Short Communication Milk Thistle, a Herbal Supplement, Decreases the Activity of CYP3A4 and Uridine Diphosphoglucuronosyl Transferase in Human Hepatocyte Cultures

Milk thistle extract is one of the most commonly used nontraditional therapies, particularly in Germany. Milk thistle is known to contain a number of flavonolignans. We evaluated the effect of silymarin, on the activity of various hepatic drug-metabolizing enzymes in human hepatocyte cultures. Treatment with silymarin (0.1 and 0.25 mM) significantly reduced the activity of CYP3A4 enzyme (by 50 and 100%, respectively) as determined by the formation of 6-b-hydroxy testosterone and the activity of uridine diphosphoglucuronosyl transferase (UGT1A6/9) (by 65 and 100%, respectively) as measured by the formation of 4-methylumbelliferone glucuronide. Silymarin (0.5 mM) also significantly decreased mitochondrial respiration as determined by MTT reduction in human hepatocytes. These observations point to the potential of silymarin to impair hepatic metabolism of certain coadministered drugs in humans. Indiscriminate use of herbal products may lead to altered pharmacokinetics of certain drugs and may result in increased toxicity of certain drugs. Milk thistle extract is one of the most commonly used nontraditional therapies, particularly in Germany. The annual sale of this product is about $180 million in Germany alone (Cowley, 1995). Milk thistle [Silybum marianum (L.) Gaertn. (Fam. Asteraceae)] is known to contain a number of flavonolignans, compounds that are produced in plants by radical coupling of a flavonoid and a phenylpropanoid (Dewick, 1997). A mixture of these flavonolignans, termed silymarin, was first isolated from an extract of milk thistle fruit [from which the pappus (feathery tuft) had been removed] some three decades ago. Silymarin is known to be composed of mainly silybin (about 50–70%), but also contains silychristin, silydianin, and other closely related flavonolignans (Wagner et al., 1985). A standardized extract of milk thistle contains at least 70% silymarin (Schulz et al., 1998; Foster and Tyler, 1999). Silymarin/silybin is reported to protect the liver against CCl4, acetaminophen-, amanitin-, thioacetamide-, and D-galactosamine-mediated hepatotoxicity in rats (Schriewer et al., 1973; Vogel et al., 1984; Mourelle et al., 1989; Muriel et al., 1992; Chrungoo et al., 1997a,b). Silymarin has been reported to inhibit certain hepatic enzymes such as aminopyrine demethylase, benzopyrene hydroxylase, hexobarbital hydroxylase, and ethoxy coumarin O-deethylase in rats (Letteron et al., 1990). Silybin is primarily conjugated and excreted in the bile and urine in rats (Lorenz, 1982). Silymarin is known to deplete the pool of uridine diphosphoglucuronic acid (UDPGA) in hepatocytes and decrease glucuronidation of bilirubin in rats (Chrungoo et al., 1997b). The above observations would suggest that there would be a competition between silymarin and other drugs that are metabolized by various cytochrome P450 (CYP) enzymes or conjugated to a glucuronide in the liver. Our objective was to evaluate the effect of acute and chronic exposure of silymarin on the activity of CYP3A4 and uridinediphosphoglucuronosyl transferase (UGT1A6/9) in primary cultures of human hepatocytes. Materials and Methods Chemicals. Williams E medium, medium supplements, dexamethasone, and insulin were obtained from BioWhittaker (Walkersville, MD). Gentamycin was obtained from Life Technologies Inc. (Grand Island, NY). Silymarin, 4-methylumbelliferone (4-MU), and testosterone were purchased from Sigma (St. Louis, MI). 6-b-Hydroxy (OH) testosterone was obtained from Steraloids (Wilton, NH). MTT was obtained from Bio-Rad (Richmond, CA). Hepatocyte Culture and Treatment. Hepatocytes were isolated from human donor livers by a three-step collagenase perfusion technique as described previously (Strom et al., 1996, 1998). The viability of the cells obtained was measured by Trypan blue exclusion test. Only cells that are .80% viable were used for further studies. The hepatocytes (1.5 3 10) were plated on six-well culture plates previously coated with rat-tail collagen. The hepatocytes were plated in Williams E medium supplemented with 0.1 mM dexamethasone, 0.1 mM insulin, 0.05% gentamicin, and 10% bovine calf serum. Cells were allowed to attach for 4 to 6 h, at which time the medium was This work was supported in part by University of Pittsburgh Pharmacy Associates grant from the School of Pharmacy (R.V., B.J.K.), by Pfizer undergraduate summer Research Fellowship (B.J.K.), and by National Institutes of Health N01-