Reactivities of Flavonoids with DifferentHydroxyl Substituents for the Cleavage of DNAin the Presence of Cu(II)

Aparna Jain,1 M. C. Martin, 2 Nazneen Parveen,3 N. U. Khan,3 J. H. Parish2 and S. M. Hadi1*1Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh - 202 002 (UP), India2Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK3Department of Chemistry, Aligarh Muslim University, Aligarh - 202 002 (UP) India

DNA strand scission reactions of flavonoids in the presence of Cu(II) have been extended by using flavo-noids with a variety of patterns of hydroxyl substitution. In particular we have examined for the firsttime a flavonoid (7,8-dihydroxyflavone) that lacks the possibility of forming a complex involving the oxy-gen at position 4. By comparing the reactivities of several flavonoids, including data from the literature,we draw generalizations for the correlation of structure and activity and present evidence for at leastthree different modes of action of flavonoids as genotoxic agents. Copyright# 1999 John Wiley & Sons,Ltd.

Keywords:flavonoids; hydroxyl substituents; DNA cleavage.

INTRODUCTION

Flavonoids are widely distributed dietary constituentsderived from plants and are genotoxic agents (Bjeldanesand Chang, 1977; Brown, 1980; Nagaoet al., 1981); theyhave a wide spectrum of pharmacological properties(Beretzet al., 1977), the mechanisms of which remain, toa large extent, unknown. In addition to their putativeenvironmental and dietary hazard, flavonoids have beenimplicated as novel antiviral, antiinflammatory andantitumour compounds (Van Hoofet al., 1984; Vrijsenet al., 1987; Brackeet al., 1988). Flavonoids have beenreported to have antioxidant properties and act asscavengers of oxygen radicals such as superoxide anion,singlet oxygen and hydroxyl radicals (Puppo, 1992). Onthe other hand flavonoids can have prooxidant effectsunder some reaction conditions. We have shownpreviously that quercetin, and certain other flavonoids,cause strand scission in DNA in the presence of Cu(II)and that this reaction is associated with transientreduction of Cu(II) to Cu(I) and the generation of activeoxygen species (Rahmanet al., 1989, 1990). However,different flavonoids result in differences in the rates ofDNA degradation, stoichiometries of Cu(II) reductionand extents of generation of active oxygen species(Ahmadet al., 1992).

The general formulae for flavonoids and relatedcompounds are shown in Fig. 1.Salmonella typhimuriummutagenicity data (Nagaoet al., 1981) had earlier

revealed that the most important structural feature formutagenicity of flavonoids is the 3-OH group of theflavonol structure (Fig. 1a). Markham and Mabry (1975)have concluded that in quercetin the favoured sequestra-tion of the metal ion is at 3–4 (not 4–5) but that 3–4 andortho chelation of different metal ions can occursimultaneously. The former conclusion is confirmed bythe pattern of oxidative fragmentation of the Cu(II)complex (Utaka and Takeda, 1985). In the light of thesefindings and the complexity of the reaction withquercetin, we have extended our study to severalpreviously unstudied flavonoids and in particular choseone synthetic flavonoid (7,8 dihydroxyflavone) for whichthe mode of binding of a metal ion, Cu(II) in thesestudies, is stereochemically unambiguous.

MATERIALS AND METHODS

7,8-Dihydroxyflavone was synthesized by the methodof Baker (1933). Quercetin, chrysin and kaempferolwere from Sigma Chemical Co., genistein and 6 and7-hydroxyflavones were from the Aldrich and SigmaChemical Co. and luteolin was from Carl Roth, GmbHKarlsruhe, Germany. These flavonoids were stored in adesiccator. Stock solutions in dimethyl sulphoxide(DMSO) were prepared immediately prior to theexperiments. Cu(II) was added as a stock solution ofCuCl2. Calf thymus DNA (Sigma; 2–3 mg/mL) wasdissolved in Tris-HCl (10 mM, Tris-HCl, 1 mM NaCl),pH 7.4. The ratio of A260/280 was 1.9, concentrationswere expressed in terms of mol base pair/L. DenaturedDNA was prepared by heating such a solution to 100°C(10 min) and rapid cooling in ice. Breakage of calfthymus DNA by flavonoids and Cu(II) as assayed byS1 nuclease hydrolysis and the reaction with super-

PHYTOTHERAPY RESEARCHPhytother. Res.13, 609–612 (1999)

CCC 0951–418X/99/070609–04 $17.50Copyright# 1999 John Wiley & Sons, Ltd.

