytes was first described by Strong et al. (40). To further demonstrate the potential of this technology, a study was conducted in our laboratory using primary human primary hepatocytes cultured on a 96-well plate precoated... 

D Cultures and Primary hepatocytes from human liver remain the gold standard for... 

Human primary hepatocytes remain as the best cell model for human liver, and the recent advances in 3D culture techniques have solved some of the problems with rapid decline in metabolic competence. However, the limited availability of human liver remains as a botdeneck. Therefore there is a clear need for more reliable and consistent source of metabolically competent hepatocytes. Previously in this chapter we have discussed the pros and cons of the HepaRG cell line, which currently represents the best... 

The metabolism of ochratoxin A was studied in cultured rat and human primary hepatocytes incubated with non-cytotoxic concentrations of PH]ochratoxin A ranging from 10 to 10 mol/l for 8 h. In rat hepatocytes, ochratoxin A was metabolized to small amounts of three products. In addition to 4-hydroxy-ochratoxin A, which is a known product of ochratoxin A biotransformation, two novel metabolites were detected and tentatively identified as hexose and pentose conjugates of ochratoxin A. In vitro induction with 3-methylcholanthrene increased the formation of 4-hydroxy-ochratoxin A but did not alter the formation of the conjugated metabolites (Gross-Steinmeyer et al., 2002). 

Gross-Steinmeyer, K., Weymann, J., Hege, H.G. Metzler, M. (2002) Metabolism and lack of DNA reactivity of the mycotoxin ochratoxin A in cultured rat and human primary hepatocytes. J. Agric. Food Chem. 50, 938-945. 

Chelators of iron, which are now widely applied for the treatment of patients with thalassemia and other pathologies associated with iron overload, are the intravenous chelator desferal (desferrioxamine) and oral chelator deferiprone (LI) (Figure 19.23, see also Chapter 31). Desferrioxamine (DFO) belongs to a class of natural compounds called siderophores produced by microorganisms. The antioxidant activity of DFO has been studied and compared with that of synthetic hydroxypyrid-4-nones (LI) and classic antioxidants (vitamin E). It is known that chronic iron overload in humans is associated with hepatocellular damage. Therefore, Morel et al. [370] studied the antioxidant effects of DFO, another siderophore pyoverdin, and hydroxypyrid-4-ones on lipid peroxidation in primary hepatocyte culture. These authors found that the efficacy of chelators to inhibit iron-stimulated lipid peroxidation in hepatocytes decreased in the range of DFO > hydroxypyrid-4-ones > pyoverdin. It seems that other siderophores are also less effective inhibitors of lipid peroxidation than DFO [371],... 

Comparative Metabolism. Since the liver is the major organ involved in the biotransformation of xenobiotics, primary hepatocyte cultures provide an excellent model for in vitro metabolism studies. Primary hepatocyte cultures provide useful tools with which to study the comparative metabolism of xenobiotics by both humans and laboratory animals. 

Green et al. (1986) compared the metabolism of amphetamine in isolated hepatocyte suspensions from rat, dog, squirrel, monkey, and human livers. The metabolite profile of hepatocytes from each species corresponded to the profile of urinary metabolites identified previously. These results indicate that species-specific differences in the metabolic activation of compounds seen in vivo can be reproduced in vitro by the utilization of primary hepatocyte cultures. 

Primary hepatocyte cultures have been used in vitro to metabolically activate toxins for evaluation with target tissues. Cocultures of rat embryos with hepatocytes have been used to study the role of metabolism in teratogenesis (Oglesby et al., 1986). Lindahl-Kiessling et al., (1989), in an attempt to bring test conditions closer to in vivo conditions, developed an assay utilizing primary rat hepatocytes and human peripheral lymphocytes to detect metabolism-mediated mutagenesis. 

Immortalization of human hepatocytes would help to overcome the hmited availability of human cells, thereby avoiding the use of mahgnant-derived cell hnes however, care will still be required with these cells. A better approach would be to develop methods for the culture of primary human hepatocytes using hormonally defined media containing growth factors in which cells are stimulated to undergo division. 

