Introduction

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UDP-Glucuronosyltransferase (UGT)¹ is a superfamily of enzymes that catalyze the glucuronidation of both endogenous toxins, e.g., bilirubin, as well as xenobiotics such as carcinogens, in general rendering them biologically inactive (Burchell and Coughtrie, 1989; Miners and Mackenzie, 1991; Mackenzie et al., 1997; de Wildt et al., 1999; Radominska-Pandya et al., 1999). Induction of these enzymes, which has long been known to occur (Burchell and Coughtrie, 1989; Miners and Mackenzie, 1991; Bock et al., 1999), could therefore be considered beneficial. On the other hand, increased glucuronidation of drugs would be expected to result in diminished pharmacologic response. Although most inducers of the UGTs are potentially harmful chemicals or drugs (Burchell and Coughtrie, 1989; Miners and Mackenzie, 1991; Bock et al., 1999), more recent observations suggest that UGT inducers may be found among dietary components (Miners and Mackenzie, 1991), including dithiolthiones in cruciferous vegetables (Grove et al., 1997; Clapper, 1998) and phenylethylisothiocyanate in watercress (Hecht et al., 1999).

Recent studies of the metabolism of the dietary flavonoid chrysin (Fig. 1) by human intestinal and hepatic cell lines as well as rat hepatocytes demonstrated glucuronidation and sulfation to be the rate-limiting metabolic reactions (Galijatovic et al., 1999). In a further study, we demonstrated efficient induction of one or several isoforms of the UGT1A subfamily by chrysin in the human intestinal cell line Caco-2 (Galijatovic et al., 2000). The induction resulted in as much as a 14-fold increase in the glucuronidation of chrysin by the cell homogenate. The response was linear with concentration of flavonoid over the range of 5 to 50 µM and was maximal after pretreatment for 3 to 4 days. As recently shown by Strassburg et al. (1999), the UGT1A isoforms are differentially expressed in human hepatic and intestinal tissue, raising the question whether the induction response is different in a hepatic as compared to an intestinal cell line.

In the present study, we examined the effect of chrysin on glucuronic acid conjugation in the human hepatic cell line Hep G2. Studies with recombinant enzymes and isoform-specific antibodies were performed to establish which UGT1A isoform(s) were induced. The finding of induction of UGT1A1 was confirmed by Northern analysis, and its functional significance explored for several substrates, such as bilirubin and the oral contraceptive drug ethinylestradiol.

	Materials and Methods

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Chemicals. Chrysin (5,7-dihydroxyflavone) (Fig. 1), bilirubin (mixed isomers), ethinylestradiol, uridine 5'-diphosphoglucuronic acid (UDPGA), and Williams' Medium E were purchased from Sigma Chemical Co (St. Louis, MO). Fetal bovine serum was obtained from Summit Biotechnology (Fort Collins, CO). Other cell culture supplies were prepared by Mediatech (Herndon, VA) and obtained from Fisher Scientific (Pittsburgh, PA). [³H]17-Ethinylestradiol (50 Ci/mmol) and [-³²P]dCTP (3000 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Chemical structure of the flavonoid chrysin.

Cell Culture. Hep G2 cells were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in Williams' Medium E with 10% fetal bovine serum, L-glutamine, and antibiotic/antimycotic solution in a humidified 37°C incubator with 5% carbon dioxide. When cells in 100-mm dishes or 6-wells were about 90% confluent (4-6 days after seeding), they were treated with 25 µM chrysin in dimethylsulfoxide (0.3% final volume) or the same volume of dimethylsulfoxide for 3 days. The medium was changed every 24 h, and the cells were used for in situ metabolism assays, homogenate or microsomal preparation, or RNA isolation at 24 h after the last medium change. For in situ evaluation of the conjugation activity of induced and control Hep G2, the cells grown in 6-wells were incubated for 6 h with 3 ml of medium containing 25 µM chrysin. The medium was then collected, subjected to solid phase extraction with Oasis cartridges (Waters, Milford, MA), as previously described (Galijatovic et al., 1999), and subjected to HPLC analysis.