* Correspondence to: S. M. Hadi, Department of Biochemistry, Faculty ofLife Sciences, Aligarh Muslim University, Aligarh - 202 002, UP, India.Contract/grant sponsor: UGC.Contract/grant sponsor: CSIR.Contract/grant sponsor: British Council (India).Contract/grant sponsor: EC DG XII (Brussels).

Received 22 November 1998Accepted 26 June 1999

coiled plasmid DNA have been describedpreviously(Rahman et al., 1989). Assay of the production ofsuperoxideanionhasbeendescribedearlier(Fazalet al.,1990).Absorptionspectrawere obtainedusing a Beck-man DU-40 spectrophotometerat ambient temperature(Rahmanetal., 1990).Dataanalysisby Jobplotsof Cu(I)productionat pH 7.4followed themethodsof Rahmanetal. (1989).

RESULTS

Flavonoidsand related compoundsin this study

Table1 summarizesthecompoundstestedandreviewedin this paper.They areclassifiedaccordingto the typesof metalchelationthat might, in principle, be found. Inthe case of compoundswith more than one of thepatterns3–4and4–5(Fig. 1), thechelation(3–4) that islikely to be favouredis listed. The compoundsare putinto groups:Group I (the most reactive) has 3–4 andortho patterns.Group II contains the 3–4 pattern ofchelation. Group III has potential for 4–5 and orthochelation, one member of this group (rutin) may beanomalousas it can hydrolyse to form the highlyreactive group I compound,quercetin (Ahmad et al.,1992); group IV comprisescompoundsfor which 4–5chelationis theonly possiblemethodof metalbinding:ofthis group, genistein may be anomalousas it is anisoflavonoid.GroupV containscompoundsthatareonlycapable of ortho chelation and, of these the mostimportant is 7,8-dihydroxyflavoneas the others havethe reducedring system.Group VI are predictedto beinactive.To this could be addedflavoneitself found to

be inactive in the Fe(III)-Fenton reaction by Puppo(1992).In additionto the possiblechelatorpositionandthe predictedcomplexesthe tablealsogivesthe relativeefficiency of Cu(II) reductionandDNA degradationby

Table 1. Summary of the activities of severalflavonoidsand related compounds.The first column (Group) is discussedin thetext. In column 2 (name (ring type)), the letter in brackets correspondsto one of the panelsfrom Fig. 1. The OHgroups(column 3) and possiblechelatepositions(column 4) are definedin Fig. 1.The predicted complexes(column 5)are explainedin the text. The data in column 6 (Cu(II) stoich)are estimatesof the stoichiometry of the mostCu-richcomplexesthat can lead to the reduction of Cu(II) to Cu-(I) from Job plots (Rahman et al., 1989) (ND no Cu(I)detected).Columns 7 and 8 (S1 sensitivity and pBR322 assays)summarize results of two different matrices forassayingDNA damage(Ahmad et al., 1992;Rahmanet al., 1989).The data on promotion of OH from Fe-EDTA andH2O2 (column 10) are summarized from Puppo (1992).NT not tested

1 2 3 4 5 6 7 8 9

Group Name (ring type) OH group(s)Possible chelate

positionsPredicted

complex(es)Cu(II)stoich S1-sensitivity

pBR322 assay(%)

OH from Fe-EDTAand H2O2

I Myricetin (a) 3,5,7,2',3' 4', 3-4, 4-5, ortho 3-4, ortho 6 55.0 ��� �����I Quercetin (a) 3,5,7,3'4' 3-4 4-5, ortho 3-4, ortho 5 41.8 ��� ����I Fisetin 3,3',4' 3-4, ortho 3-4, ortho NT NT ��� NTII Galangin (a) 3,5,7 3-4, 4-5 3-4 3 24.8 � NTII Morin (a) 3,5,7,2',4' 3-4, 4-5 3-4 3 NT �� ��II Kaempferol (a) 3,5,7,4' 3-4, 4-5 3-4 3 24.9 �� �III Rutin (a) [3-OR]b 5,7,3',4' 4-5, ortho 4-5, ortho 5 35.8 �� NTIII Luteolin (a) 5,7,3',4' 4-5, ortho 4-5, ortho 4 14.0 # NTIV Chrysin (a) 5,7 4-5 4-5 5 16.6 # NTIV Genistein (b) 5,7,4' 4-5 4-5 5 11.4 ÿ NTIV Apigenin (a) 5,7,4' 4-5 4-5 4 35.8 � NTV (ÿ) epicatechin (c) [R1 = OH] 5,7,3',4' ortho ortho 4 41.3 �� NTV (�) catechin (c) [R2 = OH] 5,7,3',4' ortho ortho NT NT NT ���V 7,8-dihydroxy¯avone (a) 7,8 ortho ortho 3 19.6 �� NTVI 7-hydroxy¯avone (a) 7 ± ± ND 3.0 ÿ NTVI 6-hydroxy¯avone (a) 6 ± ± ND 4.0 ÿ NT

