Chemoprotective and toxic potentials of synthetic and natural chalcones and dihydrochalcones in...

Toxicology 208 (2005) 81–93

Chemoprotective and toxic potentials of synthetic and naturalchalcones and dihydrochalcones in vitro

Hana Forejtnıkovaa, Kamila Lunerovaa, Renata Kubınovaa, Dagmar Jankovskaa, RadekMarekb, Radovan Karesb, Vaclav Suchya, Jan Vondracekc, Miroslav Machalac,∗

a Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republicb National Center for Biomolecular Research and Department of Organic Chemistry, Faculty of Science, Masaryk University,

Brno, Czech Republicc Veterinary Research Institute, Brno, Czech Republic

Received 6 September 2004; received in revised form 5 November 2004; accepted 8 November 2004Available online 22 December 2004

Abstract

Cytochrome P4501A activity, oxidative stress and inhibition of gap junctional intercellular communication (GJIC) are involvedin metabolic activation of promutagens and tumor-promoting activity of various xenobiotics, and their prevention is consideredto be an important characteristic of chemoprotective compounds. In this study, a series of 31 chalcones and their correspondingdihydroderivatives, substituted in 2,2′-, 3,3′-, 4- or 4′-position by hydroxyl or methoxy group, were tested for their abilityt e( lial WB-F the range0 bitedh halconess y onlya eatmento ded fromae to suggestt

, 7-e zoliumb

0

o inhibit Fe(II)/NADPH-enhanced lipid peroxidation and cytochrome P4501A-dependent 7-cethoxyresorufin-O-deethylasEROD) activity in rat hepatic microsomes. Effects of the compounds on GJIC were determined in rat liver epithe344 cells. Most of the chalcones and dihydrochalcones inhibited EROD activity in a dose-dependent manner at.25–25�M, which was comparable to model flavonoid inhibitors�-naphthoflavone and quercetin. The chalcones exhiigher inhibition activity than the corresponding dihydroderivatives. Mono and dihydroxylated chalcones, and dihydrochowed none or only a weak antioxidant activity; trihydroxyderivatives inhibited in vitro lipid peroxidation significantlt 50�M concentration. Potential adverse effects, namely inhibition of GJIC and/or cytotoxicity were detected after trf WB-F344 cells with a number of chalcone and dihydrochalcone derivatives, suggesting that they should be excludditional screening as chemoprotective compounds. Chalcones and dihydrochalcones substituted at 4- and/or 4′-position, whichlicited no inhibition of GJIC, were further tested for the potential enhancing effects on GJIC. The present data seem

hat 4-hydroxy, 2′,4′-dihydroxy-3-methoxy, 2,4,4′-trihydroxy, and 2′,4,4′-trihydroxychalcone, 2′,4-dihydroxy and 2′-hydroxy-

Abbreviations: �-NF, �-naphthoflavone; BHT,tert-butylhydroxytoluene; CYP1A, cytochrome P4501A subfamily; ERODthoxyresorufin-O-deethylase; GJIC, gap junctional intercellular communication; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetraromide; TPA, 12-O-tetradecanoylphorbol-13-acetate

∗ Corresponding author. Tel.: +42 533331813; fax: +42 541211229.E-mail address:[email protected] (M. Machala).

300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2004.11.011

82 H. Forejtnıkova et al. / Toxicology 208 (2005) 81–93

3,4-dimethoxydihydrochalcone might be promising chemoprotective compounds against CYP1A activity, and partly also againstoxidative damage without inducing adverse effects, such as GJIC inhibition. In general, determination of potencies of testedcompounds to inhibit GJIC should be involved in any set of methods for the in vitro screening of chemoprotective characteristicsof potential drugs, in order to reveal their potential adverse effects associated with tumor promotion.© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords:Chalcones; Cytochrome P450; Lipid peroxidation; Gap junctional intercellular communication

1. Introduction

Chemical carcinogenesis is a multi-step process in-volving both genotoxic (initiating) and non-genotoxic(promoting) events. Many genotoxic effects of xeno-biotics result from metabolic activation of promuta-gens by cytochromes P4501A (CYP1A) and other CYPforms (Guengerich and Shimada, 1998; Parke et al.,1990), or from an increased production of reactive oxy-gen species (Collins et al., 2004). Non-genotoxic eventsassociated with tumor-promoting mechanisms may al-ter expression of the genomic information at the tran-scriptional, translational or post-transcriptional levels(Trosko and Ruch, 1998). The molecular mechanismsof tumor promotion can involve cellular oxidativestress (Dalton et al., 1999), or inhibition of gap junc-tional intercellular communication (GJIC) leading toderegulation of cell proliferation, differentiation and/orapoptosis (Trosko and Ruch, 1998; Roberts et al.,

focused mostly on inhibition of CYP1A-dependentmetabolic activation of promutagens and antimuta-genic and antioxidant activity (Cao et al., 1997;Edenharder et al., 1993; Chae et al., 1991), since anti-inflammatory effects are often associated with antiox-idative characteristics leading to inhibition of lipoxyge-nase or cyclooxygenase activities (Sogawa et al., 1993).Contrary to that, modulations of GJIC have been rarelystudied, and this mechanism of toxic or chemopro-tective effect is generally underestimated (Trosko andRuch, 2002).

