Effects of steatosis on drug-metabolizing capability of primary human hepatocytes.
The suitability of liver grafts discarded for transplantation because of macrosteatosis for preparing human hepatocyte cultures for in vitro drug metabolism studies has been examined. Lower cell viability and yield of isolation procedure were obtained from fatty livers (>40% steatosis) with respect to normal tissue. Significant reductions in 7-ethoxycoumarin O-deethylation (ECOD) and testosterone oxidations were found in hepatocytes prepared from steatotic livers. The potential impact of lipid accumulation on P450 enzymes was studied in vitro by incubation of cultured hepatocytes with long chain free fatty acids (FFA). Treatment of cells with 0.25-3mM FFA induced dose-dependent accumulation of lipids in the cytosol. Decreased ECOD and testosterone oxidation were found after 14h of exposure to 1mM or 2mM FFA (about 60-70% and 30-60% of control, respectively). The effects of fat-overloading on individual P450s were analyzed both at activity and mRNA level. CYP1A2, CYP2C9, CYP2E1 and CYP3A4 activities were reduced after hepatocyte incubation with 1mM (to 45-65% of control) or 2mM (to 20-50%) FFA for 14h. Reductions in P450 transcripts were also found in hepatocytes treated with 1mM FFA. Our findings showed a general down-regulation of P450s involved in drug metabolism in fat-overloaded hepatocytes. The results suggest that, despite their reduced P450 function, human hepatocytes obtained from donors with steatosis are metabolically competent and could be used for drug metabolism studies.
Donato
M T
MT
Departamento de Bioquímica y Biología Molecular, Universidad de Valencia, Spain. donato_mte@gva.es
Jiménez
N
N
Serralta
A
A
Mir
J
J
Castell
J V
JV
Gómez-Lechón
M J
MJ
D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005234:Fatty Liver / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D013739:Testosterone / Q000378:metabolism
D005230:Fatty Acids, Nonesterified; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System