WO2001058410A2 - A method of treating colon cancer by administering apigenin, luteolin, diosmetin and crysin - Google Patents

Abstract

The invention provides a method of treating any condition of elevated levels of unconjugated bilirubin in adults or children, such as Gilbert's syndrome or liver cirrhosis. The method calls for the administration of chrysin or chrysin analogs via dietary or other means. Also provided is a method of increasing glucuronidation of bilirubin in a subject, comprising administering to the subject an amount of chrysin effective to increase the glucuronidation of bilirubin. A method of increasing the expression of UDP-glucuronosyltransferase in a subject is provided, comprising administering to the subject an amount of chrysin effective to increase the UDP-glucuronosyltransferase. Upregulation of the expression of UDP-glucuronosyltransferase levels is expected to be beneficial in a number of areas: 1) increased detoxification of ingested carcinogens, such as the cooked-food mutagens (chrysin is an aromatase inhibitor in cells); 2) decreasing the conversion of androgens to estrogens, potentially building muscle mass or for prevention of breast cancer in women. In the method of upregulating UGT, UDP-glucuronosyltransferase can be a UGT1A isoform. A method of treating colon cancer is provided, comprising administering an effective amount of a flavone, whereby the administration of the flavone treats the colon cancer.

Description

A METHOD OF TREATING COLON CANCER BY ADMINISTERING APIGENIN, LUTEOLIN, DIOSMETIN AND CHRYSIN

This application claims priority to U.S. No. 60/182,072, filed February 11, 2000, and U.S. No. 60/232,771, filed September 15, 2000, and which applications are incorporated by reference herein.

The work described herein was carried out, in part, under National Institutes of Health grants CA69138 and GM55561. The government therefore may have certain rights in the invention.

BACKGROUND

As emphasized during the continuing development of the Caco-2 cell line as a model for human intestinal absoφtion, these cells in culture morphologically resemble small intestine absoφtive cells with many of its typical enzymes and transporters (1-4). Although Caco-2 cells in the past have not been considered to express high levels of drug metabolizing enzymes (5, 6), conjugation reactions have previously been described. This includes sulfation of dopamine and »-nitrophenol (7) and L-a- methyldopa (8). The model substrate >-nitrophenol has also been shown to be glucuroni dated by Caco-2 cells (5, 9), as has L-a-methyldopa (8).

This report describes for the first time the induction of UGT by chrysin and the ubiquitous dietary flavonoid quercetin (Fig. 1) in Caco-2 cells.

UDP-Glucuronosyltransferase (UGT) 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. Most inducers of the UGTs are potentially harmful chemicals or drugs (Burchell and Coughtrie, 1989; Miners and Mackenzie, 1991; Bock et al., 1999).

SUMMARY OF THE INVENTION

The invention provides a method of treating any condition of elevated levels of unconjugated bilirubin in adults or children, such as Gilbert's syndrome or liver cirrhosis. The method calls for the administration of chrysin or chrysin analogs via dietary or other means.

Also provided is a method of increasing glucuronidation of bilirubin in a subject, comprising administering to the subject an amount of chrysin effective to increase the glucuronidation of bilirubin.

A method of increasing the expression of UDP-glucuronosyltransferase in a subject is provided, comprising administering to the subject an amount of chrysin effective to increase the UDP-glucuronosyltransferase. Upregulation of the expression of UDP-glucuronosyltransferase levels is expected to be beneficial in a number of areas: 1) increased detoxification of ingested carcinogens, such as the cooked- food mutagens (chrysin is an aromatase inhibitor in cells); 2) decreasing the conversion of androgens to estrogens, potentially building muscle mass or for prevention of breast cancer in women. In the method of upregulating UGT, UDP-glucuronosyltransferase can be a UGT1 A isoform. The UGT1 A isoform can be UGT1 Al . The UGT can be UGT1A9. A method of treating colon cancer is provided, comprising administering an effective amount of a flavone, whereby the administration of the flavone treats the colon cancer. The flavone can be a 5,7-dihydroxyflavone, for example one selected from the group consisting of chrysin, apigenin, luteolin and diosmetin.

Chrysin analogs can be used in the methods and compositions of the invention. A chrysin analog having a flavonoid structure is contemplated. A chrysin analog having a flavone structure is contemplated. A chrysin analog having a 5,7-substituted flavone structure is further contemplated. Chrysin and its analogs can be administered as a dietary supplement or in a food. Examples of chrysin analogs having a 5,7- substituted flavone

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1. Chemical structures of chrysin and quercetin.

Fig. 2. Chrysin metabolism by Caco-2 cells after pretreatment with 50 μM chrysin for 4 days. Open bars designate chrysin glucuronide and hatched bars chrysin sulfate. Mean values ± SEM are shown (N = 9). * Significantly higher than Control, p < 0.0001. ** Significantly lower than Control, p < 0.01.

Fig. 3. Chrysin glucuronidation by Caco-2 cell homogenates prepared from untreated cells (u) and cells pretreated with 50 μM chrysin for 4 days (s). Mean values ± SEM from 3 experiments are shown. The error bars for the untreated cells are contained within the symbols. Data were fit to the Michaelis-Menten equation.

Fig. 4. Immunoblot analysis of Caco-2 cell microsomes from cells pretreated with 50 mM chrysin for 4 days (lanes 4 and 6, 50 and 100 mg loaded) and from untreated Control cells (lanes 3 and 5, 50 and 100 mg loaded). UGT1A6 microsomal protein (50 mg loaded) was used as a positive control in lanes 1 and 2.

Fig. 5. Chrysin glucuronidation by intact Caco-2 cells after pretreatment of the cells with 10 μM quercetin or chrysin for 5 weeks. Medium containing solvent (Control), quercetin or chrysin was replaced every other day. After the last treatment, all cells were incubated with 50 μM chrysin for 24 hr for metabolite determinations. Mean values ± SEM are shown (N = 3-6).

Fig. 6. 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 ± SEM are shown (N = 11). * Significantly higher than Control (P < 0.01).