Dietary intake of natural polyphenolic compoundscan modulate drug-metabolizing enzymes, inhibit ox-idative stress or enhance GJIC suppressed in trans-formed cells or by model tumor promoters (Di Carloet al., 1999; Wattenberg, 1985). Research in thisfield has focused on flavonoids, as common com-ponents of the human diet. Nevertheless, both cit-rus fruits and apples are rich dietary sources of chal-

undsotaloredorslayingan-

1997).Induction of CYP1A activity, production of ox-

idative stress and inhibition of GJIC by chemicalshave been recognized, among other characteristics, asparameters reflecting adverse effects of xenobiotics(Rosenkrantz et al., 2000; Siess et al., 1995). For thisreason, studies on chemoprevention use detection ofinhibition of CYP1A or oxidative damage as suit-

cones and dihydrochalcones and these compocould make even a greater contribution to the tdaily intake of natural polyphenolics than the mextensively studied flavonoids (Tomas-Barberen anClifford, 2000). Chalcones are biogenetic precursof the flavonoids in higher plants and they dispa wide variety of pharmacological effects, includcytotoxicity towards cancer cell lines, antitumor,

able tools for screening chemoprotective compounds(Halliwell et al., 1995; Wattenberg, 1985). Severalchemoprotective compounds have been also reported toe ibi-t ro-m ate(R

m-p es ofa tingp far

timitotic, antimutagenic, and antitumor-promoting ac-tivities; antibacterial, antiviral, anti-inflammatory, an-tiulcerative, and hepatoprotective activities (Arty eta vae t al.,1 ica eenrb al-c onlyft trong

nhance GJIC in transformed cells or to prevent inhion of GJIC after exposure to prototypical tumor poters such are 12-O-tetradecanoylphorbol-13-acet

TPA) or epidermal growth factor (EGF) (Trosko anduch, 2002).Many natural and synthetic polyphenolic co

ounds have been found to block at least some modction associated with genotoxic or tumor-promorocesses. In vitro and in vivo studies have so

l., 2000; Dimmock et al., 1999; Dinkova-Kostot al., 1998; Sogawa et al., 1994; Wattenberg e994; Batt et al., 1993). Although antimutagenction of hydroxy and dihydroxychalcones has beported previously (Dimmock et al., 1999), the inhi-ition of major bioactivating enzyme CYP1A1 by chones and dihydrochalcones has been reportedrom several studies (Machala et al., 2001). Amonghem, 2-hydroxychalcone has been reported as a s

H. Forejtnıkova et al. / Toxicology 208 (2005) 81–93 83

inhibitor of CYP1A-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity (Machala et al.,2001).

Since several chalcones inhibit various enzymes in-volved in the generation of reactive oxygen species,such as 5-lipoxygenase, 12-lipoxygenase or cyclooxy-genase, many of their pharmacological properties aresupposed to be related to their antioxidative effect(Sogawa et al., 1993). 2′,5′-Dihydroxychalcones havebeen reported to be potent anti-inflammatory chem-icals (Ko et al., 2003; Hsieh et al., 1998), how-ever, a prototypical derivative 2′,5′-dihydroxychalconehas induced down-regulation of GJIC in endothelialcells (Lee et al., 2002). Modulations of GJIC byother hydroxylated chalcones has not been investi-gated. Another class of chalcone derivatives, 2′- and4′-hydroxylated chalcones, have been found to in-hibit aromatase and 17�-hydroxysteroid dehydroge-nase, two key enzymes of steroidogenesis, and elicitedtherefore effects potentially leading to suppression ofdevelopment of estrogen-sensitive breast cancer cells(Le Bail et al., 2001). 2′,4′-Dihydroxy and 2′,4,4′-trihydroxychalcones inhibit ovarian cancer cell growth(De Vincenzo et al., 1995) and possess antitumor ac-tivities (Shibata, 1994). Therefore, both chemopro-tective and adverse effects of 2′-, 4′-, as well as of2- and 4-hydroxylated derivatives are of particularinterest.

