Review Article Heterocyclic Aromatic Amines - downloads

Hindawi Publishing CorporationISRN BiophysicsVolume 2013 Article ID 740821 11 pageshttpdxdoiorg1011552013740821

Review ArticleHeterocyclic Aromatic Amines Heterocomplexation withBiologically Active Aromatic Compounds and Its PossibleRole in Chemoprevention

Anna Woziwodzka Grzegorz GoBuNski and Jacek Piosik

Laboratory of Biophysics Intercollegiate Faculty of Biotechnology UG-MUG Kładki 24 80-822 Gdansk Poland

Correspondence should be addressed to Jacek Piosik piosikbiotechuggdapl

Received 29 January 2013 Accepted 24 March 2013

Academic Editors L Loura M P Ponomarenko M Ruiz-Gomez and S Taneva

Copyright copy 2013 Anna Woziwodzka et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Food-borne heterocyclic aromatic amines (HCAs) are known mutagens and carcinogens present especially in Western populationdiet which contains large amount of meat and its products HCAs are capable of interacting with DNA directly through theformation of covalent adducts however this process requires biological activation in liver mainly by cytochrome P450 enzymesThis process may produce mutations and in consequence may contribute to the development of cancer However there are manystudies showing that several biologically active aromatic compounds (BACs) may protect against genotoxic effects of HCAs Directinteractions and noncovalent heterocomplexes formation may be one of the most important mechanisms of such protection Thiswork describes several BACs present in human diet which are capable of molecular complexes formation with HCAs and protectcells as well as whole organisms against HCAs action

1 Introduction

Several human mutagens and carcinogens belong to aro-matics Polycyclic hydrocarbons (PAHs) aflatoxins ethidiumbromide acridine dyes such as ICR170 and ICR191 anticancerdrugs daunomycin doxorubicin and mitoxantrone andfood-derived heterocyclic aromatic amines (HCAs) can serveas examples Among them vast attention is paid to HCAswhich might be responsible for the increase in gastroin-testinal tract cancers observed particularly in Western dietpopulations with high meat intake (Figure 1)

The mutagenic activity of thermally processed meatwas firstly observed in 1939 [1] and subsequently in thelate 1970s [2] These observations led to the isolation andcharacterization of a new class of potent food-borne muta-genscarcinogens heterocyclic aromatic amines (HCAs) [3]Basing on their structures HCAs can be divided into twoclasses [4] 2-Amino-3-methylimidazo[45-f ]quinoline (IQ-)type HCAs are produced during heat processing of crea-tinecreatinine sugars and free amino acidsmixtures in tem-peratures below 300∘C in the Maillard reaction The secondclass non-IQ-typeHCAs are formed at higher temperatures

above 300∘C by pyrolysis of amino acids and proteins AllHCAs have at least one aromatic and one heterocyclic ringin their structure

HCAs are potent mutagens in the Salmonellareversionassay as well as proven carcinogens in laboratory ani-mals producing multiorgan tumors for example in thecolon mammary glands and prostate [5] Moreover a closerelationship between consumption of thermally processedprotein-rich food (mainly meat and fish) and cancer occur-rence has been observed in many studies [6ndash9] indicating asignificant role of HCAs in human carcinogenesis

It is believed that HCAs exhibit mutageniccarcinogenicactivity only after their metabolic activation involving phaseI N-hydroxylation by different isoenzymes of cytochromeP450 (mainly CYP1A2) and phase II esterification of theexocyclic amino group Products of both phase I and IIactivation can be spontaneously converted to arylnitreniumions (R-NH+) capable of forming covalent adducts withDNA [10]

HCAs are considered as important factors in a lifestyle-connected carcinogenesis according to IARC classified as

2 ISRN Biophysics

N

NN

N

NN

N

NN

N N

N

NH

NN

N

N

IQ MeIQ MeIQx

PhIP Trp-P-2 Glu-P-1

CH3

NH2NH2

CH3

CH3

H3C

NH2

CH3

NH2

CH3

NH2

CH3

CH3

NH2

Figure 1 Examples of heterocyclic aromatic amines (HCAs) chemical structures IQ 2-amino-3-methylimidazo[45-f ]quinolineMeIQ 2-amino-34-dimethylomidazo[45-f ]quinoline MeIQx 2-amino-38-dimethylimidazo[45-f ]quinoxaline PhIP 2-amino-1-methyl-6-phenylimidazo[45-b]pyridine Trp-P-2 3-amino-1-methyl-5H-pyrido[43-b]indole Glu-P-1 2-amino-6-methyldipyrido[12-a 3101584021015840-d]imidazole