Fig. 7. Chrysin glucuronidation by Hep G2 cell homogenates prepared from untreated cells (m) and cells pretreated with 25 μM chrysin for 3 days (1). Mean values from 2 experiments are shown. The data were fitted to the Michaelis-Menten equation using Microsoft Excel.

Fig. 8. Western blot analysis of recombinant UGT isoforms (2B7, 1 Al and 1 A6; 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.

Fig. 9. Northern blot analysis of UGTl Al 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 UGTl Al (Ritter et al., 1999) and autoradiography. 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.

Fig. 10. Bilirubin glucuronidation by Hep G2 homogenates prepared from untreated cells (m) and cells pretreated with 25 μM chrysin for 3 days (1). Mean values from 2 experiments are shown. The data were fitted to the Michaelis-Menten equation using Microsoft Excel. Significantly higher than Control, p < 0.05.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "a" or "an" can mean one or more than one. For example "a cell" can mean a single cell or multiple cells.

The invention provides a method of treating any condition of elevated levels of unconjugated bilirubin in adults or children, such as Gilbert's syndrome or liver cirrhosis. The method calls for the administration of chrysin or chrysin analogs via dietary or other means.

For example, a method of treating jaundice in a neonate is provided, comprising administering to the neonate an amount of chrysin effective to treat jaundice. A method of treating hyperbilirubinemia in a neonate is thus provided, comprising administering to the neonate an amount of chrysin effective to reduce the amount of bilirubin in the neonate.

Also provided is a method of increasing glucuronidation of bilirubin in a subject, comprising administering to the subject an amount of chrysin effective to increase the glucuronidation of bilirubin. A method of increasing the expression of UDP-glucuronosyltransferase in a subject is provided, comprising administering to the subject an amount of chrysin effective to increase the UDP-glucuronosyltransferase. Upregulation of the expression of UDP-glucuronosyltransferase levels is expected to be beneficial in a number of areas: 1) increased detoxification of ingested carcinogens, such as the cooked-food mutagens (chrysin is an aromatase inhibitor in cells); 2) decreasing the conversion of androgens to estrogens, potentially building muscle mass or for prevention of breast cancer in women. For example, upregulation of the expression of UDP- glucuronosyltransferase levels is associated with neutralization of colon carcinogens which include, but are not limited to, N-OH-PhlP.

In the method of upregulating UGT, UDP-glucuronosyltransferase can be a UGTl A isoform. The UGTl A isoform can be UGTl Al . The UGT can be UGTl A9.

A method of treating colon cancer is provided, comprising administering an effective amount of a flavone, whereby the administration of the flavone treats the colon cancer. The flavone can be a 5,7-dihydroxyflavone, for example one selected from the group consisting of chrysin, apigenin, luteolin and diosmetin.

Chrysin analogs can be used in the methods and compositions of the invention.

A chrysin analog having a flavonoid structure is contemplated. A chrysin analog having a flavone structure is contemplated. A chrysin analog having a 5,7-substituted flavone structure is further contemplated. Chrysin and its analogs can be administered as a dietary supplement or in a food. Examples of chrysin analogs having a 5,7- substituted flavone structure include, but are not limited to, quercetin, apigenin, luteolin and diosmetin.

The compositions of the present invention are dietary supplements and can thus

be administered orally. For oral administration, fine powders or granules may contain

diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension

wherein suspending agents may be included, in tablets wherein binders and lubricants

may be included, or in a suspension in water or a syrup. Where desirable or necessary,

flavoring, preserving, suspending, thickening, or emulsifying agents may be included.

Tablets and granules are preferred oral administration forms, and these may be coated.

The present invention is more particularly described in the following examples

which are intended as illustrative only since numerous modifications and variations

therein will be apparent to those skilled in the art.

EXAMPLE 1

Flavonoid Induction of Glucuronidation IN Caco-2 Cells

Dietary flavonoids have been reported to be potent inhibitors of drug

metabolizing enzymes. In the present study the inducing effect of three of these

compounds, chrysin, quercetin and genistein, on UDP-glucuronosyltransferase (UGT)

in the human intestinal cell line Caco-2 were examined. The induction of UGT by

flavonoid pretreatment was studied both in the intact cells and cell homogenates,

measured as the glucuronidation of chrysin, and by immunoblot analysis of the UGT

1A protein. Exposure of Caco-2 cells to 50 μM chrysin resulted in a 3.8-fold increase

in chrysin glucuronidation in intact cells (p < 0.0001) with a 38% decrease in sulfation (p < 0.01). In the cell homogenate the induction was much larger, 14-fold. The

induction was slow to develop with maximum induction after 3-4 days. Interestingly,

the isoflavonoid genistein was without effect. Immunoblot analysis of Caco-2 cell

microsomes with a UGTl A subfamily-selective antibody showed a markedly increased

band at about 59 kDa, consistent with induction of one or more UGTl A isoforms. A 5-

week exposure of Caco-2 cells to low concentrations (10 μM) of chrysin or quercetin

also showed markedly increased glucuronidation activity. Diet-mediated induction of

intestinal UGT may be important for the bioavailability of carcinogens and other toxic

chemicals as well as therapeutic drugs.

MATERIALS AND METHODS

Materials

Chrysin, quercetin, genistein, uridine 5'-diphosphoglucuronic acid (UDPGA)

and protease inhibitors were purchased from Sigma Chemical Co. (St. Louis, MO).

Trifluoroacetic acid was of spectrophotometric grade from Aldrich Chemical Co.

(Milwaukee, WI). Hanks' balanced salts solution (HBSS) was obtained from Cellgro,

Mediatech, Fisher Scientific (Pittsburgh, PA). Recombinant human UGTl A6 and the

corresponding Western blotting kit were purchased from Gentest Coφ. (Woburn, MA).

Polyclonal anti-human UGTl A antibodies, raised against 15 amino acids in the

common C-terminal end of the UGTl A proteins, were a generous gift from Gentest.