In this study, in vitro effects of a series of31 chalcones and dihydrochalcones, hydroxylated ormC H-i esw ibitG he-l lyu s as-s ;T

hydroxylated chalcone derivatives are potent inhibitorsof CYP1A activity, however, a significant number ofcompounds also inhibited GJIC and might be classi-fied as potential tumor promoters, not suitable to beused as chemoprotective agents.

2. Materials and methods

2.1. Chemicals

2,3,7,8-Tetrachlorodibenzo-p-dioxin was pur-chased from Cambridge Isotope Laboratories (An-dover, MA, USA). 7-Ethoxyresorufin was obtainedfrom Molecular Probes (Eugene, OR, USA). Resorufin,�-naphthoflavone, quercetin,tert-butylhydroxytolu-ene, NADPH, thiobarbituric acid, malondialdehyde,12-O-tetradecanoylphorbol-13-acetate (TPA), epi-dermal growth factor (EGF), lucifer yellow, cellculture media, and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were suppliedby Sigma-Aldrich (Prague, Czech Republic). All otherchemicals used were of the highest purity available.

2.2. Synthesis and structural identification ofchalcones and dihydrochalcones

4-Hydroxy, 4′-hydroxy, 2-hydroxy, and 2′-hydroxychlacone were purchased from Fluka (Prague,Czech Republic), while other chalcone derivativesu midtc riatead f thea ,1 ew ofD edp

chalco

ethoxylated in the positions 2, 2′, 3, 4, or 4′, onYP1A-dependent EROD activity and Fe/NADP

nduced lipid peroxidation in rat hepatic microsomere investigated. The relative potencies to inhJIC were determined in rat liver stem-like epit

ial cell line WB-F344, an in vitro model frequentsed for screening this adverse effect of xenobioticociated with tumor promotion (Machala et al., 2003rosko and Ruch, 1998). We found that 2-, 2′-, 4- or 4′-

Fig. 1. Synthesis of the

nder study were prepared using the Claisen–Schondensation of acetophenone with appropromatic aldehyde (Davis and Chen, 1993). The dihy-rochalcones were obtained by hydrogenation oppropriate chalcones using 10% Pd/C (Di Carlo et al.999) (seeFig. 1). 2,4,4′-Trihydroxydihydrochalconas isolated from dragon’s blood, the resinracaena cinnabariBalf. (Agavaceae) as reportreviously (Gonzales et al., 2000).

nes and dihydrochalcones.


Table 1In vitro effect of chalcones (expressed as % of control activities)

Chalcone Inhibition of ERODactivity (at 25�M)

Inhibition of lipidperoxidation (at50�M)

Inhibition of GJICIC50 (�M)

CytotoxicityIC50 (�M)

Synthesis and spectraldata (references)

4-OH 16± 2 85 ± 4 n.i. >50 a

4′-OH 14 ± 3 87 ± 9 n.i. 38 a

2-OH 14± 2 78 ± 3 8.5 27 a

2′-OH 6 ± 1 84 ± 0 8.1 37 a

2′,3-diOH 10± 2 54 ± 4 9.1 >50 Hsieh et al. (1998)2′,4-diOH 20± 3 64 ± 4 49.2 78 Severi et al. (1996)2′-OH-3-OMe 6± 3 79 ± 0 34.1 >50 Freeman et al. (1981)2′-OH-4-OMe 15± 1 68 ± 4 83.0 82 Freeman et al. (1981)2′,3′,3-triOH 30± 2 37 ± 10 181.1 51 Severi et al. (1998)2,4,4′-triOH 26 ± 5 36 ± 5 n.i. >250 Figueiredo et al. (1994)2′,4′,4-triOH 23± 1 45 ± 4 n.i. >50 Saitoh et al. (1978)4′-OH-2,4-diOMe n.a. n.a. 190 752′-OH-2,4-diOMe n.a. n.a. 32.6 >2502′-OH-3,4-diOMe 8± 2 65 ± 5 75.6 81 Hsieh et al. (1998)2′,4-diOH-3-OMe n.a. n.a. 146.4 702′,4′-diOH-3-OMe 10± 0 59 ± 1 n.i. >50 Severi et al. (1998)�-NF 0.8± 1Quercetin 0.9± 1 13 ± 0BHT 6 ± 1Fluoranthene 9

n.a.: not analysed; n.i.: no significant inhibition of GJIC observed.a Supplied by Fluka (Prague, Czech Republic).

The products were purified by TLC, HPLC and re-crystallization from methanol. All compounds wereidentified by the UV, IR, MS,1H- and13C -NMR spec-tra. The data reported previously for the compoundswere compared with the reference values (for refer-ences, seeTables 1 and 2).