Glucuronidation Assays with Cell Homogenates and Microsomes. Confluent Hep G2 cells grown in 100-mm dishes and treated as above with chrysin or solvent for 3 days were rinsed with cold phosphate-buffered saline and harvested by scraping the cells into ice-cold 0.15 M KCl in 10 mM phosphate buffer (pH 7.4), containing the protease inhibitors phenylmethylsulfonyl fluoride, antipain, aprotinin, benzamidine, leupeptin, and pepstatin (Sigma) (5 ml/10 dishes of cells). The cells were sonified for 3 × 5 s on ice with 30-s cooling periods. The homogenates were stored in aliquots at 80°C and used for catalytic assays of glucuronidation of chrysin and bilirubin. These incubations were carried out for 1 h at 37°C with 0.1 to 100 µM substrate concentrations, 1 mM UDPGA and cell homogenate (0.7-2 mg of protein) in 0.5 ml of 100 mM Tris·HCl buffer, pH 7.4, with 5 mM MgCl₂. Control samples were incubated without UDPGA. The glucuronidation reactions for chrysin were terminated by putting the samples on ice and subjecting them to solid phase extraction, as described above. Bilirubin incubations were done under argon, and the samples were protected from light, using modifications of a method suggested by Chris Patten at Gentest. The reactions were terminated by putting the samples on ice and adding ascorbic acid (10 mg/ml) and an equal volume of acetonitrile to precipitate proteins. The supernatant, after vortex mixing and centrifugation at 14,000g, was used for HPLC analysis.

Immunoblot Analyses of UGT Proteins. Microsomes prepared from chrysin-treated and control Hep G2 cells and human liver as well as recombinant standard proteins were heated at 90°C for 5 min with an equal volume of sample buffer and loaded on 12% SDS polyacrylamide gels. After electrophoresis the proteins were transferred to nitrocellulose membranes, blocked, and incubated with primary and secondary antibodies as previously described (Galijatovic et al., 2000). The following primary polyclonal antibodies against human UGT proteins were obtained from Gentest: anti-UGT1A, anti-UGT1A6, and anti-UGT2B7. In addition, anti-UGT1A1 was prepared and used as recently described (Ritter et al., 1999) with the exception that incubations with primary and secondary antibodies were done in the presence of 5% nonfat milk.

Northern Blot Analyses of UGT mRNAs. Total RNA was isolated from chrysin-treated and control Hep G2 cells (five 100-mm dishes each) and 1 g of human liver. The cell monolayers were washed with PBS, and the cells were digested with RNAzol B (Tel-Test, Inc., Friendswood, TX) lysis buffer (1 ml/plate). The digests were treated with chloroform to remove protein and DNA, and the RNA was precipitated from the aqueous phase with isopropanol and washed with 70% ethanol according to the manufacturer's protocol. The liver tissue was treated identically after homogenization with RNAzol (6 ml) in a Potter-Elvehjem homogenizer. All samples were reconstituted in 500 µl of 0.1% SDS (3-4 µg/µl, determined by optical density at 260 nm) and, after denaturation at 65°C for 15 min with 3 volumes of RNA sample loading buffer, 20 µg of RNA was loaded on a 1% agarose gel with 3% formaldehyde and ethidium bromide for visualizing the RNA. After electrophoresis in MOPS/EDTA buffer, the RNA was transferred to nylon filters (Hybond-N, Amersham Pharmacia Biotech, Piscataway, NJ). The filters were hybridized overnight at 65°C to a ³²P-labeled probe prepared from a 0.7-kbp XhoI-EcoRI restriction fragment pSK-UGT1A1 containing the 5' end of UGT1A1 (Ritter et al., 1999). The probe was labeled by random primed synthesis, using [-³²P]dCTP and a kit from Amersham Pharmacia Biotech. After repeated washes with SDS/EDTA in phosphate buffer, the blots were subjected to autoradiography, using Hyperfilm MP (Amersham). The same membranes were then stripped and reprobed with a 1-kbp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Ambion, Austin, TX) to confirm equal loading between control and chrysin-treated cell samples.