Secondary antibody, HRP-conjugated goat anti-rabbit IgG, was purchased from

Kirkegaard & Perry Laboratories (Gaithersburg, MD). SuperSignal Chemiluminescent

Substrate was from Pierce (Rockford, IL). Electrophoresis and blotting supplies and prestained molecular weight markers were purchased from Bio-Rad Laboratories

(Hercules, CA).

Caco-2 Cell Culture

The human colon adenocarcinoma cell line Caco-2 was obtained from

American Type Culture Collection (Rockville, MD). The Caco-2 cells were cultured in

Eagle's Minimum Essential Medium with Earle's salts and L-glutamine (Cellgro,

Mediatech, Fisher Scientific, Pittsburgh, PA), supplemented with 1% non-essential

amino acids (Cellgro), 10% fetal bovine serum (Summit Biotechnology, Ft. Collins,

CO) and penicillin/streptomycin (Sigma Chemical Co.). Cells were grown in

humidified air with 5% CO₂ and subcultured in 6-well plates for incubation

experiments and 100 mm petri dishes for homogenate and microsomal preparation.

The cells were used at passage 35-75.

Chrysin Metabolism by Pretreated and Control Caco-2 cells

Four days after seeding Caco-2 cells in 6-wells, pretreatment with 50 mM

chrysin or 50 mM genistein in complete cell culture medium was started. The medium

was changed every 24 hr. Chrysin was dissolved in ethanokDMSO (80:20, v/v) at a

final concentration not exceeding 0.5%). Control cells were treated with the same

volume of solvent. After 4 days of pretreatment, 50 μM chrysin was added to both

chrysin-treated, genistein-treated and control cells. The medium was harvested 24 hr

later for analysis of chrysin and metabolites. Conjugated metabolites and unmetabolized chrysin were separated from the cellular medium using Oasis HLB 3cc

extraction cartridges (Waters, Milford, MA). The methanol eluates were taken to

dryness and the samples were reconstituted in 250 ml mobile phase and analyzed by

reversed phase HPLC, using a Symmetry C18 column (Waters) with 55% methanol and

0.3% trifluoroacetic acid as the mobile phase at 0.9 ml/min with 268 nm UV detection

(11). The cells were digested with 0.5 M NaOH and analyzed for protein content (15).

In another set of experiments, Caco-2 cells in 6-wells were pretreated with 50

μM chrysin for 1, 2, 3 or 4 days. The medium was analyzed for chrysin metabolites 24

hr after the last addition of chrysin (8 days after plating). The effect of flavonoid

pretreatment concentration on the glucuronidation was investigated using 0, 5, 10, 25

and 50 μM chrysin for 4 days.

Preparation of Caco-2 cell Homogenate and Microsomes

Caco-2 cells were plated in 100-mm petri dishes. On day 6 the cells were

incubated with 50 mM chrysin or solvent. The treatment with chrysin or solvent

continued for 4 days every 24 hr, as described above. Twenty-four hr after the last

medium change, the cell monolayers were washed twice with HBSS, scraped off the

dishes into 0.15 M KC1 in 10 mM sodium phosphate buffer (pH 7.4) (1 ml/dish). The

cells were disrupted by sonication on ice (5 x 5 sec). The cell homogenate was either

used for the catalytic assays or centrifuged at 9000 for 20 min at 4°C to obtain the

supernatant fraction (S10). The residual concentrations of chrysin glucuronide and

chrysin in the chrysin-pretreated cell homogenate were < 0.5 mM, as determined by HPLC. The S 10 fraction was further centrifuged at 100,000g for 60 min at 4°C. The

microsomal pellets were resuspended in 300 μl of homogenization buffer with protease

inhibitors (2 mM PMSF, 50 mg/ml antipain, 2 mg/ml aprotinin, 0.2 mg/ml

benzamidine, 0.5 mg/ml leupeptin and 1 mg/ml pepstatin). Homogenates and

microsomes were stored in aliquots at -80 °C.

Determination of UGT Protein Levels by Immunoblotting

Microsomal samples were heated at 90 °C for 5 min with an equal volume of

sample buffer and loaded on a 12% SDS-polyacrylamide minigel together with

molecular weight markers and positive and negative controls. After electrophoresis

(16), the proteins were transferred to a nitrocellulose membrane (17). The membrane

was blocked with 5% nonfat milk in 10 mM Tris/150 mM NaCl/0.05% Tween 20

(TBST) for 1 hr and incubated overnight at 4°C with anti-human UGTl A primary

antibody in 5% nonfat milk in TBST at a 1:100 dilution. The blot was washed with 5%

nonfat milk in TBST for 3 times 20 min and incubated with the secondary antibody,

HRP-conjugated goat anti-rabbit IgG, at a 1:5000 dilution in 5% milk in TBST for 1 hr.

After washing for 3 times 20 min with TBST, SuperSignal chemiluminescent substrate

was added and the membrane was exposed to Hyperfilm ECL (Amersham, Piscataway,

NJ) for 5 min. Chrysin Glucuronidation by Caco-2 Cell Homogenates

Chrysin (1 - 20 mM) and 100 ml of pretreated or control Caco-2 cell

homogenate (1.0 mg of protein) in 100 mM Tris.HCl buffer (pH 7.4) with 5 mM MgCl₂

were prewarmed for 10 min at 37 °C. The reactions were initiated by the addition of

0.5 mM UDPGA. The reaction mixtures (500 μl) were incubated at 37°C for 60 min.

The samples were cooled on ice and centrifuged for 2 min at 10,000g. The supernatant

was subjected to solid-phase extraction and HPLC analysis, as described above.

Kinetic parameters were obtained by fitting the data to the Michaelis-Menten equation

in Microsoft Excel.

