Diosmin and Diosmetin Are Agonists of the Aryl Hydrocarbon Receptor That Differentially Affect Cytochrome P450 1A1 Activity Henry P

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ICANCERRESEARCHss.2754-2?Â«).July1.1998] Diosmin and Diosmetin Are Agonists of the Aryl Hydrocarbon Receptor That Differentially Affect Cytochrome P450 1A1 Activity Henry P. Ciolino,1 Thomas T. Y. Wang, and Grace Chao Yeh

Cellular Defense and Carcinogenesis Section Â¡H.P. C.. G. C. Y.Â¡,and Laboratory of Nutritional und Molecular Regulation [H. P. C., T. T. Y. W., G. C. Y.I, Division of Basic Sciences, National Cancer Institute'-Frederick Cancer Research and Development Center. NIH. Frederick, Maryland 21702-1201

ABSTRACT chemically induced models of carcinogenesis (16, 17). Biochemical activities of individual flavonoids have not been fully investigated and We investigated the effect of the chemopreventive compound diosmin may fundamentally differ. and its aglycone form, diosmetin, on the carcinogen activation pathway Recent studies have shown that diosmin (3',5,7-trihydroxy-4'-me- mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 human thoxyflavone-7-rutinoside), a flavonoid found in a variety of citrus fruits, breast epithelial cancer cells. Treatment of the cells with diosmin caused a dose-dependent increase in the metabolism of the mammary carcinogen inhibits chemically induced cancers in rodents, including N-melhyl-N- 7,12-dimethylbenz(a)anthracene (DMBA), as assessed by increased for amylnitrosamine-induced esophageal cancer (18), 4-nitroquinoline 1-ox mation of DMBA-DNA adducts and by DMBA-induced cytotoxicity. In ide-induced oral cancer (19), and azoxymethane-induced colon cancer contrast, treatment of the cells with diosmetin decreased both parameters. (20). The biochemical mechanism(s) underlying the chemopreventive Diosmetin, but not diosmin, directly inhibited cytochrome P450 1A1 activity of diosmin, however, has not been identified. Furthermore, one (CYP1A1) activity in a noncompetitive manner in microsomes isolated pharmacokinetic study (21) indicates that the primary circulating form of from DMBA-treated cells, as assayed by ethyoxyresorufin-O-deethylase the flavonoid after oral administration of diosmin in humans is its agly activity. Treatment of the cells with diosmin or diosmetin, on the other cone form, diosmetin (3',5,7-trihydroxy-4'-methoxyflavone; for struc hand, caused a dose- and time-dependent increase in CYP1A1 activity in tures, see Ref. 21 or the Merck Index), the cleavage of diosmin's sugar intact cells that was comparable to that induced by DMBA or by the aryl moiety perhaps resulting from bacterial action in the intestine (22). Thus, hydrocarbon benzols )pyrene. Both diosmin and diosmetin caused an increase in the transcription of the CYPJAJ gene, as measured by in when evaluating the effect of diosmin on biochemical processes, it is creased levels of (')l'I.\Â¡ mRNA. Both compounds caused the activation important to also examine diosmetin. of the DNA-binding capacity of the AhR for the xenobiotic-responsive Chemical induction of carcinogenesis often depends on the meta element of CYP1AI. These results indicate that diosmin and diosmetin are bolic activation of the chemical in question. For example, DMBA, a natural dietary agonists of the AhR, causing a potent increase in O'I'IM PAH that induces mammary tumorigenesis in rodents (23), is acti transcription and CYP1A1 activity; however, only diosmetin is capable of vated by a pathway mediated by the AhR, a cytosolic transcription inhibiting CYP1A1 enzyme activity, thus inhibiting carcinogen activation. factor. Upon binding aryl hydrocarbons or polychlorinated biphenyls such as 2,3,7,8-tetrachlorodibenzo-/>dioxin, the AhR translocates to the nucleus, where it binds a second protein, aryl hydrocarbon nuclear INTRODUCTION translocase. This heterodimer binds enhancer sequences termed the Numerous epidemiological