Ageing is associated with increased expression but decreased activity of CYP2E1 in male Wistar rats
Introduction
Cytochrome P450s (CYPs) constitute a superfamily of heme-proteins that play an important role in the detoxification of numerous xenobiotics as well as endogenous compounds such as steroids, fatty acids, prostaglandins, and leukotrienes (Guengerich, 1990, Gonzalez, 1988, Nebert et al., 1991). In addition, they also play an important role in the maintenance of bile acid and cholesterol homeostasis (Chawla et al., 2000, Chawla et al., 2001). The activity of these enzymes may be influenced by various genetic or non-genetic factors. Among the latter, ageing may affect these enzymes thus leading to alterations in the biotransformation of drugs and, consequently, their therapeutic efficacy and safety. The mechanisms underlying such effects are not totally elucidated.
To study the effect of ageing on P450 enzymes, we selected the ageing rat model as an experimental system and focused our investigation on the CYP2E1 isoform. Indeed, different facts underline the important toxicological implication of this enzyme: CYP2E1 is constitutively expressed in several tissues; it shares high homology with the human isoform; it catalyses the oxidation of a wide variety of compounds including certain drugs, solvents and environmental pro-carcinogens (Tanaka et al., 2000, Fang et al., 1998); it is induced by ethanol (Clot et al., 1996, Fang et al., 1998) and it forms reactive oxygen species (Ekstrom and Ingelman-Sundberg, 1989, Goasduff and Cederbaum, 1999).
The regulation of P450 genes occurs at different levels via various mechanisms that are quite diverse and not well understood (Akiyama and Gonzalez, 2003). The analysis of promoter sequences contained in numerous genes that exhibit liver-specific expression reveals the presence of binding sites for liver-enriched transactivating factors, such as hepatocyte nuclear factor (HNF) 1, HNF3, HNF4, HNF6 or C/EBPα (Cereghini, 1996). In vitro transcription and transactivation studies have suggested that CYP2E1 is controlled in part by HNF1α (Liu and Gonzalez, 1995, Ueno and Gonzalez, 1990).
The aim of this work was to study the influence of ageing on CYP2E1 at the level of activity as well as its mRNA and protein content in both adult (9 months) and senescent (24 months) male Wistar rats. The oxidation of chlorzoxazone to 6-OH-chlorzoxazone was measured as an index of CYP2E1 enzyme activity (Kobayashi et al., 2003). The amount of both CYP2E1 proteins and mRNA was determined by immunoblotting and quantitative PCR, respectively. Due to the influence of oxidative stress in age-related processes, certain standard markers of an oxidant insult were measured; the levels of aldehydes (by both spectrophotometry and histochemistry), protein carbonyls (by both colorimetry and immunoblotting detection). The chymotrypsin-like proteasome activity was also recorded. Moreover, the activities of major antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSHpx) and catalase (CAT) and the content of reduced glutathione (GSH) were also measured in both adult and senescent rats. Finally, since HNF1α is involved in the expression of CYP2E1, we decided to investigate its potential role on the regulation of CYP2E1 by measuring its expression by RT-PCR.
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Animals
Animal care and experiments were performed according to the Biosafety and Ethical rules in application in Belgium as adopted by the Bioethical Committee of the Université Catholique de Louvain. Male Wistar rats 9 and 24 months of age were purchased from Harlan (Horst, The Netherlands) and housed in individual cages in a temperature- and light-controlled room (12 h/12 h dark/light cycle) during 1 week before their sacrifice. They received a standard diet (A03; UAR, Epinay sur Orge, France) and
Results
In order to know the influence of ageing on a general index of drug metabolism, we determined the P450 content in male rats at different ages ranging from 9 to 24 months. Fig. 1 shows that the total cytochrome P450 content of liver microsomes from male Wistar rats remained relatively constant at about 0.8–1.0 nmol/mg protein between the ages of 9 to 21 months. A significant decrease of 35% was observed in rats of 24 months as compared to rats of 9 months. Indeed, the content of P450 decreased
Discussion
CYP2E1 is a constitutively expressed isoform that has received much attention because of its importance in the activation of chemicals to cytotoxic products and its potential role in ethanol-induced liver toxicity. In agreement with previous reports showing age-related reductions in certain cytochrome P450 activities (Kamataki et al., 1985, Wauthier et al., 2004, Warrington et al., 2004a, Dhir and Shapiro, 2003), the results of the present study demonstrate a decreased CYP2E1 activity in
Acknowledgements
We thank Véronique Allaeys and Nicole Maton for their excellent technical assistance. The authors express their gratitude to Valérie Van Kerckhove and Vincent Haufroid (Unité TOXI, UCL) for their valuable contribution in the quantitative PCR determination of CYP2E1 mRNA.
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