Chronic Caco-2 Cell Flavonoid Treatment

Caco-2 cells were cultured as above and subcultured at a seeding density of

80,000 cells/cm² every 7 days. Fresh medium containing 10 μM chrysin, 10 μM

quercetin or DMSO was added on days 2, 4 and 6 after each subculturing. After 5

weeks of pretreatment, chrysin metabolism experiments were done in 6-wells at 24 hr

after the last flavonoid addition. The growth rate and viability were identical for

flavonoid-treated and control cells.

Statistical analysis used student's unpaired t-test with a significance level of p <

0.05. RESULTS

When the metabolism of chrysin by Caco-2 cells was analyzed by reversed-

phase HPLC, a glucuronic acid and a sulfate conjugate were identified by MS and

enzymatic techniques (11). After pretreatment of the cells for 4 days with 50 mM

chrysin, the concentration of unchanged chrysin was reduced, whereas that of the

glucuronic acid conjugate was markedly increased, with minimal change in the sulfate

conjugate concentration. The results from 9 experiments, with and without

pretreatment of Caco-2 cells with chrysin for 4 days are summarized in Fig. 2.

Whereas the glucuronic acid conjugate increased 3.8-fold (p < 0.0001), the sulfate

conjugate decreased 38% (p < 0.01). Although an increase in glucuronidation occurred

as early as after a 1-day pretreatment, the maximum response to chrysin was reached

after a 3- to 4-day pretreatment (Table I). The 30-50 % reduction in the formation of

the sulfate conjugate did not appear to be related to extensive consumption of the

substrate by the glucuronidation pathway. When examining the influence of chrysin

concentration on glucuronic acid conjugation, there appeared to be a linear increase

with concentration up to 50 mM (Table II). Whereas low chrysin concentrations did

not influence sulfate conjugation, 50 mM chrysin reduced this pathway similar to that

in Table I. In separate experiments also examined was the effect of pretreatment with

50 mM genistein, an isoflavonoid, on chrysin metabolism. There was no effect on

glucuronidation (126 ± 12 vs. 113 ± 12 pmol/mg protein/hr; treated vs. control), whereas sulfation was slightly decreased (226 ± 25 vs. 150 ± 19 pmol/mg protein/hr; p

< 0.05)(n=3).

We next examined the induction response in the Caco-2 cell homogenate after

careful washing of the cells prior to the homogenization. The increase in

glucuronidation after chrysin pretreatment was now as high as 14-fold compared to

control (Fig. 3). Because of the relatively low level of chrysin glucuronidation in the

homogenate of uninduced cells, accurate kinetics could not be determined. However,

the apparent K-,, value for glucuronidation of chrysin in the induced Caco-2 cell

homogenate was estimated to be about 0.8 mM.

In the next series of experiments, whether chrysin pretreatment of Caco-2 cells

produced an increase in UGT protein was determined. For these experiments,

microsomes of pretreated and untreated cells were prepared and an antibody, under

development by Gentest (Woburn, MA), which detects all proteins in the UGTl A

subfamily (Fig. 4) was used . There was a substantial increase in the intensity of a ~ 59

kDa band in the induced vs. uninduced control cells. Recombinant UGTl A6 protein

served as a positive control, producing a band with a migration identical to that of the

Caco-2 cell microsomes. Thus, chrysin pretreatment of Caco-2 cells resulted in

markedly increased expression of one or more UGTl A isoforms. In contrast,

experiments using an antibody specific for UGTl A6 failed to demonstrate induction of

this isoform. Finally, experiments were designed in which Caco-2 cells were exposed to

flavonoids for a period of 5 weeks. In addition to chrysin, quercetin, the most common

flavonoid in our diet (18) was also studied. Flavonoid concentrations of only 10 mM

were used in these experiments (Fig. 5). There was a more than 3-fold increase in the

glucuronidation of chrysin in the intact Caco-2 cells after chrysin pretreatment, similar

to the effect of 50 mM pretreatment in the acute experiments (Fig. 2). After quercetin

pretreatment, there was an about 2-fold increase in the glucuronidation activity.

This is the first report on induction of members of the UGT family by dietary

flavonoids in the human intestinal Caco-2 cells. This might have implications with

respect to the oral bioavailability of carcinogens as well as other toxicological agents

and commonly used drugs.

Pretreatment of human intestinal cells with chrysin markedly induces the

metabolism of this flavonoid through the glucuronidation but not the sulfation pathway.

The maximum induction response in the intact Caco-2 cells was a 3.8-fold increase in

chrysin glucuronidation (Fig. 2) which was magnified to a 14-fold increase in the Caco-

2 cell homogenate (Fig. 3). The lower apparent response in the intact cells may be due

to high accumulation of substrate and product, as demonstrated in a previous study for

quercetin (21), resulting in substrate/product inhibition. In contrast, both substrate and

product in the homogenate were efficiently removed, as evidenced by HPLC.

Maximum induction appeared to occur after 3-4 days of pretreatment and was linear with chrysin concentration in these short-term experiments. The inhibitory effect of

chrysin on the sulfation pathway at 50 μM concentration (Tables I and II) appears

similar to a previous observation for quercetin (22).

In order to test the effect of a chronic treatment schedule, the Caco-2 cells were

exposed to either chrysin or quercetin for up to 5 weeks, using only 10 μM

concentrations of each flavonoid. The induction response to chrysin was identical to

that of the short-term exposure; quercetin, the most abundant flavonoid in our diet (18),

was somewhat less effective. In terms of the flavonoid concentrations needed to

produce a significant induction response, the 10-50 μM concentrations used in the

present study are very likely to be achieved in the intestinal lumen after normal dietary

exposure to these compounds (10).

A different measure of the induction response was obtained by SDS-PAGE of

the microsomal proteins and immunoblotting. The use of a UGTl A subfamily-

selective antibody clearly demonstrated increased levels of one or several isoforms of

this group of enzymes (Fig. 4). Interestingly, experiments with a UGTlA6-specific

antibody showed no evidence of induction. UGTl A6 (23-25) as well as UGT1A9 and

UGT2B7 (25) have so far been identified in the Caco-2 cells.