The HPLC analyses and separations and the UVspectra were carried out using the HPLC/UV appara-tus HP 1100 equipped with a diode-array detector (Ag-ilent Technologies, Waldbromn, Germany) using theSupel cosil

TMABZ + Plus column (150 mm× 4.6 mm,

particle size 3�m, Supelco, Bellefonte, PA, USA) andthe following parameters: linear gradient starting from10% acetonitrile and 90% of 40 mM HCOOH to 100%acetonitrile over 65 min; flow rate of 1 mL/min; columntemperature of 40◦C; detection at 280 and 330 nm. TheHPLC isolation of 2,4,4′-trihydroxydihydrochalconewas performed using a semi-preparative column Supel-cosilTM ABZ + Plus, 250 mm× 10 mm, particle size5�m and isocrate elution within 13 min using a mix-ture of 39% acetonitrile and 61% of 40 mM HCOOH.

The IR spectra were recorded on a Nicolet FTIRImpact 400D (Nicolet, Madison, WI, USA) using KBr

pellets. The mass spectra were obtained using a TRIO1000 quadrupole mass spectrometer Finnigan MAT(Fisons Instruments, San Jose, CA, USA); the directinsertion probe (DIP) was used in the EI+ ionizationmode, applying electron energy of 30 eV. The mass-monitoring interval was from 40 to 400 amu. The NMRspectra were recorded in CDCl3 or CD3OD by usingBruker Avance DRX 500 and Avance DPX 300 spec-trometers at 303 K. The13C spectra and were refer-enced to the solvent signals of CDCl3 (77.00 ppm),CD3OD (49.15 ppm) and the1H spectra to the residualCHCl3 (7.26 ppm) or CHD2OD (3.31 ppm) in the re-spective solvents. The individual resonances were as-signed on the basis of COSY, NOESY (mixing time800 ms), GHSQC (J= 145 Hz), GSQMBC (J= 7.5 Hz),and GHMBC (J= 7.5 Hz) methods (for experimentaldetails, seeSeckarova et al., 2002; Marek et al., 1999).

2′,4-Dihydroxydihydrochalcone: C15H14O3; mp:106–107◦C; UVmax: 212, 253, 325 nm; IR (KBr):3417 (OH), 1637 (CO), 1612, 1518, 1495, 1259, 1200,1169, 831, and 765 cm−1; m/z (EI, 30 eV, rel. int.)242 (M+, 31%), 224 (M+ − H2O, 18), 223 (21), 121(C6H4(OH)CO+, 100), 107 (C6H4(OH)CH2

+, 60), 93


Table 2In vitro effects of dihydrochalcones (expressed as % of control activities)

Dihydrochalcone Inhibition of ERODactivity (at 25�M)

Inhibition of lipidperoxidation (at50�M)

Inhibition of GJICIC50 (�M)

CytotoxicityIC50 (�M)

Synthesis and spectraldata (references)

4-OH 24± 4 82 ± 2 w.i. >250 Bunce and Reeves (1989)4′-OH 16 ± 4 75 ± 1 n.i. 59 Chiba et al. (1996)2-OH 12± 4 80 ± 1 w.i. 99 Chambers et al. (1992)2′-OH 11 ± 4 77 ± 6 13.2 >50 Cimarelli and Palmieri (1998)2′,3-diOH 20± 0 68 ± 1 73.9 75 Murphy and Wattanasin (1980)2′,4-diOH 22± 1 70 ± 2 n.i. >502′-OH-3-OMe 10± 2 83 ± 6 60.2 >250 Kirchiakarian et al. (1989)2′-OH-4-OMe 26± 2 78 ± 2 50.7 >250 Kirchiakarian et al. (1989)2′,3′,3-triOH 42± 0 27 ± 2 n.a. n.a.2,4,4′-triOH 56 ± 2 53 ± 2 n.a. n.a. Gonzales et al. (2000)2′,4′,4-triOH 44± 1 42 ± 9 n.i. >50 Severi et al. (1998)4′-OH-2,4-diOMe n.a. n.a. w.i. 902′-OH-2,4-diOMe n.a. n.a. 40.2 >2502′-OH-3,4-diOMe 15± 2 70 ± 8 n.i. >250 Kirchiakarian et al. (1989)2′,4-diOH-3-OMe n.a. n.a. n.a. n.a.2′,4′-diOH-3-OMe 29± 4 75 ± 0 n.i. >250�-NF 0.8± 1Quercetin 0.9± 1 13 ± 0BHT 6 ± 1Fluoranthene 9

n.a.: not analysed; n.i.: no significant inhibition of GJIC observed; w.i.: weak inhibition – IC50 could not be estimated due to significant GJICinhibition but lower than 50% of control.