Species comparisons of hepatic peroxisomal proliferation have also included studies of human and non-human primate primary hepatocyte cultures. Hepatocytes isolated from Wistar-derived rats (180-220 g), male Alderley Park guinea-pigs (400-500 g), male marmosets (350-500 g) and three human liver samples (renal transplant donors) were treated with 0-0.5 mM mono(2-ethylhexyl) phthalate for 72 h (Elcombe Mitchell, 1986). While there was a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes, no induction was observed in guinea-pig or human hepatocytes and only small non-concentration-dependent effects were observed in marmoset hepatocytes. Metabolite VI induced cyanide-insensitive palmitoyl-CoA oxidation and lauric acid hydroxylation in cultured... 

Unscheduled DNA synthesis was not induced in either mouse or hirman primary hepatocyte cultures with mono(2-ethylhexyl) phthalate, and neither this metabolite nor 2-ethylhexanol induced mutations in mouse lymphoma cells in vitro. Mono(2-ethyl-hexyl) phthalate induced sister-chromatid exchange in Chinese hamster V79 cells and chromosomal aberrations in Syrian hamster embryo cells. It also induced transformation in Syrian hamster embryo cells, but not in mouse C3H10T /2 cells. Gap-junctional intercellular communication was inhibited by mono(2-ethylhexyl) phthalate in Syrian hamster embryo cells and in Chinese hamster V79 cells. As reported in an abstract (Baker et al., 1996), this function was also inhibited in rat and mouse hepatocytes, but not in Syrian hamster or human hepatocytes. 

Hepatic Effects. No studies were located regarding hepatic effects in humans after oral exposure to DEHP. Limited information on hepatic effects in humans exposed to DEHP is available from studies of dialysis patients and cultured human hepatocytes. In one individual there was an increased number of liver peroxisomes after 1 year, but not after 1 month of treatment (Ganning et al. 1984, 1987). A serious limitation of this observation is that repeat biopsies were not obtained from the same patient, so that an appropriately controlled analysis is not possible. Additionally, analysis of liver biopsies from patients receiving other kinds of hypolipidemic drugs has not yielded any evidence for peroxisomal proliferation (Doull et al. 1999). Recognizing some limitations of using primary hepatocytes in vitro because of their tendency to lose some metabolic capabilities (Reid 1990), in cultured human hepatocytes there were no changes in the activities of peroxisomal palmitoyl-CoA oxidase and/or carnitine acetyltransferase when... 

Strong KL, Rushmore TH, Richards KM. Quantitation of human CYP3A4, 3A5 and 3A7 RNA levels in cultured primary hepatocytes using real-time RT-PCR. ISSX Proc 1999 15 69. 

Immortalized hepatocytes A major drawback of the use of primary hepatocytes is that hepatocytes can not be expanded in culture. To overcome this problem, researchers have embarked on the immortalization of primary hepatocyte cultures (e.g. Bayad et al. 1991 Li et al. 2005). Currently, immortalized human hepatocyte cell lines are available commercially and may represent convenient screening experimental systems for enzyme induction studies. Unfortunately, at the time of this writing, there are no peer-reviewed publications on the application of human immortalized hepatocytes in induction studies. It is important to ensure that the induced isoforms in the cell lines are the mature P450 isoforms rather than the embryonic forms. For instance, the use of HepG2 cells may not be appropriate as the embryonic P450 isoforms CYP1A1... 

LeCluyse EL, Alexandre E, ffamilton GA, et al. Isolation and culture of primary human hepatocytes. Method Mol Biol. 2005 290 207-229. 

Ullrich A, Stolz DB, Ellis EC, Strom SC, Michalopoulos GK, Hengstler JG, Runge D (2009) Long term cultures of primary human hepatocytes as an alternative to drug testing in animals. ALTEX 26(4) 295-302... 

Blazak W, Stewart B, DiBiasio-Erwin D, et al. 1985. Induction of sister chromatid exchanges (SCE) by dimethylnitrosamine (DMN) in Chinese hamster cells co-cultured with primary human hepatocytes (PHH) [abstract]. Environ Mutagen. 7 32. 

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