There are a number of UGT isoforms, which could potentially use chrysin and

other flavonoids as substrates; these include UGT1A1 (26), UGT1A3 (27), UGT1A8

(28), UGTl A9 (29) and UGT2B15 (30, 31). As recently shown by Strassburg et al. (32), UGT1A1, UGTl A3, UGT1A8 and UGT1A9 are all expressed in the normal

human colon in addition to UGTl A4, UGTl A6 and UGTl A10.

Whereas UGTl Al, the isoform that metabolizes bilirubin, may be induced by

phenobarbital (33, 34), another UGT induction response is triggered by dioxin and

polyaromatic hydrocarbons via an effect on the Ah-receptor. This has been shown to

involve mainly UGT1A6 (24, 25) but also UGTl A9 (25). A recent study indicated that

the antioxidant t-butylhydroquinone induced UGT1A6, 1A9 and 2B7 (25). These

observations suggesting that UGT1A6 may not be involved (see above) also suggest

that the induction response produced by chrysin is different from that by dioxin and the

polyaromatic hydrocarbons, i.e. may not involve the Ah receptor.

These studies have suggested that the intestinal UGTs play an important role in

the detoxification of carcinogens and cytotoxic agents in general.

Throughout this application, various publications are referenced. The

disclosures of these publications in their entireties, as well as the references cited in

these publications, are hereby incoφorated by reference into this application in order to

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Table 1. Effect of Pretreatment Time with Chrysin (50 mM) on the Formation of

Chrysin Glucuronide and Sulfate

Pretreatment Time Metabolite Formation⁰

(days) (pmol/mg protein/hr)

Glucuronide Sulfate

0 115 ± 11 109 ± 8

1 182 ± 15* 72 ± 8*

2 2 2 27711 ±± 4499** 58 ± 16*

3 427 ± 66* 75 ± l^b

4 450 ± 20* 73 ± 6^b

^a Mean values ± SD (N=3)

* Significantly different from 0 days (p < 0.05)

Table 2. Effect of Pretreatment Chrysin Concentration on the Formation of Chrysin

Glucuronide and Sulfate

Pretreatment Concentration Metabolite Formation"

(mM) (pmol/mg protein/hr)

Glucuronide Sulfate

0 115 ± 11 109 ± 8

5 135 ±7 115 ± 5

10 187 ±13* 108 ± 10

25 330 ±46* 94 ± 15

50 445 ± 29* 60 ± 5*

Incubations were done for four days

^a Mean values ± SD (N=3)

* Significantly different from 0 days (p < 0.05)

EXAMPLE 2

Chrysin Induction of UDP-Glucuronosyltransferase 1 Al in Hep G2 Cells

The UDP-glucuronosyltransferases (UGTs) have long been known to be

inducible by various chemicals, including drugs, although the extent of induction in general has been modest. In the present study the ability of the dietary flavonoid

chrysin to induce UGT activity, protein and mRNA were determined. When

pretreating human hepatoma Hep G2 cells with 25 μM of chrysin, the glucuronidation

of chrysin itself increased 4.2-fold when measured in the intact cell and 14-fold in the

cell homogenate, i.e. autoinduction. Microsomes from chrysin-treated cells probed

with specific antibodies in Western analyses showed marked induction of the UGTl A

family of proteins. Iso form-specific induction of the important hepatic UGTIAI

protein was observed, but not of UGTl A6 or UGT2B7. The strong induction of

UGTIAI was confirmed by Northern analyses of total RNA as well as mRNA, using a

specific probe. UGTIAI message as well as protein were detectable also in untreated

Hep G2 cells. In catalytic activity assays with recombinant UGTIAI, 1 A4, 1A6 and

1A9, chrysin was found to be a high affinity substrate for UGTIAI (K_m 0.35 μM).

Catalytic activity was also found for UGTl A9 and 1 A6 but not for 1 A4. Further

studies demonstrated a 20-fold induction of the glucuronidation of bilirubin by the

chrysin-treated cells and an 7.9-fold induction of the glucuronidation of the oral

contraceptive drug ethinylestradiol, two of the best known and specific UGTIAI

substrates, demonstrating the potential importance of this induction. In view of these

findings, it is expected that these results will apply to other dietary flavonoids.

Example 1 demonstrated efficient induction of one or several isoforms of the

UGTl A subfamily by chrysin in the human intestinal cell line Caco-2. 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

5-50 μM and was maximal after pretreatment for 3-4 days. As recently shown by

Strassburg et al. (Strassburg et al., 1999), the UGTl A 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 Example, the effect of chrysin on glucuronic acid conjugation in

the human hepatic cell line Hep G2 was examined. Studies with recombinant enzymes

and isoform-specific antibodies were performed to establish which UGTl A isoform(s)

were induced. The finding of induction of UGTl Al 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

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]17a-Ethinylestradiol (50 Ci/mmol) and [a³²P]dCTP

(3000 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). Cell Culture

Hep G2 cells were obtained from American Type Culture Collection (Rockville,

MD). 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 just confluent (4-6

days after seeding), they were treated with 25 μM chrysin in dimethylsulfoxide

(DMSO; 0.3%) of final volume) or the same volume of DMSO for 3 days. The medium

was changed every 24 hr and the cells were used for in situ metabolism assays,

homogenate or microsomal preparation or RNA isolation at 24 hr 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 hr 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 phosphate-buffered saline and harvested

by scraping the cells into 0.15 M KC1 in 10 mM phosphate buffer (pH 7.4, 1 ml/dish).

The cells were sonified for 3 x 5 sec. 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 hr at 37°C with 0.1-100 μM substrate concentrations, 1 mM UDPGA and cell homogenate (0.7-2 mg protein) in 0.5 ml 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.

HPLC analyses of chrysin incubates were done with 55% methanol and 0.3%

trifluoroacetic acid on a Symmetry C18 column (Waters), as previously described

(Galijatovic et al., 1999). Bilirubin sample HPLC analyses also used a Symmetry C18

column with a linear solvent gradient from 0.1% trifluoroacetic acid in water to 100%

acetonitrile and detection at 450 nm.