(C6H5OH+, 11), 77 (14), 65 (15);1H -NMR (CDCl3)δ (ppm): 12.35 (2′-OH), 7.74 (H-6′), 7.46 (H-4′), 7.11(H-2 and H-6), 6.99 (H-3′), 6.89 (H-5′), 6.78 (H-3 andH-5), 5.04 (bs, 4-OH), 3.29 (CH2-�), 3.00 (CH2-�);13C -NMR (CDCl3) δ (ppm): 205.70 (CO), 162.37 (C-2′), 154.02 (C-4), 136.36 (C-4′), 132.79 (C-1), 129.87(C-6′), 129.51 (C-2 and C-6), 119.29 (C-1′), 118.97 (C-5′), 118.52 (C-3′), 115.43 (C-3 and C-5), 40.26 (CH2-�), 29.27 (CH2-�).

2′,4′-Dihydroxy-3-methoxydihydrochalcone:C16H16O4; mp: 71–73◦C; UVmax: 213, 277, and313 nm; IR (KBr): 3143 (OH), 1629 (CO), 1593,1464, 1326, 1283, 1170, 1143, and 791 cm−1; m/z (EI,30 eV, rel. int.) 272 (M+, 100), 254 (M+ − H2O, 19),137 (C6H3(OH)2CO+, 97), 135 (C6H4(OCH3)C2H4

+,43), 134 (11);1H -NMR (CDCl3) δ (ppm): 12.75(2′-OH), 7.63 (H-6′), 7.22 (H-5), 6.83 (H-6), 6.79(H-2), 6.77 (H-4), 6.37 (H-3′), 6.36 (H-5′), 5.9 (bs,4′-OH), 3.80 (3-OMe), 3.23 (CH2-�), 3.03 (CH2-�);13C -NMR (CDCl3) δ (ppm): 203.68 (CO), 165.17(C-2′), 162.55 (C-4′), 159.73 (C-3), 142.44 (C-1),132.22 (C-6′), 129.59 (C-5), 120.78 (C-6), 114.29(C-2), 113.85 (C-1′), 111.59 (C-4), 107.78 (C-5′),

103.61 (C-3′), 55.22 (3-OMe), 39.55 (CH2-�), 30.39(CH2-�).

2′,4,4′-Trihydroxydihydrochalcone: C15H14O4;mp: 134–135◦C, UVmax: 213, 277, and 313 nm; IR(KBr): 3329 (OH), 1644 (CO), 1593, 1454, 1352,1225, and 1147 cm−1; m/z (EI, 30 eV, rel. int.) 258(M+, 44), 240 (9), 138 (7), 137 (C6H3(OH)2CO+,100), 121 (C6H4(OH)C2H4

+, 14), 120 (11);1H -NMR(methanol-d4) δ (ppm): 7.68 (H-6′), 7.1 (H-5), 6.71(H-6), 6.70 (H-2), 6.65 (H-4), 6.39 (H-5′), 6.29 (H-3′),3.18 (CH2-�), 2.90 (CH2-�); 3-OH, 2′-OH and 4′-OHnot observed;13C -NMR (methanol-d4) δ (ppm):206.21 (C O), 166.12 (C-4′), 165.68 (C-2′), 157.56(C-3), 143.74 (C-1), 134.08 (C-6′), 130.70 (C-5),121.10 (C-6), 116.27 (C-2), 114.22 (C-4), 113.99(C-1′), 109.69 (C-5′), 103.81 (C-3′), 40.12 (CH2-�),31.66 (CH2-�).

2.3. Preparation of hepatic microsomal fraction

The hepatic microsomal fraction was isolated by ho-mogenization and differential centrifugation from the42-day-old male Wistar rats (Charles River Laborato-


ries, Prague, Czech Republic), exposed to a single i.p.dose of 2,3,7,8-tetrachlordibenzo-p-dioxin (1 ng/g).The microsomes forming the 105.000×g pellet werewashed, resuspended in 0.05 M Tris–HCl buffer of pH7.5 containing 20% of glycerol and 0.1 mM EDTA, andstored at−80◦C until use.

2.4. In vitro inhibition of CYP1A activities andlipid peroxidation in rat hepatic microsomes

The in vitro potency of a series of chalcone deriva-tives to inhibit CYP1A-dependent EROD activity wasdetermined in rat hepatic microsomes using direct flu-orimetric method (Prough et al., 1978). The reactionmixture (2 mL) consisted of microsomes (equivalentto 0.2 mg protein), 2�M ethoxyresorufin, 0.1 M phos-phate buffer of pH 7.6, 0.2 mM NADPH (final concen-trations), and 10�L of dimethylsulfoxide as solvent ordissolved chemical under study.�-NF and quercetinwere used as reference inhibitory compounds in con-centrations corresponding with chalcones and dihy-drochalcones under study.