studies on the relationship between diet XREs or dioxin-responsive elements and induces transcription of a and carcinogenesis have demonstrated a protective effect of the con variety of genes. The best-characterized response to the AhR at the sumption of fruits and vegetables against various forms of cancers molecular level is the induction of CYP1A1, a gene that encodes the (1-3). A number of dietary components, both nutrients and nonnutri- enzyme CYP1A1 (24). This enzyme catalyzes the oxidative catabo- ents, have been shown to inhibit or prevent chemical carcinogenesis in lism of DMBA, generating genotoxic metabolites that may covalently animal models. Among the nonnutrient dietary components believed bind specific residues of DNA and form adducts (25), unless they are to exert a chemopreventive effect are flavonoids, polyphenolic deriv detoxified by Phase II enzymes. DNA adduci formation is thought to atives of benzo(y)pyrone that are widely distributed in edible plants be the major cause of carcinogenesis induced by DMBA and other (4). There are several major classes of flavonoids, which may occur as aryl hydrocarbons. Decreased metabolic activation of carcinogens, glycosides or aglycones. Total dietary intake of flavonoids has been either through direct inhibition of CYP1A1 activity or through inhi estimated as high as 1 g/day, equivalent to 50,000 ppm in the diet (5), bition of the AhR pathway leading to transcriptional induction of the although more recent studies have indicated that intake varies widely gene, may be an important mechanism of cancer prevention. (6). Flavonoids have been reported to have a wide variety of biochem In the present study, we have investigated the interaction of diosmin ical effects in vitro, including metal chelation (7), free radical scav and diosmetin with the carcinogen activation pathway mediated by the enging (8), inhibition of a number of enzymes (9-12), inhibition of AhR in MCF-7 cells, a human breast epithelial carcinoma cell line in cellular proliferation (13), and induction of apoptosis (14). In vivo, which the AhR pathway is well characterized (26-28). We found that various flavonoids are effective chemopreventive agents against diosmetin but not the glycone diosmin inhibited CYP1A1 activity, DMBA2-induced mammary cancer in rodents (15) as well as other inhibited the formation of DMBA-DNA adducts, and reduced the cytotoxicity of DMBA in proliferating cells. Diosmin, on the other hand, increased DMBA-DNA adduci formation and the cytotoxicity Received 1/13/98; accepted 4/30/98. The costs of publication of this article were defrayed in part by the payment of page of DMBA. Both compounds induced CYP1A1 mRNA levels and charges. This article must therefore be hereby marked advertisement in accordance with CYP1A1 activity and caused an increase in activated AhR DNA 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Laboratory of Nutritional and binding. These results indicate that diosmin and diosmetin activate Molecular Regulation. National Cancer Institute-Frederick Cancer Research and Devel AhR-mediated transcription and thus may be natural dietary agonists opment Center. Building 560/Room 12-05, P. O. Box B, Frederick. MD 21702-1201. Phone: (301) 846-5160; Fax: (301) 846-6093; E-mail: [email protected]. of the AhR; however, only diosmetin inhibits CYP1 Al activity. 2 The abbreviations used are: DMBA. 7.12-dimethylbenz(rt)anthracene; AhR. aryl hydrocarbon receptor; CYP1 Al. cytochrome P450 1A1; EMSA. electrophoretic mobility shift assay; EROD. ethyoxyresorufin-0-deethylase; G3PDH. glyceraldehyde-3-phosphate MATERIALS AND METHODS dehydrogrenase: RT-PCR. reverse transcription-PCR; TBE. Tris-borate EDTA; XRE. xenobiotic-responsive element; PAH. polycyclic aromatic hydrocarbon; BP. benzo- Materials. RPMI 1640. glutamine. fetal bovine serum, trypsin/EDTA. (o)pyrene. PBS. and TBE buffer were from BioFluids (Rockville, MD). Diosmin, dios- 2754