a There are two possible ortho pairings, 2'3' or 3'4', of which the latter is likely to be favoured (Markham and Mabry, 1975)b In rutin, R is rutinose (a disaccharide).# Some open circular DNA was formed after 6h incubation.

Figure 1. Structure of (a) a ¯avonoid showing the A and Brings and the conventional numbering of the carbon atomsand (b) an iso¯avonoid. Arrows point to points of substitutionby OH groups in the compounds discussed in this paper anddouble arrows indicate three possible type of chelationposition: 3-4 and 4-5 are speci®c to those positions; orthorefers to any catechol-like position (3'-4' in ring B is shown asan example). The molecules shown (lacking hydroxyl sub-stituents) are called (a) ¯avone and (b) iso¯avone. Panel (c)shows part of the structure of a reduced ¯avonoid: one of thegroups R1 and R2 is OH and the other is H (see Table 1).

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variousflavonoids–Cu(II)systems.Thestoichiometryofthe flavonoid-Cu(II) complex that generatesCu(I) ismuch higher than the number of possible chelatepositionsin variousflavonoids.

Interaction betweenflavonoid and Cu(II)

Table 2 summarizesspectral changesobservedwhenCu(II) is added to solutions of certain flavonoids.Quercetin,kaempferolandluteolin havesimilar spectralchanges(large hypsochromicand hypochromicshifts)characteristicsof the formation of charge transfercomplexes(Rahmanet al., 1990) but thesedecay atdifferent rates possibly reflecting the different (butunknown) decompositionpathways of the flavonoid.We do know in the caseof quercetinthat the decom-position requires oxygen and is acceleratedby thepresenceof DNA (Rahmanet al., 1990). The simplestexplanationfor the seeminglyanomalousbehaviourofchrysin is that a complex is formed but decomposesessentiallyinstantaneously.Genistein(a Group IV iso-flavonoid)seemsnot to form a chargetransfercomplex.7,8-dihydroxyflavonedoes not have the characteristicflavonoid absorptionin the 320–390nm region and thehyperchromicshift of the catecholpeak at 292nm ispossiblya featureof Cu(II)-chelation.Although we didnot refer to it at the time, thereis hyperchromicshift in

this region of the quercetinspectrum(Rahmanet al.,1990).

We havemeasuredthe apparentstoichiometryof theflavonoids–Cu(II) complex that generatesCu(I) fromreducedJob plots. The data (Table 1) emphasizethatthis methodgiveshigh valuesthatdo not directly reflectthe tightly bound or chelatedCu(II). In the case of7,8-dihydroxyflavone for which there is only onechelationsite,thestoichiometryis ashigh as3 (Table1;Fig. 2).

By usingpreviouslyestablishedconditions(Rahmanetal., 1989)we havemeasuredthecleavageof calf thymusDNA (assessedby its sensitivityto S1-nuclease)andalsothe sensitivity of pBR322plasmidDNA (Fig. 3 for anexample)to additionalflavonoids(Table1). The resultsobtainedby the two methodsare essentiallysimilar interms of relative DNA degradation.However, the S1-nucleaseassayis moresensitivebut theplasmidassayisusefullyconfirmatoryandhastheadditionaladvantageofbeingsuitedto quantitativeanalysisof thedamage.

Table 2. Summary of formation of possiblechargetransfer complexeswith Cu(II).The experimentswere performed aspreviously (Rahman et al., 1990)andthe data for quercetin are taken from the spectrain (Rahmanet al., 1990).7,8-DHF is 7,8-dihydroxyflavone.The groups refer to thoseof Table I. lmax [1] is the absorbancemaximum of flavonoid; l max [2] is the new lmax after addition of Cu(II) (ratio of Cu(II) to flavonoid was 2.0) and‘ratio’ is the absorption at l max [2] of the complexwith Cu(II) divided bythe absorption at l max [1] of the free flavonoid. The value of t1/2 is anestimate of the half-life of the decay of the complex to decompositionproduct(s)

Compound Quercetin Kaempferol Luteolin Chrysin Genistein 7,8-DHF

Group I II III IV IV Vl max [1] 370 370 368 330 324 292l max [2] 450 412 400 412 324 292Ratio 0.68 0.39 1.04 1.03 1.0 1.25t1/2 (min) 0.5 30 >30 >15 >15 >15Notes Slow decay No decay No decay No decay

Figure 2. Job plots of the production of Cu(I) (see Materialsand Methods) were obtained for 20 mM (*) of 10 mM (*) 7,8-dihydroxy¯avone. All points represent triplicate samples andmean values are plotted.

