Establishment Of Fibrotic Rat Precision-cut Liver Slice Technique And Its Application In Drug Metabolism

Use of precision-cut liver slices (PCLS) as a valuable tool for studies of xenobiotic (drug and toxicant)metabolism and toxicity in vitro is increasing in recent years. However, the metabolizing characteristic of pathologic PCLS such as fibrotic PCLS has not been reported until now. This study was undertaken to grope the optimal culture condition, test the stability and inducibility of phase I and phase II enzymes of drug metabolism, and use this technique to study and compare the metabolism of a calcium antagonist-verapamil in both normal and fibrotic rat liver slices.I. Establishment of fibrotic rat precision-cut liver slice technique and inducibility in vitro of drug-metabolizing enzymesComplex factors (higher fat diet, alcohol drinking and CCl₄ injection) were administered in rats for 3 weeks to make liver fibrosis. Fibrotic PCLS were prepared and incubated. Lactate dehydrogenase (LDH) leakage, glutathione S-transferase (GST) activity and MTT reduction were chosen as indexes to assess the viability of fibrotic PCLS. Meanwhile, PCLS were treated with specific cytochrome P450 (CYP450) inducers, and the activities of CYP1A1, CYP2E1, CYP3A1/2, GST and UDP-glucuronosyltransferase (UDPGT) were monitored. Rats were in the earlier status of hepatic fibrosis after treatment of complex factors for 3 weeks. When the thickness of slices was 300 μm and pH of medium was 7.0, the LDH leakage, GST activity and MTT reduction of fibrotic slice could maintain on a steady level during 6 h of culture. Meanwhile, there were no significant changes in all the enzymatic activities after 6 h incubation; while the induction of CYP2E1 by ethanol (50 mM) and CYP3A1/2 as well as UDPGT by phenobarbital (0.5 mM) was demonstrated in the time-dependent manners, cresting at 6 h, which were 2.3, 4.4- and 1.6-folds, respectively, of their controls (P < 0.05). A 300 urn of the thickness, 7.0 of the medium pH and 6 h of culture time are the optimal slicing and culturing conditions for fibrotic liver slice. Phase I and phase II enzymes of fibrotic PCLS could maintain the steady levels, and the activities of CYP2E1, CYP3A1/2 and UDPGT could be induced invitro by inducers within 6 h incubation.II. Metabolism of verapamil by normal and fibrotic rat precision-cut liver slicesNormal and fibrotic rat precision-cut liver slices were prepared and incubated individually in the presence of 10 uM verapamil. After 2, 5, 15, 30, 45, 60, 90, 120, 240 and 360 min of incubation, the incubation medium was removed for analysis. The concertration of verapamil from the medium was determined by HPLC. Subsequent to the incubation, each slice was removed and homogenized by hand and its protein content was determined. Intrinsic clearance (CLints) was calculated as the ratio of its initial concentration to the area under concentration-time curve (AUC), and correlated to the average protein content. The AUC was model-independently calculated by employing WinNonlin Standard Edition Version 4.0.1 (Pharsight, USA). Significant differences in the CLjnts (|il.min"1.nig¹) obtained with normal (9.7 ± 1.8) and fibrotic (5.6 ± 1.4) rat liver slices were observed (P < 0.01). The result demonstrates that the rate of verapamil metabolism with rat liver slices was affected by status of fibrosis. These findings add further support to the use of fibrotic liver slices as a tool for studying drug metabolism and instructing clinical medication.