diosmetin for 6 h. Cells were washed twice with cold PBS. trypsinized, and pelleted. Nuclei were isolated by incubating the cells for 10 min on ice in 10 min Tris-HCl (pH 7.5) with 320 mM sucrose, 5.0 mM MgCl,, and 1% Triton X-100. The nuclei were pelleted by centrifugation at 800 X g for 10 min at 4Â°C.Genomic DNA was then isolated by the method of Miller et al. (29). The amount of DNA was measured by spectrophotometry, and the adducts were quantified by scintillation counting in Aquasol (Beckman). Measurement of Cell Growth. MCF-7 cells were plated at 25,000 cells/ well in 24-well plates. After 24 h, the medium was changed to medium containing 5 Â¡J.Mdiosmin or diosmetin. After an additional 24 h, the medium was again changed with medium containing 5 Â¡Â¿Mdiosminor diosmetin in the presence of the indicated concentrations of DMBA. After 3 days, the total cell growth was assessed by sulforhodamine (30). CYPIAI Activity in Microsomes or Intact MCF-7 Cells. MCF-7 cells were treated with 1 /XM DMBA for 24 h to induce CYPIAI activity. Microsomes were isolated as follows: the cells were washed twice with PBS; trypsinized; and pelleted by centrifugation at 500 X g for 5 min at 4Â°C.The pellet was washed in PBS and repelleted. The pellet was resus- 1 2.5 5 pended in 0.25 M sucrose and 10 mM Tris-HCl (pH 7.5) with protease [Diosmin] |iM inhibitors (100 jig/ml phenylmethylsulfonyl fluoride. 300 (xg/ml EDTA, 0.5 /xg/ml leupeptin, 0.5 fig/ml aprotinin, and 0.7 /ng/ml pepstatin A). The cells were sonicated for 30 s on ice using a Branson sonifier at setting 2. The sonicate was then centrifuged at 10,000 X g for 10 min at 4Â°C.The B supernatant was subjected to centrifugation at 500,000 x g for 15 min at 4Â°C. The resulting microsomal pellet was resuspended in the above- â€¢¿5o mentioned buffer, and the protein was assayed by the method of Bradford A 0.8- (31). Aliquots of microsomes were stored at -80Â°C until use. The EROD activity of 10 ng of microsomes was assayed using 5 /XMor the indicated Oc 0.6- concentrations of ethoxyresorufin in 100 p\ of PBS (pH 7.2) in a CytoFlor < s II plate reader with an excitation wavelength of 530 nm and an emission SQs wavelength of 590 nm. Fluorescence was measured every 10 min for 60 11 0.4- min, and the rate of fluorescence increase was calculated. A standard curve was constructed using resorufin. For assays of EROD activity in intact cells, cells in 24-well plates were â€”¿ 0.2 - treated with diosmin or diosmetin for the times and concentrations indicated. The EROD activity of CYPIAI was determined as described by Kennedy and Jones (32). The medium was removed, the cells were washed once with PBS, and 1 ml of growth medium containing 5 JIM ethoxyresorufin and 1.5 mM O 2.5 salicylamide was added to prevent the reduction of resorufin by conjugating [Diosmetin] (IM enzymes. Fluorescence was monitored as described above. RT-PCR for CYPIAI. Confluent MCF-7 cells were treated with dios Fig. 1. Effect of diosmin (A) or diosmetin (ÃŸ)on the formation of DMBA-DNA min or diosmetin in growth medium for 24 h at the concentrations indi adducts. MCF-7 cells were treated with the indicated concentrations of diosmin or diosmetin for 24 h. The cells were then exposed to 0.1 fig/ml |'H]DMBA for 6 h. cated. The cells were washed twice with PBS. and total RNA was isolated Genomic DNA was isolated, and the adduci formation was quantified. Each bar represents using Trizol reagent as directed. cDNA was synthesized from 10 pig of total the average of four determinations Â±SE. Please note the difference in scales in A and B. RNA using a RT-PCR kit (Stratagene) as instructed. PCR was performed metin, DMBA. HEPES, EDTA. DTT, glycerol. polyfdeoxyinosinic-deoxycyti- 1.2-, dylic acid), sodium molybdate. ethyoxyresorufm. resorufin, Tris-HCl, sulfor hodamine, salmon sperm DNA. and protease inhibitors were from Sigma (St. Louis, MO). a-Napthoflavone was from Indofine (Somerville, NJ). [32P]CTP and [32P]ATP were from DuPont New England Nuclear (Boston, MA). RT- PCR was performed with a kit from Stratagene (La Jolla, CA). TBE gels, TBE 1 0.8- running buffer, and high-density sample buffer were from Novex (San Diego, M CA). Primers for G3PDH PCR were from Clontech (Palo Alto, CA). The 0.6- Bradford protein assay kit was from Bio-Rad (Hercules, CA). [3H]DMBA was from Amersham (Arlington Heights. IL). Aquasol was from Beckman (Palo 1 0.4- Alto, CA). Trizol reagent was from Life Technologies. Inc. (Gaithersburg, Â« Control MD). Polyclonal antibody to AhR was a generous gift from Dr. Alan Poland Si (University of Wisconsin. Madison, WI). 0.2- Diosmetin Cell Culture. MCF-7 cells were grown in RPMI 1640 supplemented with 2 mM glutamine and 10% fetal bovine serum. Cell were subcultured weekly Diosmin using 0.25% trypsin and 0.05% EDTA. All experiments were carried out on confluent cultures in growth medium, unless otherwise noted. 200 400 600 800 DMBA-DNA Adduct Formation. Confluent cultures of MCF-7 cells in [DMBA] nM 75-cm2 flasks were treated with diosmin or diosmetin at the doses indicated for 24 h. Cells were then exposed to 0.1 jug/ml [3H]DMBA in the presence of Fig. 2. Effect of diosmin or diosmetin on DMBA-induced cylotoxicity. The cytotox- diosmin or diosmetin for 6 h. Confluent cultures of MCF-7 cells in 75-cm2 icity of the indicated concentrations of DMBA in the presence of 5 (ÃŒMdiosminor diosmetin was assessed by measuring relative cell growth after 3 days in culture using flasks were exposed to 0.1 /Â¿g/ml[3H]DMBA in the presence of diosmin or sulforhodamine staining. Each point represents the average of four determinations Â±SE. 2755