In vitro Fe(II)/NADPH-enhanced lipid peroxidationwas determined in the rat hepatic microsomal frac-tion (Machala et al., 2001). tert-Butylhydroxytolueneand quercetin were used as reference antioxidant com-pounds in the same concentrations as tested chal-cones and dihydrochalcones. The reaction mixture(1 mL) consisted of microsomes (equivalent to 0.4 mgprotein), 0.1 M Tris–HCl buffer of pH 7.4, 2 mMaN s-s ando singt ,1

2

,1 me tionso sup-p S,aac es aw

2.6. Cytotoxicity

In vitro cytotoxicity was measured by thetetrazolium-based colorimetric assay (MTT assay) asreported previously (Mosmann, 1983) in WB-F344cells in two independent experiments. Briefly, thecells were incubated on 96-well plates with variousconcentration of each compound. After 24-h incuba-tion, 25�L MTT (2.5 ng/mL) was added to each welland the plates were incubated for 90 min at 37◦C.The water-insoluble formazan crystals were dissolvedwith sodium dodecylsulphate and optical density at540 nm was measured by a spectrophotometric mi-croplate reader (Labsystems iEMS Reader, Labsys-tems, Turku, Finland). The percentage of the cellgrowth inhibition was calculated as cell growth inhibi-tion (%) = (1−T/C) × 100, whereCwas OD540 valueof the control andT was OD540 value of tested chem-ical. The 50% inhibitory concentrations (IC50 values)were obtained from dose–response curves.

2.7. Inhibition of GJIC

The confluent rat liver epithelial WB-F344 cells,grown in 24-well plates, were exposed to variousconcentrations of chalcones or dihydrochalcones for30 min. Exposure to fluoranthene (40�M) and DMSOwere used as positive and negative controls, respec-tively. The concentrations of DMSO did not exceed0.5% (v/v). After the exposure, a modified protocol ofst ,2r asa pedw omt .5P ra-t itha on,J ulart mu-n werec wellw ivet ed inp trics d

denosine diphosphate, 0.12 mM Fe(NO3)2, 0.1 mMADPH and 10�L of compound under study diolved in DMSO. The resulting malondialdehydether reactive lipid metabolites were measured u

he thiobarbituric acid assay (Uchiyama and Mihara978).

.5. Cell culture

WB-F344 rat liver epithelial cell line (Tsao et al.984) were cultured in modified Eagle’s minimussential medium with 50% increased concentraf essential and non-essential amino acids, andlemented with pyruvate (110 mg/L), 10 mM HEPEnd 5% fetal bovine serum, at 37◦C in humidifiedtmosphere containing 95% air and 5% CO2. Theells were passaged by trypsinization three timeek.

crape-loading method (El-Fouly et al., 1987) was usedo assess GJIC as described previously (Machala et al.003). Cells were washed twice with 0.5× PBS, fluo-escent dye (Lucifer Yellow, 0.05% (w/v) in PBS) wpplied onto cell monolayer, and the cells were scraith a surgical blade. After 3 min incubation at ro

emperature, the cells were washed twice with 0×BS and fixed in 4% (v/v) formaldehyde. The mig

ion of the dye from the scrape line was quantified wn epifluorescence microscope (Nikon Inc., Nikapan). The distance of dye migration perpendico the scrape represents the ability of cells to comicate via GJIC. Three independent experimentsarried out in duplicate; at least three scrapes perere evaluated. The ratio of GJIC inhibition relat

o the negative control was evaluated and expressercentage (fraction of control, FOC). Non-parametatistical methods, Kruskal–Wallis ANOVA followe


by the Mann–Whitney test, were used for assessmentof significance. The concentrations of the referenceinhibitor of GJIC (fluoranthene) and the tested com-pounds causing 50% inhibition of GJIC (IC50) weredetermined by the non-linear logit regression, and 95%confidence intervals for IC50 were estimated; relativeerror did not exceed 15%.

2.8. Co-treatment with inhibitors of GJIC

Confluent cell monolayers were treated with TPA (4and 20 ng/mL final concentration) or EGF (0.1�g/mLfinal concentration) for 30 min. Control cultures weretreated with DMSO (5�L/mL). The cells were pre-treated with chalcones or dihydrochalcones (25, 50,100�M final concentrations) for 1, 5 or 24 h prior toTPA or EGF exposure.

