Potential impact of steatosis on cytochrome P450 enzymes of human hepatocytes isolated from fatty liver grafts.
Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of "cellular steatosis" by incubation of cultured hepatocytes with increasing concentrations (0.25-3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45-65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.
Donato
M Teresa
MT
Unidad de Hepatología Experimental, Hospital Universitario La Fe, Avenida Campanar 21, 46009, Valencia, Spain. donato_mte@gva.es
Lahoz
Agustín
A
Jiménez
Nuria
N
Pérez
Gabriela
G
Serralta
Alfonso
A
Mir
José
J
Castell
José V
JV
Gómez-Lechón
M José
MJ
D000328:Adult; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D005234:Fatty Liver / Q000201:enzymology* / Q000473:pathology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000473:pathology; D008297:Male; D008862:Microsomes, Liver; D008875:Middle Aged; D010078:Oxazines / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
D005230:Fatty Acids, Nonesterified; D010078:Oxazines; D012333:RNA, Messenger; D013739:Testosterone; C076580:7-methoxyresorufin; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human