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Fig. 3. Effect of diosmin or diosmetin on DMBA-induced CYPlAl EROD activity. A, microsomes were isolated from MCF-7 cells that had been treated with l Â¿AMDMBAfor 24 h to induce CYP1 Al. The EROD activity of 10 Â»igofmicrosoma] protein in the presence of DMSO (control) or the indicated concentrations of diosmin or diosmetin was measured as described (n = 4 Â±SE). B, MCF-7 cells were incubated with I JIM DMBA for 24 h. The medium was removed, the cells were washed in PBS. and fresh medium containing the indicated concentrations of diosmetin was added. EROD activity in the intact cells was measured after 30 min. C. the EROD activity of 10 ^g of microsomes in the presence of 0 (DMSO), 12.5, or 25 nM diosmetin and increasing amounts of substrate was determined, and double-reciprocal plots were generated (n = 4).

using the primer sequences and method of Dohr et al. (27). except that 27 RESULTS cycles were used in the presence of 1.5 fiCi of [32P]dATP. PCR of G3PDH was carried out using primers from Clontech for 19 cycles as instructed. Effect of Diosmin and Diosmetin on the Metabolic Activation of After PCR, 5 /u.1of sample buffer (Novex) were added to the samples, and DMBA in MCF-7 Cells. DMBA-DNA adduci formation in MCF-7 they were subjected to electrophoresis on a 10% TBE gel (Novex) in IX cells after 24 h of pretreatment with various concentrations of TBE running buffer. The gel was dried, and the result was visualized and diosmin (Fig. 1A) or diosmetin (Fig. IÃŸ)was determined. In quantified on a BioRad GS-363 molecular imaging system. The relative control cultures, 1549 Â±41 fmol/mg DNA of [SH]DMBA-DNA amount of CYPIAI mRNA was normalized using the amount of G3PDH adducts were formed. Treatment with diosmin caused a dose- mRNA. dependent increase in the amount of adducts formed (up to a 7-fold EMSA. Confluent cultures of MCF-7 cells were treated with diosmin or increase in adducts at 5 JUMdiosmin). In contrast, 5 /AMdiosmetin diosmetin in growth media for 3 h. Nuclear protein was isolated, and EMSA was performed by the method of Denison et al. (33). Synthetic inhibited adduci formation, reducing the adducts formed by 65% at oligonucleotides containing the AhR-binding site of the XRE (34) were 5 /J.M.Furthermore, we measured the effect of diosmin or diosme labeled with [12P]dCTP. The binding reactions were carried out for 30 min tin on the inhibition of cell growth by DMBA (Fig. 2). At 5 /J.M, and contained 5 Â¿tgof nuclear protein, 1 ;u.g of poly(deoxyinosinic-deoxy- diosmin increased the cytotoxicity of DMBA, shifting the IC50 of cytidylic acid), 500 ng of salmon sperm DNA, and â€”¿50,000cpm of labeled DMBA from an estimated 1.2 Â¡J.Mto400 nM, whereas 5 /J.M probe in a final volume of 20 jul of binding buffer [25 m.MTris (pH 7.9), diosmetin afforded complete protection from DMBA-induced cy 50 mM KC1, 1 mM MgCl2, 1.5 mM EDTA, 0.5 mM DTT, and 5% glycerol]. totoxicity at the concentrations tested. Neither diosmin nor dios To determine the specificity of binding to