Glucuronidation Assays with Recombinant Enzymes and Human Liver Microsomes. Chrysin and bilirubin (0.1-100 µM) were used as potential substrates for recombinant UGT isoforms, i.e., 1A1, 1A4, 1A6, and 1A9, supplied as microsomes of human lymphoblast-expressed enzymes (Gentest, Woburn, MA) and also with human liver microsomes. Human livers were obtained from Liver Tissue Procurement and Distribution System (University of Minnesota, Minneapolis, MN) kept frozen at 80°C. The human liver microsomes were prepared by standard differential centrifugation (La Du et al., 1972). All procedures were carried out at 4°C. Briefly, 1 to 2 g of frozen liver was cut in small pieces and homogenized with a Teflon/glass homogenizer in 4 volumes of 1.15% KCl. After centrifugations at 10,000 and 100,000g, the microsomal pellet was resuspended in 100 mM potassium phosphate buffer (pH 7.4), containing 10% glycerol, EDTA, butylated hydroxytoluene, and phenylmethylsulfonyl fluoride. Microsomal suspensions were quick-frozen and stored in aliquots at 80°C. The incubations and analyses were done exactly as with the Hep G2 cell homogenates above.

Calculation of Enzyme Kinetic Parameters. Apparent K_m and V_max values were obtained from the Henri-Michaelis-Menten equation (Segel, 1975) by nonlinear regression analysis of velocity versus substrate concentration plots, using the Solver function of Microsoft Excel.

	Results

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The flavonoid chrysin is efficiently metabolized by human Hep G2 cells both by glucuronidation and sulfation (Galijatovic et al., 1999). When the Hep G2 cells were pretreated with 25 µM chrysin for 3 days, the glucuronidation of chrysin in the intact cell increased 4.2-fold (P < .01; N = 11) while the sulfation remained unaffected, Fig. 2. The next experiments focused on glucuronidation only, using the cell homogenate rather than the intact cell, to monitor chrysin glucuronidation, after addition of the cofactor UDPGA. In these experiments, chrysin pretreatment resulted in a 14-fold increase in activity compared to untreated cells (Fig. 3). The apparent enzyme kinetic parameters determined in these experiments demonstrated that the increase in chrysin glucuronidation efficiency (V_max/K_m) was mainly due to an increase in the V_max value, although the K_m value of 4.9 µM was reduced after induction (1.9 µM).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Chrysin metabolism by Hep G2 cells after pretreatment with 25 µM chrysin for 3 days. Open bars denote chrysin glucuronide and hatched bars chrysin sulfate. Mean values ± S.E.M. are shown (N = 11). *, significantly higher than control (P < .01).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Chrysin glucuronidation by Hep G2 cell homogenates prepared from untreated cells () and cells pretreated with 25 µM chrysin for 3 days (). Mean values from two experiments are shown. The kinetic parameters were calculated from the Henri-Michaelis-Menten equation, using nonlinear regression analysis of the velocity versus substrate concentration with the Solver function of Microsoft Excel.

In the next series of experiments, four antibodies were used in Western analyses of microsomes isolated from chrysin-treated and untreated Hep G2 cells (Fig. 4). An antibody recognizing all of the UGT1A isoforms demonstrated a low expression in untreated cells but a marked induction in the chrysin-pretreated Hep G2 cells (Fig. 4, top panel). Antibodies recognizing the common hepatic UGT1A6 and 2B7 isoforms showed that these isoforms were expressed in Hep G2 cells but were not induced by chrysin (Fig. 4, middle panels). In contrast, an antibody recently shown to specifically recognize UGT1A1 (Ritter et al., 1999) detected a high level of induction of this isoform by chrysin in the Hep G2 cells with virtually no staining in the control cells (Fig. 4, bottom panel).

View larger version (66K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of recombinant UGT isoforms (2B7, 1A1, and 1A6; lanes 1, 2 and 3, respectively) and microsomes from untreated (control) Hep G2 cells (lane 4) and cells pretreated with 25 µM chrysin for 3 days (lane 5). Four different immunoblots were done with antibodies specific for UGT 1A, 1A6, 2B7 and 1A1, respectively. The arrows designate the UGT bands.