Figure 3. Plasmid pBR322 was incubated for 2 h with 0.2 mM

Cu(II) and 0.1 mM ¯avonoid; the products were fractionatedby agarose gel electrophoresis and stained with ethidiumbromide. From the left the tracks on the gel are (1) controlplasmid and treatment with (2) quercetin, (3) kaempferol, (4)chrysin, (5) 7,8-dihydroxy¯avone, (6) 7-hydroxy¯avone and(7) genistein. The fast running band is supercoiled DNA; thetop band (seen faintly in the control) is open circular DNA andthe band just ahead of that (visible only in the quercetin track)is linear DNA. We regard as positive the tracks in which themajority of the DNA has been converted to the open circularform. Minor quantitative differences in tracks 1,4,6 and 7could re¯ect differences in loading and are thereforeconsidered negative.

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DISCUSSION

Our study provides an opportunity of reviewing theinfluenceof the patternsof hydroxyl substitutionin aflavonoidfor themolecule’scapacityto degradeDNA inthepresenceof Cu(II). Thispapercontainsthefirst reportthataflavonoidthatcanonly form anorthostructure(7,8-dihydroxyflavone)is activein DNA strandcleavage;thepreviousexampleof suchagroupV compound(Table1)was (ÿ) epicatechinwhich is not a true flavonoid andcouldthereforebearguedasapoorexample.Weattempthere to generalize about substituentsand the DNAreactivity of the compounds.Although the study ischemicallyincompletebecauseof thelack of availabilityor accessibilityto synthesisof sometestcompounds,thefollowing generalizations(i–iv) would accountfor thedatapresented(Table1).

(i) All threeformsof complexes,3–4,4–5andorthocangeneratespeciesthat attackDNA, the 3–4 complexis moreactive than the other two which we do notrank.

(ii) The presenceof ortho dihydroxy groupsenhancesthe activity of a compoundwith the 3–4 configura-tion. We takeit to beaxiomaticthat a 3–4complexcannotalsoform a 4–5complexfor stereoelectronicreasons.

(iii) The effectsof 4–5 and ortho configurationsin thesamemoleculearenot additive.

(iv) Thepresenceof additionalOH groupsin a flavonoidenhancesthe reactivity.

If we acknowledgethatrutin is anomalousbecauseof itslikely conversion to quercetin, comparison of 7,8-

dihydroxyflavone(group V), chrysin (group IV) andgalangin(groupII) support(i). Comparisonof quercetinandgalangin(groupI andII) supports(ii). Generalization(iii) is supportedby comparisonof apigenin(groupIV)and luteolin (group III). Apigenin is an interestingexamplein severalrespects:its considerablereactivitytowards DNA confirms that the 4–5 complex can beactiveandthelower reactivityof luteolinmightbeduetothe preferential sequestrationof Cu(II) by the orthoconfiguration.It is thuspossiblethatCu(II) hasa higheraffinity for thelessreactiveorthostructure.Theenhancedreactivity of additionalOH groups(iv) is evidencedbymyricetin and quercetin (group I) and chrysin andapigenin(groupIV).

We thereforeproposethat there are three differentcomplexes (3–5, 4–5 and ortho) whereby a metal–flavonoid complex can fragmentDNA. The formationof a charge-transfercomplexis not involved in at leastone of thesemechanisms.We are unableat presenttocommenton whetherour generalization(iv) is due tonon-specific effects due to (for example) a greaterinstability of an intermediateor whether the marginalactivity of the simple phenolic flavonoid, 7-hydroxy-flavone(groupVI) is a genuineactivity. Thefact thatwedid not detectCu(I) formation in this casesupportstheformer hypothesis.

Acknowledgements

This work was supportedby the UGC, CSIR (Grant 37 10982/98/EMR-II) and the British Council (India) and by the EC DG XII(Brussels).

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