Assays of the glucuronidation of ethinylestradiol used microsomes of cells

treated as above. The microsomes were prepared by centrifugation of the cell

homogenates above for 1 hr at 100,000 g and 4°C and resuspension of the pellets in

homogenization buffer. A substrate concentration of 100 μM with 0.5 μCi of

[ H] ethinylestradiol per 0.5-ml sample was used. The incubations were carried out as

for chrysin above and terminated with an equal volume of 0.25 M Tris buffer, pH 8.7.

The samples were extracted twice with toluene to remove unreacted substrate (Zhu et al., 1998) and aliquots of the aqueous phase were subjected to liquid scintillation

spectrometry after the addition of ScintiSafe Econo2 (Fisher) scintillant.

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 in Example 1.

The following primary polyclonal antibodies against human UGT proteins were

obtained from Gentest: anti-UGTl A, anti-UGTlA6 and anti-UGT2B7. In addition,

anti-UGTl Al 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 (5 100-

mm dishes each) and 1 g 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 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 RNA was loaded on a 1%

agarose gel with 3% formaldehyde. 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 Xhol-EcoRI restriction fragment pSK-UGTl Al containing the

5' end of UGTIAI (Ritter et al., 1999). The probe was labeled by random primed

synthesis, using [a³²P]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 beween control and

chrysin-treated cell samples.

In order to improve sensitivity in detecting UGTIAI mRNA, poly(A)⁺ RNA

was also isolated from chrysin-treated and control Hep G2 cells, using a FastTrack 2.0

kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Five 100-

mm dishes of confluent cells were used for each preparation. The mRNA was

dissolved in 25 μl buffer (~ 0.7 μg/μl) and 5 μg was loaded on a gel. Electrophoresis

and hybridization with the UGTIAI and GAPDH probes were done as for the total

RNA 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., 1 Al, 1 A4, 1 A6 and 1A9, supplied as microsomes of

human lymphoblast-expressed enzymes (Gentest, Wobum, MA), and also with human

liver microsomes prepared by standard differential centrifugation (La Du et al., 1972).

Human livers were obtained from Liver Tissue Procurement and Distribution System

(University of Minnesota, Minneapolis, MN) kept frozen at -80°C. The incubations

and analyses were done exactly as with the Hep G2 cell homogenates above.

Results

The flavonoid chrysin is efficiently metabolized by human Hep G2 cells both by

glucuronidation and sulfation. 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

< 0.01; N = 11) while the sulfation remained unaffected, Fig. 6. 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. 7. The apparent enzyme kinetic parameters determined in these

experiments demonstrated that the increase in chrysin glucuronidation efficiency

(N_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. 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. 8. An

antibody recognizing all of the UGTl A isoforms demonstrated a low expression in

untreated cells but a marked induction in the chrysin-pretreated Hep G2 cells (Fig. 8,

top panel). Antibodies recogmzing the common hepatic UGTl A6 and 2B7 isoforms

showed that these isoforms were expressed in Hep G2 cells but were not induced by

chrysin (middle panels). In contrast, an antibody recently shown to specifically

recognize UGTIAI (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. 8, bottom panel).

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 UGTIAI mRNA

with the expected size (2.3 kb) in the chrysin-treated cells but only trace amounts in the

control cells (Fig. 9, lanes 3 and 2, respectively). As expected, total RNA isolated from

human liver gave a positive signal (lane 1). 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 20-30-fold more mRNA on the gel,

UGTIAI mRNA could be clearly detected also in the control cells and was strongly

induced in the chrysin-treated cells (Fig. 9, lanes 4 and 5, respectively). Again,

reprobing with GAPDH cDNA showed similar loading. When comparing the catalytic activities towards chrysin of recombinant UGT

isoforms 1A1, 1A6, 1A9 and 1A4, all of which are hepatic isoforms, the isoform that

demonstrated the highest activity (V--,- /!^), interestingly, was the inducible form,

UGTIAI, Table 3. Its K_m value for chrysin glucuronidation was low, 0.35 μM, and its

V_max value high, 360 pmol/min/mg protein. A high catalytic efficiency for UGTl A9,

about 70%) of that for UGTIAI was also found, but relatively low efficiency for

UGTl A6, the isoform that is known to use simple planar phenols as substrates was

found, Table 3. Chrysin was not a substrate for UGTl A4.

In view of the findings of the induction of the UGTIAI isoform, the

glucuronidation of compounds which have been shown to be specific substrates for this

isoform was examined, using the homogenates of chrysin-induced and non-induced

Hep G2 cells. The best recognized substrate, bilirubin, was glucuronidated both by the

non-induced 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.

Total glucuronidation of bilirubin by the cell homogenates is shown in Fig. 10.

Although activity was clearly measurable at all bilirubin concentrations, 0.1 - 5 μM,

accurate enzyme kinetic parameters could not be determined for the non-induced 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 protein. For comparison, total bilirubin glucuronidation by recombinant UGTIAI yielded a K--, value of 0.23 μM and by liver microsomes from two human

donors K-,, values of 0.78 and 0.79 μM.

The oral contraceptive drug ethinylestradiol has also been shown to be a

substrate for UGTIAI (Ebner and Burchell, 1993). When using this drug to assay

glucuronidation activity of the non-induced Hep G2 cell homogenate, the activity was

barely above background, 1.4 pmol/min mg protein. In the homogenate from the

chrysin-induced cells, the activity increased 7-fold to 9.7 ± 2.8 (SEM; N = 4)

pmol/min/mg protein (P < 0.05).

Example 1 demonstrated induction of glucuronidation when the human

intestinal cell line Caco-2 was treated with chrysin, suggesting the possibility that the

flavonoids might induce their own elimination. In the present Example these

observations were extend 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. The lower level of induction in the intact cells may

be attributed to substrate/product inhibition, although the possibility of UDPGA

cofactor depletion in the intact cell cannot be excluded, 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 shown for Caco-2 cells,

maximal induction of glucuronidation in the Hep G2 cells occurred after pretreatment

with chrysin for 3-4 days and the minimum concentration for induction was about 10

μM.

