Effect of spironolactone on the expression of rat hepatic UDP-glucuronosyltransferase
Introduction
UGTs are a superfamily of membrane-bound enzymes that metabolize both endogenous compounds (such as bilirubin, bile acids and steroid and thyroid hormones), and exogenous compounds (such as food additives, therapeutic drugs and environmental pollutants), thereby facilitating their excretion from the body [1], [2]. Based on the nucleotide and amino acid sequences, UGT isoforms in mammals are grouped in two major families termed 1 and 2. The structure of the UGT1 gene contains several first exons encoding isoform-specific sequences that confer aglycone specificity, followed by a set of common exons (2–5) encoding the C-terminal sequence that is identical in all UGT1 isoforms [3], [4], [5]. The transcript then undergoes alternative splicing. In contrast, UGT family 2 isoforms are each derived from an individual gene and are responsible for the glucuronidation of steroids and opioids [6], [7].
UGT activity is affected by many factors including aging, diet, hormones, diseases and enzyme inducers [1], [8], [9]. Glucuronidation is under complex regulation. The first control may occur either at the gene transcription level, resulting in changes in mRNA and protein levels, and/or at the level of post-translational processing. The second one is exerted on the modulation of the functional state (or activity per molecule) of the enzyme, because of the association of UGT with the lipid environment [10], the restriction of the cosubstrate UDP-glucuronic acid (UDPGA) to access the enzyme active site [11], [12] and/or the protein–protein interactions derived from oligomer formation [13], [14], [15].
Different forms of UGT are selectively induced by the administration of various chemical compounds [16]. The induction of individual enzymes of UGT in rat liver has been extensively studied for classical inducers such as phenobarbital, clofibrate, 3-methylcholanthrene and dexamethasone [3], [17], [18], revealing transcriptional regulation. SL, a diuretic widely used in patients with edema or ascites [19], also induces the activity of several hepatic microsomal enzymes including UGT [20], [21], [22]. This could result in increased metabolism of drugs coadministered with the diuretic; e.g. drugs suffering phase I metabolism have a shorter half-life in cirrhotic patients receiving SL [23].
Characterization of SL effect on the different isoforms of UGT has been performed only in terms of activity toward the model substrates reacting with the UGT1 family isoforms. Thus, hepatic UGT activity toward bilirubin was reported to be substantially increased in rats pre-treated with SL [20], [21] whereas the conjugation rate of planar phenols was not affected [22]. Because of overlapping of substrate specificity among the different isoforms and multifactorial regulation of UGT activity, previous results on enzyme activity do not necessarily reflect changes in protein and/or mRNA levels. Microsomal lipids were shown to influence UGT activity [10]. It is also known that K+ canrenoate (an active metabolite of SL) induces an increase in the cholesterol/phospholipid molar ratio and a decrease in plasma membrane fluidity in rat liver [24]. Consequently, it is possible that these steroids affect UGT activity by modulating enzyme environment, in addition to gene expression.
In the present study we analyzed the effect of SL administration on expression of UGT family 1 isoforms (1A1, 1A5 and 1A6) and on UGT2B1, a relevant family 2 isoform, in rat liver. Protein and mRNA levels as well as activity toward the classical substrates of the different isoforms were systematically determined. The effect of SL on UGT latency and on lipid composition and physicochemical properties of the microsomal membrane was also evaluated.
Section snippets
Chemicals
17α-[6,7--N]Ethynylestradiol (49.1 Ci/mmol) was from New England Nuclear. SL, bilirubin, UDPGA (ammonium salt), UDP-N-acetylglucosamine (UDP-N-AG), p-nitrophenol, mannose 6-phosphate (disodium salt), d-saccharic acid 1,4-lactone, alamethicin, 1,6-diphenyl-1,3,5-hexatriene (DPH) and non-labeled ethynylestradiol were purchased from Sigma Chemical Co. All other reagents were of the highest grade commercially available.
Animals and treatment
Adult male Wistar rats (300–360 g) were used throughout. They were maintained ad
UGT activities
First, we evaluated the functional integrity of microsomal vesicles. Hepatic microsomes from control group exhibited a latency of mannose 6-phosphatase of 89–95% (N=7) that was not affected by SL treatment (88–94%, N=11). Thus, changes in UGT activity determined in native or UDP-N-AG-activated microsomes in response to SL administration could not be attributed to artifacts of the isolation procedure, and any eventual change in the functional features of UGT, detected by activation analysis,
Discussion
UGT isoforms may be selectively induced by the administration of chemical compounds. UGT1A1 is a constitutively expressed form involved in conjugation of the endogenous substrate bilirubin and also in conjugation of the synthetic estrogen ethynylestradiol in position 3-OH [32]. It was reported that its transcription is stimulated by administration of dexamethasone or clofibrate to rats [3], [18]. In the same report the authors found that UGT1A5, which belongs to bilirubin cluster [18], is
Acknowledgements
We express our gratitude to Drs. José M. Pellegrino, Luis M. Veggi and Silvina Pessino for their technical assistance and valuable suggestions. This work was supported by Research Grants from Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET) and from Universidad Nacional de Rosario, Argentina.
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