3. Results

3.1. In vitro inhibition of CYP1A activity in rathepatic microsomes

The inhibition of CYP1A1-dependent EROD activ-ity by chalcone and dihydrochalcone hydroxyderiva-tives was determined in rat hepatic microsomes andcompared with potencies of two prototypical ERODinhibitors, �-NF and quercetin (Fig. 2). The resultsare summarized inTables 1 and 2. All chalcones andd ntlyt ns.E delC 2h ig-n a-t enc s.

3

-s m-i op-e owni af-f hem y-

droxyderivatives, however, their inhibitory potencieswere significant only at high concentrations. Resultsare summarized inTables 1 and 2. Our study revealedthat chalcones and dihydrochalcones tested are in gen-eral only weak inhibitors of lipid peroxidation and theirantioxidant potencies, evaluated as potency to blockFe/NADPH-induced lipid peroxidation are low.

3.3. Cytotoxicity and inhibition of GJIC in vitro

Two potential adverse effects of chalcones weredetermined in the non-transformed rat liver epithe-lial WB-344 cell line. General cytotoxicity was de-termined after 24-h treatment with various concentra-tions of tested compounds in MTT assay. The mosttoxic compounds were 2-hydroxy, 2′-hydroxy and 4′-hydroxylated chalcones (Table 1), however, severalother compounds showed also significant cytoxicity atmicromolar concentrations.

Acute inhibition of GJIC in rat epithelial cell line is asuitable tool to screen for potential adverse effect asso-ciated with tumor promotion (Trosko and Ruch, 2002;Rosenkrantz et al., 2000). A significant number ofchalcones and dihydrochalcones under study inhibitedGJIC in rat liver epithelial cells (Tables 1 and 2), sug-gesting that they should be excluded from the panel oftested chemoprotective compounds. Dose-dependentinhibitory effect of 2′-hydroxychalcone is presentedhere as a representative micrograph (Fig. 4). A ma-jority of 2′-hydroxylated chalcones were significantG ddi-t dl -v d bef om-pma -x torso renc howa edf ICa er-m tedt tor-i

ihydrochalcones under study inhibited significahe EROD activity at submicromolar concentratioffects of chalcones were comparable with moYP1A inhibitors. With exception of 2-hydroxy and′-ydroxy derivatives, all dihydrochalcones elicited sificantly lower inhibitory potency. Trihydroxyderiv

ives were weaker inhibitors of the EROD activity whompared with either mono or dihydroxy-derivative

.2. Microsomal lipid peroxidation assay

The inhibition of in vitro peroxidation of microomal lipids, catalyzed by Fe(II)/NADPH, was exaned in order to determine potential antioxidative prrties of chalcones and dihydrochalcones. As sh

n Fig. 3, neither chalcones nor dihydrochalconesected lipid peroxidation in a significant manner. Tost potent inhibitors of lipid peroxidation were trih

JIC inhibitors; the effect was attenuated by aional substitutions (Fig. 5). Dihydrochalcones showeower GJIC inhibitory potencies (Fig. 6). However, seeral chalcones did not affect GJIC and they coulurther studied as promising chemoprotective counds. Taken together, 4-hydroxy, 2′,4′-dihydroxy-3-ethoxy, 2,4,4′-trihydroxy, and 2′,4,4′-trihydroxych-lcone, 2′,4-dihydroxy and 2′-hydroxy-3,4-dimethoydihydrochalcone were found to be potent inhibif CYP1A, which did not inhibit GJIC and they weot cytotoxic in WB-F344 cells (Tables 1 and 2). Thosehalcones and dihydrochalcones, which did not sny GJIC inhibitory potency, were further examin

or their potential ability to prevent inhibition of GJfter co-treatment with tumor promoter TPA or epidal growth factor. However, no compound protec

he cells against the TPA or epidermal growth facnduced inhibition of GJIC (data not shown).


Fig. 2. Inhibition of EROD activity by chalcones and corresponding dihydrochalcones at concentrations 0.25�M (A), 2.5�M (B) and 25�M(C). Inhibitory potencies are compared with effects of�-naphthoflavone and quercetin; control activities were obtained in the presence of DMSO.Results are expressed as means± S.D. of three independent measurements.


Fig. 3. Inhibition of lipid peroxidation by chalcones and corresponding dihydrochalcones: A (1�M), B (10�M) and C (50�M) in comparisonwith BHT and quercetin. Results are expressed as mean± S.D. of three independent measurements.