In experiments using UGT isoform-specific antibodies and Western analyses,

microsomes from chrysin-treated vs. control Hep G2 cells were probed to identify

which isoforms may have been induced. An antibody selective for all UGTl A, as

opposed to UGT2B, isoforms demonstrated a high degree of induction. Interestingly, a

major hepatic isoform, UGTl A6, was not induced and this was also the case with

UGT2B7. In contrast, a band with migration identical to that of UGTIAI was

markedly induced. This was detected using a newly developed antibody highly specific

for this isoform (Ritter et al., 1999). The finding of UGTl Al induction points to a

highly selective induction response of the UGTs to chrysin.

Strongly supporting the UGTIAI 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 UGTIAI 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 UGTIAI.

Consistent with induction of chrysin glucuronidation, chrysin was a substrate

for UGTIAI, in fact was a highly efficient substrate for this isoform. In the present

study, the enzyme kinetic properties, demonstrating a very low K--, value of 0.35 μM

for the recombinant protein were also established. Chrysin was also an efficient

substrate for UGTl A9, with a low K--, value of 1.7 μM, but a poor substrate for

UGT1A6 (K-_π 12.8 μM) and not a substrate for UGT1A4. This suggests that UGTl A9

may also be induced by chrysin. The finding that chrysin is a substrate for UGTl A6,

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,-, value for chrysin glucuronidation by recombinant UGTIAI.

This observation could suggest that additional UGT isoforms may be induced.

Previous work has identified several other UGT isoforms which could potentially use

chrysin or other flavonoids as substrates. These include UGTl A3 (Green et al., 1998),

1 A8 (Cheng et al., 1998), 1A9 (Ebner and Burchell, 1993) and 2B15 (Green et al.,

1994; Levesque et al., 1997). As recently shown by Strassburg et al. (Strassburg et al., 1999), UGTIAI, 1 A3, 1 A4, 1A6 and 1 A9 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 UGTIAI 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--, for the Hep

G2 cell homogenate compared to recombinant UGTIAI.

The induction of UGTl Al by chrysin has important biological implications.

The best known substrate for UGTIAI is the endogenous toxin bilirubin (Chowdhury

et al., 1995; Ritter et al., 1999). Induction of UGTl Al therefore suggests that bilirubin

glucuronidation is increased, as clearly shown in the present example, 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 UGTIAI and human liver microsomes of 0.23-1.4 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 UGTIAI, 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 the present Example. The induction by 3-methylcholanthrene

suggests the involvement of the aryl hydrocarbon receptor (Ritter et al., 1999).

However, UGTl A6, inducible by arylhydrocarbon receptor agonists in Caco-2 cells

(Abid et al., 1995; Bock et al, 1999; Mϋnzel et al., 1999), was not affected by chrysin.

The mechanism of chrysin induction thus needs to be investigated.

Finally, the induction of UGTIAI may affect metabolism of some drugs. Thus,

the glucuronidation of the synthetic estrogen ethinylestradiol, which has been shown to

be a substrate for UGTIAI (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.

Chrysin is not one of the major flavonoids in our diet, but its ability to inhibit

the enzyme aromatase (Kao et al., 1998), catalyzing the conversion of testosterone to

17b-estradiol, suggests its use in lowering breast cancer risk. These findings are likely

to apply to other flavonoids, especially those present at higher levels in various fruits

and vegetables. Example 1 already shows that quercetin can produce the same

response when cells are exposed for several weeks to this flavonoid (Galijatovic et al.,

2000). Throughout this application, various publications are referenced. The

disclosures of these publications in their entireties, as well as the references cited in

these publications, are hereby incoφorated by reference into this application in order to

more fully describe the state of the art to which this invention pertains.

References

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Caco-2 human intestinal cell line: Induction of UDP-glucuronosyltransferase 1*6.

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Table 3. Chrysin glucuronidation by recombinant UGTl A isoforms. Apparent

enzyme kinetic parameters were obtained by fitting the data, as in Fig. 3, to the

Michaelis-Menten equation. Mean values from 2 experiments are shown.

UGT isoform Km V ^τ max /'¥ -"__T.I

(μM) (pmol/min/mg protein) (rrtl/rnin/mg protein)

UGTIAI 0.35 360 1.02

UGT1A6 12.8 157 0.012

UGT1A9 1.7 1210 0.71

UGT1A4 n.d. n.d. n.d.

n.d., Activity not detectable

EXAMPLE 3

Low Oral Bioavailability Of The Flavonoid Chrysin, An Aromatase Inhibitor.

Chrysin, a potent inhibitor of the enzyme aromatase, is currently available as a dietary supplement in health food stores. In a previous study using the human intestinal Caco-2 cells as a model, low bioavailability of chrysin was found, mainly due to extensive glucuronic acid and sulfate conjugation by the enterocytes (Biochem Pharmacol 58:431-8, 1999). In the present study we tested these findings directly in normal volunteers after oral doses (400 mg) of chrysin. Only trace levels of chrysin (<25 ng/ml) could be detected in plasma together with its sulfate conjugate (<250 ng/ml). Low levels of chrysin and the sulfate conjugate were also found in urine, accounting for <2% of the dose. The fecal excretion of unchanged chrysin, however, was very high. To be able to rationalize these observations, parallel studies were performed in bile-duct cannulated rats. Based on these studies, it is suggested that chrysin is absorbed in humans and efficiently conjugated, with the conjugates secreted both in intestine and liver by MRP2 and subsequently hydrolyzed to chrysin in the feces.

EXAMPLE 4

As shown herein, the bioflavonoid chrysin, induced UDP- glucuronosyltransferase (UGT) activity and expression in the human intestinal cell line Caco-2 (Galijatovic et al, Pharm. Res. 17:21, 2000). In the present example, the specific UGT isoform(s) that chrysin induces and whether induction of a specific isoform facilitates increased glucuronidation of the dietary colon carcinogen N-OH- PhlP were determined.