Total RNA isolated from control and chrysin-induced Hep G2 cells, subjected to agarose-formaldehyde electrophoresis and hybridized to a ³²P-labeled cDNA fragment of pBluescript SK/UGT1A1, clearly showed the presence of UGT1A1 mRNA with the expected size (2.3 kb) in the chrysin-treated cells but only trace amounts in the control cells (Fig. 5, lanes 3 and 2, respectively). As expected, total RNA isolated from human liver gave a positive signal (Fig. 5, lane 1). The size estimate of this band was based on its location in relation to the 18 S and 28 S ribosomal RNA bands visualized on the agarose gel by ethidium bromide fluorescence before transfer. The membrane was then stripped and hybridized with a GAPDH cDNA probe (lower panel), showing similar loading of control and induced cells. When poly(A)⁺ RNA was isolated from control and chrysin-treated Hep G2 cells, allowing the loading of about 20- to 30-fold more mRNA on the gel, UGT1A1 mRNA could be clearly detected also in the control cells and was strongly induced in the chrysin-treated cells (Fig. 5, lanes 4 and 5, respectively). Again, reprobing with GAPDH cDNA showed similar loading.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Northern blot analysis of UGT1A1 mRNA expression in Hep G2 cells. Total RNA (20 µg) from human liver (positive control) (lane 1), control and chrysin-treated cells (lanes 2 and 3, respectively) and poly(A)+ RNA (5 µg) isolated from control and chrysin-treated cells (lanes 4 and 5, respectively) were subjected to agarose-formaldehyde electrophoresis, hybridization with a [32P]dCTP-labeled EcoRI/XhoI cDNA fragment of UGT1A1 (Ritter et al., 1999), and autoradiography. The assignment of the UGT1A1 band was based on its location on the blot in comparison with the 18 S and 28 S ribosomal RNA bands on the ethidium bromide-stained gel. The blots were then stripped and hybridized to a GAPDH cDNA probe (lower panel) to confirm equal sample loading for control and chrysin-treated samples on each blot.

When comparing the catalytic activities toward chrysin of recombinant UGT isoforms 1A1, 1A6, 1A9, and 1A4, all of which are hepatic isoforms, the isoform that demonstrated the highest activity (V_max/K_m), interestingly, was the inducible form, UGT1A1 (Table 1). Its K_m value for chrysin glucuronidation was low (0.35 µM), and its V_max value was high (360 pmol/min/mg of protein). We found a high catalytic efficiency also for UGT1A9, about 70% of that for UGT1A1, but relatively low efficiency for UGT1A6, the isoform that is known to use simple planar phenols as substrates (Table 1). Chrysin was not a substrate for UGT1A4.

In view of the findings of the induction of the UGT1A1 isoform, we examined the glucuronidation of compounds that have been shown to be specific substrates for this isoform, using the homogenates of chrysin-induced and noninduced Hep G2 cells. The best recognized substrate, bilirubin, was glucuronidated both by the noninduced and the chrysin-treated cells, producing both isomeric monoglucuronides and the diglucuronide in a ratio of about 6:1, using a high resolution HPLC approach (data not shown). Total glucuronidation of bilirubin by the cell homogenates is shown in Fig. 6. Although activity was clearly measurable at all bilirubin concentrations (0.1-5 µM), accurate enzyme kinetic parameters could not be determined for the noninduced cells. After pretreatment of Hep G2 cells with 25 µM chrysin, total bilirubin glucuronidation increased about 20-fold with an apparent K_m value of 1.4 µM and a V_max value of 3.7 pmol/min/mg of protein. For comparison, total bilirubin glucuronidation by recombinant UGT1A1 yielded a K_m value of 0.23 µM and by liver microsomes from two human donors K_m values of 0.78 and 0.79 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Bilirubin glucuronidation by Hep G2 homogenates prepared from untreated cells () and cells pretreated with 25 µM chrysin for 3 days (). Mean values from two experiments are shown. The kinetic parameters were calculated from the Henri-Michaelis-Menten equation, using nonlinear regression analysis of the velocity versus substrate concentration with the Solver function of Microsoft Excel.

The oral contraceptive drug ethinylestradiol has also been shown to be a substrate for UGT1A1 (Ebner and Burchell, 1993). When using this drug to assay glucuronidation activity of the noninduced Hep G2 cell homogenate, the activity was barely above background (1.4 pmol/min/mg of protein). In the homogenate from the chrysin-induced cells, the activity increased 7-fold to 9.7 ± 2.8 (S.E.M.; N = 4) pmol/min/mg of protein (P < .05).