the oligonucleotide, a 200-fold metin was cytotoxic in itself at the concentrations tested (data not excess of unlabeled specific probe, a 200-fold excess of unlabeled probe of shown). the transcription factor AP-2, or anti-AhR polyclonal antibody (0.864 /xg) Effect of Diosmin or Diosmetin on DMBA-induced CYPIAI was incubated on ice with nuclear extract from diosmin (5 /j.M)-treated cells for 15 min. DNA-protein complexes were separated under nondenaturing Enzyme Activity. We isolated microsomes from MCF-7 cells that conditions on a 6% polyacrylamide gel using 0.5% TBE (45 mM Tris- had been exposed to 1 /XMDMBA for 24 h and investigated the borate, 45 mM boric acid, and 2 mM EDTA) as a running buffer. The gels effect of diosmin or diosmetin on CYPIAI activity. Diosmetin but were dried, and the DNA-protein complexes were visualized on a BioRad not diosmin potently inhibited microsomal CYPIAI enzyme ac GS-363 molecular imaging system. tivity in a dose-dependent fashion, with an IC50 of approximately 2756

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. DIOSMIN AND DIOSMETIN INDUCE CYPIAI AT measured after 30 min. Diosmetin inhibited EROD activity in intact cells in a dose-dependent manner (Fig. 3ÃŸ).The effect of

M13u.*?!IlERODles/min/oÂ«9-18-7-6-5-4- diosmetin on microsomal EROD activity in the presence of differ ent substrate concentrations was measured, and the result was plotted as a double-reciprocal (Lineweaver-Burk) plot (Fig. 3C). At the concentrations tested (12.5 and 25 nM), diosmetin decreased the Vmax from 3.33 pmol/min/10 /Mg protein in the absence of diosmetin to 1.72 pmol/min/10 Â¿Â¿gproteinin the presence of 25 nM diosmetin, whereas the Km remained unchanged at 166 nM. This is 3-2-1-OH consistent with noncompetitive inhibition of enzyme activity. Effect of Diosmin or Diosmetin on CYPIAI Activity of MCF-7 Cells. Because of the increase in the metabolic activation of DMBA caused by diosmin, we examined whether diosmin could induce CYPIAI enzyme activity in MCF-7 cells. As shown in Fig. 4, diosmin caused an increase in CYPIAI activity as measured by 12 24 36 48 60 72 EROD activity in MCF-7 cells in a time (Fig. 4A)- and dose (Fig. Incubation time, hr 4ÃŸ)-dependent fashion. Surprisingly, diosmetin also caused an in crease in enzyme activity, with a modest increase in activity in cells 251 treated with up to 2.5 /LIMdiosmetin, but with no increase in activity compared to controls in cells treated with 5 JU.Mdiosmetin. The increase in CYPIAI activity caused by diosmin or diosmetin

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[FlavonoidJiaM G-3-PDH Fig. 4. Time course (Ai and dose dependence (ÃŸ)ofCYPIAI EROD activity in MCF-7 cells treated with diosmin. A. MCF-7 cells were treated with 0.5 (Â¿Mdiosminfor the times indicated, and EROD activity in intact cells was measured as described. B. MCF-7 cells were treated with the indicated concentrations of diosmin or diosmetin for 24 h, and the EROD activity in intact cells was measured. Each point represents four determina tions Â±SE. 35 251 Diosmin

