The use of precision-cut liver slices from male Wistar rats as a tool to study age related changes in CYP3A induction and in formation of paracetamol conjugates

Abstract

Precision-cut liver slices (PCLS) offer a lot of advantages because all heterogeneity and cell-cell interactions within the original tissue matrix are maintained. This in vitro model was used to study the effect of ageing on certain aspects of drug metabolism and liver function in young (3 months), adult (9 months) and old (24 months) Wistar male rats. Protein synthesis, an important liver function, was not modified in young, adult and old rats, suggesting that ageing does not impair liver functionality but it affects some specific targets. Among them, a decrease in total P450 in liver microsomes and the loss of CYP3A23 inducibility in PCLS were clearly observed in old rats as compared to adult rats. Finally, the amount of total paracetamol conjugates was not modified between 9 and 24 months but in old rats, sulfoconjugation of paracetamol, its major route of elimination, was decreased.

Introduction

It is well know that ageing may lead to alterations in the biotransformation of drugs, and therefore their therapeutic efficacy and safety. A number of studies in male rats have documented significant age-related declines in the amounts, specific activities and rates of induction of liver microsomal mono-oxygenases (Birnbaum and Baird, 1978; McMartin et al., 1980; Schmucker and Wang, 1981), as well as an increase in inter-individual variability in most liver functions (Schmucker, 2001). To learn more about the underlying causes of this age-related alteration in drug metabolism numerous in vivo studies have been carried out in animals, especially rats. However, experiments in animals to investigate the possible causes of these alterations and especially the interpretation of the results of in vivo studies are complicated by a number of problems. Indeed, the in vivo drug metabolic clearance is not only affected by intrinsic metabolic capacity but also by organ blood flow and binding to plasma proteins, factors which may also be altered due to the increased incidence of functional abnormalities in a number of organs with ageing (Barnett et al., 1974; Messineo et al., 1983; van Bezooijen, 1984). Moreover, it is highly unlikely that ageing is affecting the various isoforms of drug metabolizing enzymes to the same extent. Although an approach looking for simultaneous measurements of different P450 isoforms can be conducted by using “special mixtures”, the possible contribution of extrahepatic tissues to the overall metabolism complicates the interpretation of the results.

In order to solve some of these inconveniences, we decided to use the in vitro model of precision-cut liver slices (PCLS) to investigate the influence of ageing on drug metabolism in rats. PCLS present a lot of advantages by maintaining cell heterogeneity and cell-cell interactions within the original tissue matrix. Indeed, PCLS have been shown to be a suitable model to study survival and cellular metabolism, drug toxicity and xenobiotic metabolism (Morales et al., 1998; Vanhulle et al., 2001; Evdokimova et al., 2001a, Evdokimova et al., 2002; Rekka et al., 2001). In the present study, some parameters related to drug biotransformation were investigated in PCLS prepared from young (3-month), adult (9-month) and old (24-month) male Wistar rats. They include the total content of P450, the capacity of CYP3A23 to be induced under in vitro conditions, and a phase II-related activity, namely the formation of paracetamol conjugates (glucuronide, sulfate and glutathione) together with SULT1A1 protein content. In addition, protein synthesis ability (a major metabolic activity of hepatocytes), was measured to assess the metabolic competence of the slices.