	Discussion

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The flavonoid chrysin is cleared exclusively by glucuronidation and sulfonation both from intestinal and liver tissue (Galijatovic et al., 1999). This appears to be a common occurrence with many dietary flavonoids (Abe et al., 1990; Boutin et al., 1993; Shimamura et al., 1993; Zhu et al., 1994; Jäger et al., 1998; Manach et al., 1998), although oxidative metabolism cannot be excluded for some flavonoids. We very recently demonstrated induction of glucuronidation when the human intestinal cell line Caco-2 was treated with chrysin (Galijatovic et al., 2000), suggesting the possibility that the flavonoids might induce their own elimination. In the present study, we extend these observations to include also the human hepatic cell line Hep G2. Thus, pretreatment of these cells for 3 days with 25 µM chrysin resulted in a 4.2-fold increase in the glucuronidation of chrysin by the intact cells with no change in sulfonation.

When chrysin glucuronidation was examined in the cell homogenate after washing of the cells to remove chrysin used in the pretreatment of the cells, the induction was as high as 14-fold. We attribute the lower level of induction in the intact cells to substrate/product inhibition, although we cannot exclude the possibility of UDPGA cofactor depletion in the intact cell, as opposed to the homogenate incubations where UDPGA was added in excess. Another possible reason for this phenomenon is that latent enzyme is activated by sonification in the cell homogenization procedure. The apparent enzyme kinetic parameters for the homogenate experiments indicated that the K_m value remained virtually the same after induction, whereas the V_max value increased dramatically. As previously shown for Caco-2 cells, maximal induction of glucuronidation in the Hep G2 cells occurred after pretreatment with chrysin for 3 to 4 days and the minimum concentration for induction was about 10 µM (data not shown).

The only previous report of a flavonoid inducing a UGT activity in a human cell involved the isoflavonoid biochanin A, which increased the glucuronidation of testosterone in an androgen-responsive prostate cancer cell line (Sun et al., 1998). However, studies in the rat had previously provided evidence that flavonoids may be inducers of UGT (Canivenc-Lavier et al., 1996; Siess et al., 1996). Flavonoids and other dietary components (Miners and Mackenzie, 1991; Grove et al., 1997; Clapper, 1998; Hecht et al., 1999), may thus have an important influence on glucuronidation of endogenous as well as exogenous chemicals.

In experiments using UGT isoform-specific antibodies and Western analyses, we probed microsomes from chrysin-treated versus control Hep G2 cells in attempts to identify which isoforms may have been induced. An antibody selective for all UGT1A, as opposed to UGT2B, isoforms demonstrated a high degree of induction. Interestingly, a major hepatic isoform, UGT1A6, was not induced, and this was also the case with UGT2B7. In contrast, a band with migration identical with that of UGT1A1 was markedly induced. This was detected using a newly developed antibody highly specific for this isoform (Ritter et al., 1999). The finding of UGT1A1 induction points to a highly selective induction response of the UGTs to chrysin.

Strongly supporting the UGT1A1 protein induction were the Northern analyses using a probe highly specific for the mRNA for this isoform (Ritter et al., 1999). In the analysis of total RNA, while there was a strong signal in the chrysin-treated cells, there was no detectable message for UGT1A1 in the control cells, consistent with previous observations (Batt et al., 1995; Ritter et al., 1999). However, in the Northern analysis of purified mRNA, the message was clearly present also in the uninduced cells and, again, strongly induced in the chrysin-treated cells. An additional band with lower molecular weight was also induced, which may be a splice variant of UGT1A1.

