The concentration of cytochrome P-450 in human hepatocyte culture

Abstract

INTRODUCTIONA problem with the study of drug metabolism and toxicity in experimental animals is extrapolating the relevance of the results to man. It is unethical to determine the toxic properties of chemicals by dosing these to humans but cell cultures prepared from human tissues could circumvent this problem. Liver cell culture is a popular model system to study mechanisms of hepatotoxicity in experimental animals (for reviews see 1 and 2). However, a limitation of this model is the rapid loss of cytochrome P-450 during the first 24 hours of hepatocyte culture (3)(4)(5). This is an obstacle since cytochrome P-450 mediated metabolism frequently determines the toxicity and carc~nogenicity of many natural as well as synthetic chemicals (6).In rat hepatocyte culture the loss of cytochrome P-450 can be prevented by including metyrapone or 1,2-cyanophenylimidazole (7) in the culture medium. Little is known about the effect of the culture conditions on the concentration of cytochrome P-450 in human hepatocytes and where results have been reported these are only on a few donors (8-10). The present study reports our experience with hepatocyte cultures prepared from seven human individuals and therefore, to the best of our knowledge, represents the largest study to be reported, The results show that, in contrast to hepatocyte cultures prepared from experimental animals (3,4) the majority (i.e. five) of the cultures prepared from human liver do not lose cytochrome P-450 during the first 24 hours of culture. Furthermore, where the loss of cytochrome P-450 did occur in culture the addition of metyrapone or 1,2-cyanophenylimidazole to the culture medium, which prevents the loss of the cytochrome in rat hepatocyte culture (7), also prevents the loss of the cytochrome in cultured human hepatocytes. MATERIALS AND METHODSIsolation of hepatocytes. Liver samples were obtained from renal transplant donors who had met traumatic death and were maintained on life support systems until the kidneys were removed. Donors were not receiving any chronic drug treatment but were given such drugs as necessary while on life support systems. After removal of the kidneys a section of liver (approximately 100 g) was removed from the organ such that the section had only one cut face. In order to remove as much of the entrapped blood as possible, the liver section was perfused with Hank's balanced salt solution (HBSS from Gibco Europe) via the larger vessels on the cut surface, using a 20 ml syringe and a flow rate of ca 20 ml/min. The procedure to isolate hepatocytes commenced within 30 minutes of removal of the liver section from the donor. A smaller section (approx 30 g) was cut, ensuring that the section had only one cut face and this piece was used for the isolation of hepatocytes. A vessel was selected, so that a cannula with a male luer end-piece fitted snugly into it. Too large a vessel resulted in leakage of perfusate, too small a vessel resulted in the vessel being damaged and both resulted in poor perfusion. The liver section was perfuse...

Scaling Factors for the Extrapolation of In Vivo Metabolic Drug Clearance From In Vitro Data: Reaching a Consensus on Values of Human Micro-somal Protein and Hepatocellularity Per Gram of Liver

“…Factors that need to be assessed in evaluating the quality of the data include the source of the livers (typically surgical samples from patients, tissue from kidney transplant donors or from healthy livers after sudden death), demographic details, and how corrections were made for loss of microsomal protein or cells during preparation of microsomes and hepatocytes [24,51]. In general, MPPGL is a theoretical estimate that accounts for the inefficiency of the standard sub-fractionation process in recovering all microsomal protein from liver samples.…”

Inter‐individual variability in levels of human microsomal protein and hepatocellularity per gram of liver

“…The value of HPGL is also useful in IVIVE when in vitro data are obtained as rate of metabolism per hepatocyte. In order to calculate the number of hepatocytes accurately in a given mass of liver, corrections must be made for the incomplete yield of cells following perfusion of the liver sample [13]. Iwatsubo et al [14] used the hepatocyte approach to determine a value of MPPGL of 52.5 mg g -1 liver.…”

Reduced mitochondrial membrane potential and metabolism correspond to acute chloroform toxicity ofin vitro hepatocytes

“…The observed effect on culture viability following a 2-h exposure in this study was expected based on previous studies (el-Shenawy and Abdel-Rahman, 1993) but the doses required to see a significant decrease were higher than the 2.5 mM reported for freshly isolated hepatocytes during a 2-h exposure (Ammann et al, 1998). The mechanism of chloroform toxicity is dependent on the P-450-dependent biotransformation of chloroform to phosgene (IPCS, 1994;Constan et al, 1999) and because cytochrome P-450 expression decreases in culture (Blaauboer et al, 1985) the effect of chloroform on freshly isolated hepatocytes may be greater than that on cultured hepatocytes. Thus, a lower P-450 expression in the cultured hepatocytes of this study could presumably explain preliminary evidence for mitochondrial damage and changes in metabolic rates that appeared to correspond to a decrease in culture viability following an exposure to chloroform.…”