Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide

Abstract

The inhibitory effects of silymarin, its main constituent silibinin and the metabolite silibinin-glucuronide on UDP-glucuronosiltransferase (UGT) were evaluated in rat hepatic microsomes. Three substrates were chosen to cover both UGT1A and UGT2B family isozymes: bilirubin (substrate of UGT1A1), p-nitrophenol (UGT1A6) and ethinylestradiol (UGT2B1 and 2B3 for position C17 and UGT1A1 for position C3). The study of p-nitrophenol and bilirubin glucuronidation indicated that silymarin (SM) and silibinin glucuronide (SB-G) were enzyme inhibitors. The kinetic analysis showed that the type of inhibition was competitive in all cases and the Ki obtained were: for p-nitrophenol glucuronidation, Ki_SB-G^app: 14 ± 1 μg/ml and Ki_SM^app: 51 ± 10 μg/ml and for bilirubin glucuronidation, Ki_SB-G^app: 16 ± 3 μg/ml. In turn, ethinylestradiol glucuronidation was not affected by any of the compounds studied suggesting that the inhibitory effect was restricted to UGT1A isozymes. Similar studies performed using human hepatic microsomes showed that SM and SB-G were also inhibitors of human UGT1A isozymes. In conclusion, administration of silymarin or its main constituent silibinin could lead to the decrease in the glucuronidation of substrates whose conjugation depends on UGT1A isozymes in a process mediated by silibinin-glucuronide, though their effect in humans needs further investigation.

Introduction

Silymarin is a purified extract from the milk thistle Silybum marianum (L.) Gaertn, composed mainly of four isomeric flavonolignans: silibinin, isosilibinin, silidianin and silichristin (Kvasnicka et al., 2003). Extracts of milk thistle have been used as medical remedies for almost 2000 years, and continue to be used as therapeutic agents for many types of acute and chronic liver diseases (reviewed by Flora et al., 1998). In addition, silymarin was shown to protect experimental animals from various hepatotoxicants, including carbon tetrachloride, acetaminophen and phalloidin (Schuppan et al., 1999) and from experimental cholestasis (Crocenzi et al., 2001, Crocenzi et al., 2003).

In vitro, silymarin has been reported to inhibit certain hepatic enzymes such as aminopyrine demethylase, benzopyrene hydroxylase, ethoxy coumarin O-deethylase (Lettéron et al., 1990) and glutathione S-transferase (Bartholomaeus et al., 1994). The effect of flavonolignans on UDP-glucuronosyltransferase (UGT) is less known. This enzyme system is a superfamily of enzymes that catalyze the conjugation of glucuronic acid to both endogenous compounds, such as bilirubin and steroid hormones, and exogenous compounds, including food additives, drugs, and environmental pollutants (for review, see Tephly and Burchell, 1990). Based on the nucleotide and amino acid sequences, mammalian UGT isozymes are grouped into two major families termed UGT1A and UGT2B (Burchell et al., 1991, Mackenzie et al., 1997). Silymarin or its main constituent, silibinin, has been reported to decrease the glucuronidation of two family 1A substrates: 4-methylumbelliferone (Venkataramanan et al., 2000) and 3-OH-benzo(a)pyrene (Chrungoo et al., 1997) both in vitro and in intact cells, pointing the experimental evidence to a direct effect of the flavonolignans or their metabolites on the enzyme molecule (Venkataramanan et al., 2000). In this work we further investigate, in vitro, the characteristics of the eventual inhibitory effect of silymarin and silibinin on UDP-glucuronosyltransferase using bilirubin and p-nitrophenol, substrates of the main expressed family 1A isoforms in liver, UGT1A1 and 1A6, respectively (Ritter et al., 1992), and ethinylestradiol, substrate of two family 2B isozymes 2B1 and 2B3 (Mackenzie, 1987, Mackenzie et al., 1997). Since silibinin is metabolized by UGTs (Kren et al., 2000), the effect of silibinin-glucuronide was additionally studied.