4. Discussion

Many chalcone derivatives show antioxidative andanti-inflammatory properties, which include theirpotency to inhibit lipoxygenase and cyclooxygenase

activities, as well as lipid peroxidation (Ko et al., 2003;Arty et al., 2000; Sogawa et al., 1994). Chalconescould also modulate sex hormone-related carcino-genic processes. Several chalcones, including 2′,4′-dihydroxychalcone and 2′,4,4′-trihydroxychalcone


Fig. 4. Dose-dependent inhibition of GJIC by 2′-hydroxychalcone.

inhibited ovarian cancer cell proliferation and estradiolbinding activity (De Vincenzo et al., 1995) and they arepotent inhibitors of aromatase activity (Le Bail et al.,2001). 2′,4′-Dihydroxy and 4,4′-dihydroxychalconeinhibited proliferation of breast cancer epithelialMCF-7 cells (Markaverich et al., 1990) and 2′,4,4′-trihydroxychalcone has also been reported to have an-titumor effects (Shibata, 1994). In our previous study,2-hydroxychalcone belonged among potent inhibitors

of CYP1A-dependent EROD activity (Machala et al.,2001). Taken together, chalcones appear to be a promis-ing group of chemoprotective compounds with varioustypes of effects. However, 2′,5′-dihydroxychalcone hasbeen recently reported to inhibit GJIC, which suggeststhat some of chalcone derivatives might also have ad-verse effects (Lee et al., 2002).

The aim of the present study was to study poten-tial chemoprotective and/or adverse properties of a se-


Fig. 5. Concentration-dependent inhibition of GJIC by selected 2′-hydroxychalcones in the WB-F344 cells (30 min exposure). Inhibi-tion of GJIC was determined as described in Section2. Results wereobtained in two independent experiments carried out in triplicates.

ries of chalcone derivatives, hydroxy and methoxy-chalcones and dihydrochalcones substituted in 2,2′, 3, 4 or 4′-positions, that were synthetized byClaisen–Schmidt condensation reaction (Davis andChen, 1993). Two possible chemoprotective proper-ties of hydroxychalcones were examined in vitro, theirpotencies to inhibit CYP1A-dependent EROD activityand Fe(II)/NADPH-enhanced lipid peroxidation in rathepatic microsomes. While antioxidant activity of hy-droxychalcones and dihydrochalcones was very low(Fig. 3), all chalcones under study were strong in-hibitors of the EROD activity (Fig. 2, Tables 1 and 2).However, the compounds with substitution in 2- or2′-position also inhibited GJIC in rat liver epithe-lial WB-F344 cells (Tables 1 and 2). Since this ac-

Fig. 6. Comparison of inhibitory effects of 2-hydroxy and2 nes(

tivity is strongly associated with tumor promotion(Rosenkrantz et al., 2000), the GJIC-inhibitory com-pounds should be excluded from the further screen-ing of chemoprotective properties. Additionally, 4′-hydroxychalcone, which is a potent inhibitor of ERODactivity with no effect on inhibition of GJIC, showeda strong cytotoxicity. This seems to favor its exclusionfrom additional studies on chemoprotectivity. Never-theless, several promising chalcone and dihydrochal-cone derivatives were identified in this study, includ-ing 2′,4,4′-trihydroxychalcone, which is known for itsantitumor effects (Shibata, 1994) and inhibition ofproliferation of ovarian cancer cells (De Vincenzo etal., 1995), and 4-hydroxychalcone, 2′,4′-dihydroxy-3-methoxychalcone, 2,4,4′-trihydroxychalcone, 2′,4-dihydroxydihydrochalcone and 2′-hydroxy-3,4-dime-thoxydihydrochalcone. These compounds inhibitedboth EROD activity and lipid peroxidation and elicitedno cytotoxicity or inhibitory effects on GJIC; they de-serve further attention as potential chemoprotectivecompounds.

In conclusion, determination of cytotoxicity and arelative potency to inhibit GJIC in WB-F344 cells al-lowed us to select hydroxylated chalcones and dihy-drochalcones, which are suitable for testing as chemo-protective agents. Although a majority of chalconesinhibited significantly CYP1A activity in rat hepaticmicrosomes, many of them, substituted in 2- or 2′-position, should not be used as chemoprotective chem-icals, due to their potential tumor-promoting or hepa-t hibitG as-s ingf

A

ofA ofE

R

A vans and7.

′-hydroxyderivatives of chalcones (CH) and dihydrochalcoDHCH) on GJIC in WB-F344 cells.

otoxic effects. Thus, assessment of potency to inJIC in model cellular system is a valuable in vitro

ay, which might be an integral part of in vitro screenor chemoprotective chemicals.

cknowledgements

This work was supported by the Czech Ministrygriculture (Grant No. 0002716201) and Ministryducation (Grant No. 163700003).

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