Potential Impact of Steatosis on Cytochrome P450 Enzymes of Human Hepatocytes Isolated from Fatty Liver Grafts

Abstract

ABSTRACT: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.

Increased Levels of Fatty Acids Contributed to Induction of Hepatic CYP3A4 Activity Induced by Diabetes — In Vitro Evidence From HepG2 Cell and Fa2N-4 Cell Lines

“…Several studies have showed that the unsaturated fatty acids, not saturated fatty acids, inhibited CYP3A4catalyzed metabolic reactions (35,36). It was reported that 14h treatment with a highconcentration fatty acids mixture (1 and 2 mM) significantly decreased CYP3A4 activity in primarily cultured human hepato cytes (37). However, data from the present study showed that 48h treatment of hepatic cells (both HepG2 and Fa2N4 cells) with PA and OA (0.1 and 0.2 mM) induced CYP3A4 activity.…”

Metabolite Formation Kinetics and Intrinsic Clearance of Phenacetin, Tolbutamide, Alprazolam, and Midazolam in Adenoviral Cytochrome P450-Transfected HepG2 Cells and Comparison with Hepatocytes and In Vivo

“…The tandem mass spectrometry analysis was performed with a triple quadrupole analyzer operating in the multiple reaction monitoring mode using argon as collision gas (Table 3). Resorufin formed during the CYP1A2 activity assay and 6␤-hydroxytestosterone (CYP3A4) were quantified fluorometrically (Donato et al, 1993) and by high-performance liquid chromatography analysis (Donato et al, 2006), respectively.…”

“…Marker activities were measured in each Ad-P450 HepG2 preparation. Data from hepatocytes are an average of at least 12 independent cultures (Donato et al, 1999(Donato et al, , 2006. For both Ad-P450 HepG2 and primary human hepatocytes, activities were assayed by direct incubation of cell monolayers with 10 M 7-ethoxyresorufin, 10 M diclofenac, or 200 M testosterone for 60, 120, or 60 min, respectively (Donato et al, 2006).…”

Homogeneous phenomenon of the graft when using different genotype characteristic of recipients/donors in living donor liver transplantation

“…It should be easier and more accurate to demonstration the SNP allele when compared with the traditional sequence ( Figure 1). The bias of our study was the lack of microdisection of the hepatocyte [11][12][13] or isolation of the hepatocyte [14,15] from the graft, because we believed that the genetic characteristic of the graft hepatocyte is belongs to the donor originally. The peripheral blood of the recipient continuous goes through into the graft but it may not be contribute the genetic characteristic of the donor graft.…”