Consistent with induction of chrysin glucuronidation, chrysin was a substrate for UGT1A1, in fact was a highly efficient substrate for this isoform. This has previously been observed (King et al., 1996), although in the present study we also established the enzyme kinetic properties, demonstrating a very low K_m value of 0.35 µM for the recombinant protein. Chrysin was also an efficient substrate for UGT1A9, with a low K_m value of 1.7 µM, but a poor substrate for UGT1A6 (K_m 12.8 µM) and not a substrate for UGT1A4. This suggests that UGT1A9 may also be induced by chrysin. However, attempts to examine this question with available antibodies were inconclusive (data not shown). On the other hand, the finding that chrysin is a substrate for UGT1A6, also shown previously (Galijatovic et al., 1999), suggests that this isoform might be responsible for chrysin glucuronidation in the uninduced Hep G2 cells. This is supported by the Western analysis, showing equal presence of UGT1A6 in induced and control cells. The induction of chrysin glucuronidation in the Hep G2 cells resulted in a reduction in the K_m value, although it was not as low as the K_m value for chrysin glucuronidation by recombinant UGT1A1. This observation could suggest that additional UGT isoforms may be induced. Previous work has identified several other UGT isoforms that could potentially use chrysin or other flavonoids as substrates. These include UGT1A3 (Green et al., 1998), 1A8 (Cheng et al., 1998), 1A9 (Ebner and Burchell, 1993), and 2B15 (Green et al., 1994; Lévesque et al., 1997). As recently shown by Strassburg et al. (1999), UGT1A1, 1A3, 1A4, 1A6, and 1A9 are all present in the liver, whereas the intestine also contains UGT1A8 and 1A10.

The finding that the K_m value for chrysin glucuronidation by the Hep G2 cell homogenate is about 5 times higher than by recombinant UGT1A1 may potentially be due to an endogenous inhibitor in the cell homogenate. This hypothesis is supported by the very similar finding with bilirubin as substrate, i.e., 6 times higher K_m for the Hep G2 cell homogenate compared to recombinant UGT1A1.

The induction of UGT1A1 by chrysin may have important biological implications. The best known substrate for UGT1A1 is the endogenous toxin bilirubin (Chowdhury et al., 1995; Ritter et al., 1999). Induction of UGT1A1 therefore suggests that bilirubin glucuronidation is increased, as clearly shown in our study, with the level of induction being as high as with chrysin glucuronidation, i.e., more than 10-fold. Also, the values for V_max were very similar for bilirubin and chrysin glucuronidation. Interestingly, the K_m value for bilirubin glucuronidation both by induced Hep G2 cells, recombinant UGT1A1 and human liver microsomes of 0.23 to 1.4 µM is considerably lower than in general reported (Radominska-Pandya et al., 1999). This may be the result of a more efficient and sensitive separation/detection system for the bilirubin glucuronides in the present study. Because of the importance of glucuronidation in the removal of the endogenous toxin bilirubin, the induction of UGT1A1, the isoform responsible for this reaction, has been of interest. Phenobarbital is suggested to be effective (Chowdhury et al., 1995; Ritter et al., 1999) and so is 3-methylcholanthrene (Ritter et al., 1999), although the inducibility appears to be considerably less than observed with chrysin in our study. The induction by 3-methylcholanthrene suggests the involvement of the aryl hydrocarbon receptor (Ritter et al., 1999). However, UGT1A6, inducible by arylhydrocarbon receptor agonists in Caco-2 cells (Abid et al., 1995; Bock et al., 1999; Münzel et al., 1999), was not affected by chrysin. The mechanism of chrysin induction thus needs to be investigated.

Finally, the induction of UGT1A1 may affect metabolism of some drugs. Thus, the glucuronidation of the synthetic estrogen ethinylestradiol, which has been shown to be a substrate for UGT1A1 (Ebner et al., 1993), was induced about 7-fold by chrysin. Such increased glucuronidation may lead to diminished contraceptive efficacy of this drug, of obvious importance.

However, much additional information is needed before the significance of the chrysin-induced UGT1A1 can be fully understood. Experiments with normal hepatocytes as well as in vivo experiments both in animal and man have already been initiated. Although chrysin is not one of the major flavonoids in our diet, its ability to inhibit the enzyme aromatase (Kao et al., 1998), catalyzing the conversion of testosterone to 17-estradiol, suggests its potential use in lowering breast cancer risk and has also led to its marketing as a high dose dietary supplement in body builders. It will, however, be important to expand our studies to other flavonoids, especially those present at higher levels in various fruits and vegetables. We have already found that quercetin can produce the same response when cells are exposed for several weeks to this flavonoid (Galijatovic et al., 2000).