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Fig. 5. Comparison of diosmin or diosmetin with aryl hydrocarbons as inducers of CYP1A1 EROD activity in intact MCF-7 cells. MCF-7 cells were treated with l Â¿IMofthe indicated compounds for 24 or 48 h, and EROD activity was measured. Each point represents the mean of four determinations Â±SE. 0 0.25 0.5 5 [Concentration] jaM 30 riM (Fig. 3A). We also examined the effect of diosmetin on Fig. 6. Effect of diosmin or diosmetin on CYPIAI mRNA levels in MCF-7 cells. DMBA-induced CYPIAI activity in intact MCF-7 cells. CYPIAI MCF-7 cells were treated with the indicated concentrations of diosmin or diosmetin for 24 h. Total RNA was isolated, cDNA was synthesized, and the amount of CYPIAI mRNA was induced by exposure of the cells to l /J.MDMBA for 24 h, was determined by RT-PCR. Data was normalized to G3PDH mRNA levels. Bars diosmetin was added to the cultures, and the EROD activity was represent the mean of three determinations Â±SE. N.D., not determined. 2757

Hours Incubation ment of the cells with the transcription inhibitor actinomycin D (5 /Â¿g/ml,added l h before diosmin treatment; data not shown). 0 6 24 48 72 Effect of Diosmin or Diosmetin on the Activation of AhR. Lane 1 234 56789 10 11 12 13 14 15 Because diosmin and diosmetin increased both CYPIAI mRNA and CYPIAI enzyme activity, we investigated whether these flavonoids CYPIA1 â€”¿â€”¿â€”¿â€¢¿Â»â€¢¿â€¢¿ â€¢¿Â«â€¢¿â€”¿â€”¿â€”¿ could cause the activation of the DNA-binding ability of the AhR. Therefore, we measured the nuclear accumulation of activated AhR by EMSA (Fig. 7). Both diosmin (Lanes 9-12) and diosmetin (Lanes G-3-PDH 5-8) caused a dose-dependent increase compared with controls in the DNA-binding capacity of nuclear extracts for an oligonucleotide containing the AhR-binding sequence of CYP1AÃŒ(Lanes 1 and 2). The amount of band shift may be compared to that induced by BP (Lanes 3 and 4). The specificity of this band shift was confirmed by the inhibition of band shift in nuclear extracts treated with 5 H.M diosmin in the presence of an unlabeled probe (Lane 13), by the lack 1Ã¬ of inhibition in the presence of an unlabeled nonspecific probe of AP-2 (Lane 14), and by the inhibition of band shift in the presence of 12- a polyclonal antibody to the AhR (Lanes 15).

AhR, causing it to translocate to the nucleus and gain DNA-binding [Diosmetin] [Diosmin] 5 W . UM ill c. Â£ activity. Control B[a]P 0.5I 5.0 I 0.5 I 5.0 I S* 7ft ?= Our studies demonstrate that diosmin and diosmetin act as agonists I I in lâ€”i iâ€”i iâ€”liâ€”i '" 4 of the AhR that are capable of activating the receptor, inducing Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 CYPÃŒA1transcription, and increasing CYP1A1 enzyme activity in MCF-1 human breast carcinoma cells. The known agonists of the AhR, with the exception of indolo[3,2-/?]carbazole [a natural com 'I pound found in some vegetables (40)] and the marine neurotoxin AhR*- brevetoxin 6 (41), are man-made contaminants such as 2,3,7,8-lelra- chlorodibenzo-p-dioxin and BP, synthetic compounds such as the drug omeprazole (42), or napthoflavones (43, 44). Our results suggest that natural dietary flavonoids such as diosmin and diosmetin may also be AhR agonists. This is supported by the findings of Siess et al. (45) and Canivenc-Lavier et a!. (46), which demonstrated that

