Effects of Thioacetamide—Induced Cirrhosis, after a Drug Washout Period, on the Michaelis–Menten Kinetics of Major Cytochrome P450 Enzymes in the Rat Liver Microsomes

Abstract

PurposeThioacetamide (TAA) is used to induce liver cirrhosis in rodents. However, the drug itself directly inhibits cytochrome P450 (CYP450) enzymes. The purpose of this study was to determine the effects of TAA‐induced liver cirrhosis, after a washout period to eliminate TAA, on the Michaelis‐Menten (MM) kinetics and protein contents of the major CYP450 enzymes CYP2D, CYP2E1, and CYP3A in rat liver microsomes.MethodsAdult, male Sprague‐Dawley rats were treated for 10 weeks with either TAA (200 mg/kg ip, 3 days per week) or vehicle (n = 7/each group). After a 10‐day washout period, animals were euthanized, and the liver was collected after cardiac perfusion with cold saline. Liver microsomes (MC) were prepared by differential centrifugation. The activities of the liver microsomal CYP2D (dextromethorphan O‐demethylation), CYP2E1 (chlorzoxazone 6‐hydroxylation) and CYP3A (midazolam 1′‐hydroxylation) were estimated by measurement of the metabolites by LC‐MS/MS. The microsomal CYP450 reductase (CPR) activity was analyzed spectrophotometry. Furthermore, the protein expressions of the isoenzymes were determined using Western blot analysis. The MM kinetic parameters maximum velocity (Vmax) and MM constant (Km) were determined using nonlinear regression analysis. Unpaired t‐test was used for statistical analysis of data, with a p value of < 0.05 as significant.ResultsWhereas the MM kinetics of dextromethorphan demethylation (CYP2D) were best fit by a two‐enzyme equation, those of chlorzoxazone hydroxylation (CYP2E1) and midazolam 1′‐hydroxylation (CYP3A) were best fit by a single‐enzyme equation. Cirrhosis resulted in significant reductions in the Vmax values for all the tested microsomal activities. The microsomal high‐ and low‐capacity Vmax values for CYP2D demethylase activity were significantly reduced by 74% (p value < 0.0001) and 51% (p < 0.05), respectively, in the TAA group. This was associated with a significant (p < 0.001) reduction (71%) in the protein contents of CYP2D in the TAA group. However, Km values were not affected by cirrhosis. For CYP2E1, cirrhosis caused a significant reduction (52%) in the Vmax value (p < 0.0001) for the chlorzoxazone hydroxylation activity. However, the Km values and protein contents of CYP2E1 remained unchanged. The Vmax and Km values of microsomal CYP3A hydroxylase activity, resulting in 1′‐hydroxylation of midazolam, were significantly reduced by 71% (p < 0.001) and 46% (p < 0.05), respectively, which was associated with a 30% reduction (p < 0.05) in the protein contents of CYP3A. Cirrhotic animals also showed a 31% decline (p < 0.05) in the microsomal CPR activity.ConclusionsTAA‐induced cirrhosis in rats resulted in a decreased liver microsomal activity of CPR and major CYP450 enzymes (CYP2D, CYP2E1, and CYP3A). Except for CYP2E1, the decreased isoenzyme activities were also associated with decreases in the protein concentrations of the isoenzymes. However, the cirrhosis‐induced decrease in the CYP2E1 activity, which was in the absence of a change in its protein levels, is possibly due to a decrease in the microsomal CPR activity.Support or Funding InformationFunding: Chapman University School of Pharmacy