Establishment of rat precision-cut fibrotic liver slice technique and its application in verapamil metabolism.
1. Liver fibrosis is the compensatory state of cirrhosis. In the long asymptomatic period, it is imperative to select a proper dosing regimen for drugs that are applicable to hepatic fibrosis. Otherwise, progressive deterioration to uncompensated cirrhosis may occur. The present study explored the characteristics of drug metabolism in fibrotic liver. 2. A rat precision-cut fibrotic liver slice (PCFLS) technique was established and the metabolism of verapamil was studied employing this technique. A rat hepatic fibrosis model was successfully induced integrating complex factors that included a high-fat diet, alcohol and CCl4. The PCFLS were incubated under different conditions and lactate dehydrogenase leakage, glutathione S-transferase activity and 3[4,5-dimethythiazole-2-yl]-2,5-diphenyltetrazolium bromide reduction were used as indices to assess PCFLS viability. Activities of phase I and phase II metabolizing enzymes were monitored following treatment with cytochrome P450 (CYP) inducers. Normal and fibrotic liver slices were incubated individually with 10 micromol/L verapamil. The concentration of verapamil in the medium was determined by high-performance liquid chromatography and intrinsic clearance (Cl(int)) was calculated on the basis of the concentration-time curve. 3. The results showed that the PCFLS viability remained steady throughout the 6 h of culture when the thickness of slices was 300 microm and pH of the medium was 7.0; CYP inducers (phenobarbital and ethanol) enhanced CYP2E1, CYP3A1/2 and uridine diphosphate-glucuronate transferase (UDPGT) activities, respectively, in a time-dependent manner. The Cl(int) (microL/min per mg) values differed significantly between normal (9.7 +/- 1.8) and fibrotic (5.6 +/- 1.4) liver slices (P < 0.01). 4. These results suggested that the PCFLS could remain viable for 2-6 h under appropriate conditions. The stability and inducibility of drug-metabolizing enzymes of PCFLS were also demonstrated. Furthermore, the metabolic rate of verapamil in PCFLS was decreased. These findings add further support to the use of PCFLS as a tool to study drug metabolism and to guide clinical medication.
Guo
Yu
Y
Department of Pharmacology, Basic Medical College of Wuhan University, Wuhan, Hubei Province, China.
Wang
Hui
H
Zhang
Chun
C
D000818:Animals; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000493:pharmacokinetics; D002470:Cell Survival / Q000187:drug effects; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D008106:Liver Cirrhosis, Experimental / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology*; D008297:Male; D050216:Metabolic Detoxication, Phase I; D050217:Metabolic Detoxication, Phase II; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D046509:Tissue Culture Techniques / Q000379:methods*; D014700:Verapamil / Q000378:metabolism* / Q000493:pharmacokinetics; D019324:beta-Naphthoflavone / Q000494:pharmacology
D002121:Calcium Channel Blockers; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D014700:Verapamil; D007770:L-Lactate Dehydrogenase; D014453:Glucuronosyltransferase; D005982:Glutathione Transferase