Free*. tangeritin, a natural flavonoid also found in Citrus fruit, induced Probe hepatic EROD activity and other enzyme activities associated with AhR-mediated signal transduction in rodents. Because increased CYP1A1 activity results in increased metabolic Fig. 8. Effect of iliiiMiiiii and diosmetin on the binding of activated AhR to the XRE activation of PAHs (and hence, genotoxicity), the established chemo- of CYP1A1. MCF-1 cells were treated with DMSO (contrai), 1 fiM BP. or the indicated preventive effect of diosmin may depend on two factors: (a) in concentrations of diosmin or diosmetin for 3 h. Nuclear protein was isolated, and the amount of activated AhR in 5 fig of protein was measured by EMSA. For competition addition to CYPÃŒAÃŒ,activationof AhR-mediated signal transduction (Limes 13-15). nuclear extract of cells treated with 5 /J.Mdiosmin (Lanes II and 12) was also causes the transcriptional activation of a number of Phase II incubated with an excess of unlabeled XRE. unlabeled AP-2 probe, or an antibody to the enzymes, including glutathione 5-transferase and NADPH-quinone AhR. reducÃase(47). In tissues, stimulation of AhR-mediated transcriplional activalion by diosmin or diosmelin may result in the simullaneous enzyme activity in a noncompetitive fashion, implying that diosmetin increase of bolh CYP1A1 and ihe Phase II enzymes, resulling in acted at an allosteric site on the enzyme. The inhibitory effect of increased detoxification of PAHs, thus producing a chemoprevenlive diosmetin accounts for the decrease in metabolic activation of DMBA effect In MCF-7 cells, however, melabolic aclivalion (as measured by in cells treated with diosmetin. adduci formalion) increases wilh increasing CYP1A1 enzyme activ The increase in adduci formation and cytotoxicity of DMBA in ity. Therefore, the parent compound diosmin may not be chemopre- diosmin-treated cells, on the other hand, could result from increased venlive; and (b) conversion of diosmin to its aglycone form diosmetin. activation of DMBA due to elevated CYP1A1 enzyme activity. Ac As mentioned earlier, Cova et al. (21) showed that diosmetin is the cordingly, we investigated the effect of diosmin on CYP1A1 activity primary circulating form of ihe flavonoid in humans, and lhal ihere is in the cells. Diosmin caused a dose- and time-dependent increase in extensive uptake of diosmetin by the tissues. Thus, the chemopreven CYP1A1 enzyme activity (Fig. 4, A and B). This increase was greater live effecl of dielary diosmin may aclually be due lo the potenl and more sustained than that induced by DMBA, although it was less inhibitory aclivily of diosmelin on melabolic aclivalion. than that induced by BP, an aryl hydrocarbon known to be a potent inducer of CYP1 AI (Fig. 5). We further demonstrated that the induc tion of CYP1A1 activity by diosmin was completely blocked by the ACKNOWLEDGMENTS protein synthesis inhibitor cyclohexamide (data not shown), indicating We thank Drs. Carmen J. Allegra and Susan N. Perkins for critical reading that the increase in enzyme activity was dependent on new CYP1A1 of the manuscript. apoprotein synthesis. Diosmin also increased the levels of CYP1A1 mRNA in a dose- and time-dependent manner (Figs. 6 and 1, respec tively). This was blocked by the RNA polymerase inhibitor actino- REFERENCES mycin D (data not shown), indicating that the increase in CYPIA1 1. 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Toxicol., 33: 1061-1080, 1995. intracellular diosmetin remains, particularly in cells treated with 5. Pierpoint. W. S. Flavonoids in the human diet. Prog. Clin. Biol. Res., 213: 125-140, higher concentrations, which would inhibit EROD activity, thus pro 1986. 6. Hertog. M. G.. Kromhout. D., Aravanis. C, Blackburn. H., Buzina, R., Fidanza, F.. ducing a biphasic response. Giampaoli. S.. Jansen, A., Menotti. A.. Nedeljkovic. S., Pekkarinen. M., Simic, B. S., Transcriptional activation of the CYPIA1 gene and the subsequent Toshima, H., Feskens, E. J. M.. Hollman, P. C. H.. and Katan, M. B. Flavonoid inlake and long-term risk of coronary heart disease and cancer in the seven countries study. increase in CYP1 Al enzyme activity in cells and tissues are mediated Arch. Intern. Med.. /55: 381-386. 1995. by the AhR. We tested whether diosmin and diosmetin could activate 7. Moran. J. F., Klucas, R. V.. Grayer. R. J.. Abian. J.. and Becana, M. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. Diosmin and Diosmetin Are Agonists of the Aryl Hydrocarbon Receptor That Differentially Affect Cytochrome P450 1A1 Activity

Henry P. Ciolino, Thomas T. Y. Wang and Grace Chao Yeh

Cancer Res 1998;58:2754-2760.

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