Western blot analysis using isoform specific antibodies showed that pretreatment of Caco-2 cells with 50 μM chrysin results in substantial induction of the UGT 1 Al isoform. Chrysin pretreatment had no effect on the expression of UGT 1 A6, UGT 1 A9 and UGT 2B7.

Northern blot analysis using UGT 1 Al specific probe showed markedly increased expression of mRNA after chrysin treatment. Incubation of 25 μM N-OH- PhlP with control and chrysin treated Caco-2 microsomes resulted in a 6.5 fold increase in glucuronidation of N-OH-PhlP. Diet-mediated induction of intestinal UGT can be an important protective mechanism in deactivating carcinogens and other toxic chemicals.

Human intestinal Caco-2 cells were exposed to 50 μM chrysin or vehicle (control) for 3 days. The cells were harvested and the microsomal fraction, containing UGT enzymes was prepared. Control and chrysin-treated microsomes were then incubated with 25 μM N-hydroxy-PhlP. The reaction mixtures were analyzed by HPLC. This is similar methodology that was used to show the increased bilirubin glucuronidation in chrysin-treated Hep G2 and Caco-2 cells.

In the presence of the cofactor UDPGA, the formation of N-hydroxy-PhIP N2- glucuronide was 6.5 times higher in the chrysin-treated compared with the control cell preparation. The formation of this glucuronide has previously clearly been linked to detoxification of N-hydroxy-PhIP. Similar detoxification may occur for other human colon carcinogens.

EXAMPLE 5

The examples above, using the human hepatic cell line Hep G2 and the human intestinal cell line Caco-2 have demonstrated induction of UGTl Al by the flavonoid chrysin (5,7-dihydroxyflavone), using catalytic activity assays as well as Western and Northern blotting (1,2). What features of the flavonoid structures were associated with the greatest induction as well as whether common drug metabolizing enzyme inducers also produce this induction were studied. The model used for these studies was intact Hep G2 cells with chrysin as the model substrate. Both glucuronidation and sulfation were measured. Hep G2 cells were pretreated for 3 days with 25 μM concentrations of each of 18 flavonoids (N = 4). Only 3 flavonoids demonstrated induction of glucuronidation similar to that of chrysin, i.e. 3-5-fold in the intact cells. These were apigenin, luteolin and diosmetin, all of which, like chrysin, are 5,7-dihydroxyflavones with varying substituents in the B-ring. 5- and 7-hydroxy-flavone produced a modest 1.5-2-fold induction, whereas all other flavonoids examined were without effect. In addition, the induction by 7 common drug metabolizing enzyme inducers at their optimal enzyme inducing concentrations was determined. Only 3-methylcholanthrene and oltipraz showed modest induction of chrysin glucuronidation, but not e.g., TCDD or phenobarbital. (Galijatovic et al., Pharmaceut Res 17:21,2000; 2. Walle et al., Drug Metab Disp, subm.)

Cultured human liver-derived Hep G2 cells were exposed to 25 μM of each of 18 flavonoids for three days. The culture medium was then removed. The UGT enzyme activity was measured as the amount of chrysin glucuronide formed after a 6- hour incubation of the intact cells with culture medium containing 25 μM chrysin.

Only 3 flavonoids demonstrated induction of glucuronidation similar to that of chrysin, i.e. 3-5-fold in the intact cells. These were apigenin, luteolin and diosmetin, all of which, like chrysin, are 5,7-dihydroxyflavones with varying substituents in the B-ring. Other flavonoids with this common structural feature can therefore be predicted to be inducers as well.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

What is claimed is:

1. A method of treating jaundice in a neonate, comprising administering to the neonate an amount of chrysin effective to treat jaundice.

2. A method of treating hyperbilirubinemia in a neonate, comprising administering to the neonate an amount of chrysin effective to reduce the amount of billirubin in the neonate.

3. A method of increasing glucuronidation of bilirubin in a subject, comprising administering to the subject an amount of chrysin effective to increase the glucuronidation of bilirubin.

4. A method of increasing the expression of UDP-glucuronosyltransferase in a subject, comprising administering to the subject an amount of chrysin effective to increase the UDP-glucuronosyltransferase.

5. The method of claim 4, wherein the UDP-glucuronosyltransferase is a UGTl A isoform.

6. The method of claim 5, wherein the UGTl A isoform is UGTIAI .

7. The method of claim 5, wherein the UGT1A isoform isUGTlA9.

8. A method of increasing the expression of UDP-glucuronosyltransferase in a subject, comprising administering to the subject an amount of apigenin effective to increase the UDP-glucuronosyltransferase.

9. The method of claim 8, wherein the UDP-glucuronosyltransferase is a UGT1A isoform.

10. The method of claim 9, wherein the UGTl A isoform is UGTIAI .

11. The method of claim 9, wherein the UGT 1 A isoform is UGT 1 A9.

12. A method of increasing the expression of UDP-glucuronosyltransferase in a subject, comprising administering to the subject an amount of luteolin effective to increase the UDP-glucuronosyltransferase.

13. The method of claim 12, wherein the UDP-glucuronosyltransferase is a UGTl A isoform.

14. The method of claim 13 , wherein the UGT 1 A isoform is UGT 1 A 1.

15. The method of claim 13, wherein the UGT1A isoform is UGT1A9.

16. A method of increasing the expression of UDP-glucuronosyltransferase in a subject, comprising administering to the subject an amount of diosmetin effective to increase the UDP-glucuronosyltransferase.

17. The method of claim 16, wherein the UDP-glucuronosyltransferase is a UGT 1 A isoform.

18. The method of claim 17, wherein the UGTl A isoform is UGTIAI .

19. The method of claim 17, wherein the UGTl A isoform is UGT1A9.

20. A method of treating colon cancer, comprising administering an effective amount of a flavone, whereby the administration of the flavone treats the colon cancer.