2AB carcinogens [11 12] therefore being extensively inves-tigated worldwide with a particular interest in findingmethods to diminish their genotoxic potential As a resultnumerous studies concerning protective effects of severaldietary components against HCAs mutagenic and carcino-genic activity have been performed It is estimated thatapproximately 600 different individual compounds as well ascomplex mixtures have been described as antimutagenic oranticarcinogenic towards HCAs representing diverse modesof action [13] Proposed mechanisms of protection involveinhibition of mutagen formation inhibition of HCAs acti-vating enzymes induction of HCAs detoxifying enzymeselectrophile-scavenging enhanced DNA repair alteration ofsignaling pathways involved in apoptosis and proliferationand direct binding of HCAs and their metabolites by inter-ceptor molecules [14]

In the studies concerning the molecular mechanisms ofchemoprevention against HCAs activity observed for severalfood components the main focus is placed on the interactionof these compounds with different metabolic pathways ofHCAs with protective effects resulting from reduction inHCAs bioactivation andor enhancement of their detoxi-cation Nevertheless taking into consideration that manynatural protective compounds have aromatic moieties intheir structure it seems possible that noncovalent directinteractions especially stacking (120587-120587) complexes formationbetween protective molecules and HCAs can at least in partcontribute to the observed chemopreventive effects Suchsequestration of mutagenmolecules in heterocomplexes withbiologically active aromatic compounds (BACs) can reducetheir bioavailability and in consequence effectively preventfrom their genotoxic effects (Figure 2)

This paper summarizes the current state of knowledgeon the importance of heterocomplexation in diminishingvarious biological effects of HCAs

2 Mechanism of Heterocomplexation

Mechanism of small aromatic molecules protective actionagainst HCAs activity is often attributed to their ability toformheterocomplexes between these compounds Formationof such complexes is dependent on hydrophobic interactionsbetween 120587-120587 electron systems from overlapping aromaticrings present in both molecules The ability of stackingheterocomplexes formation can be described by severalbiophysical parameters such as enthalpy changes (ΔH) orassociation constants (eg neighborhood association con-stant 119870AC) For example calculated values of neighborhoodassociation constants (119870AC) for HCAs-theophylline complexformation are greater than 119870AC values of HCAs-caffeine andHCAs-pentoxifylline heterocomplexes [15] Andrejuk et alreported similar relationship between association constantvalues of heterocomplexes formation for several aromaticcompounds and theophylline and other methylxanthines[16] This difference was interpreted as a consequence ofa 7-Me group absence in theophylline molecules whichis present in caffeine and can introduce steric hindrancereducing heterocomplexation possibility [16] Possibility ofhigh-order complex formation can be considered as anotherreason for differences between association constants val-ues of stacking complexes formation Formation of suchhigh-order complexes was proposed previously for theinteraction between caffeine (CAF) and anticancer drugmitoxantrone (MIT) [17] The authors suggested that theformation of MIT-(CAF)

2complex is more probable and

more thermodynamically favorable than 1 1 heterocomplexas concluded from lower values of enthalpy changes for2 1 heterocomplexes [17] Apart from stacking complexesformation direct binding of mutagen to inhibitor moleculescan involve electrostatic and H-bond interactions whichcan act independently or additionally to heterocomplexes

ISRN Biophysics 3

Cu

N

N N

N

O

ONa

ONa

ONa

O

O

N

NN

N

O

O

O

O

OOH

OH

OH

OH

OH

OH

OH

HO

O

OOH

HO

OHO

O

HO

OH

O

CHO

CHL EGCG

CAF Apigenin Xanthomonasin A

CH3

CH3

CH3

H3C

H3C

H2C

CH3

C5H11

H3C

CH3

CH3

Figure 2 Examples of biologically active aromatic compounds (BACs) chemical structures CHL chlorophyllin EGCG (minus)-epigallocatechingallate CAF caffeine

formation stabilizingpromoting120587-120587 interactions by facilita-tion of two molecules docking Semenov et al suggested thatstacking heterocomplexes may be stabilized by intermolec-ular hydrogen bonds formation which were demonstratedusing vibrational spectroscopy for flavin-mononucleotideinteractions with ethidium bromide and proflavine [18] Allabove-mentioned factors may influence the ability of severalaromatic compounds to form stacking heterocomplexes withHCAs

There are two possible mechanisms describing how for-mation of stacking heterocomplexes between small aromaticmolecules can modulate their biological activity First of allformation of heterocomplexes between HCAs and severalaromatic compounds can decrease concentration of freebiologically active form of HCAs thus diminishing theirbiological activityThis mechanism of protection is known asldquointerceptorrdquomode of CAF and othermethylxanthines actionand was proposed by Bedner et al [19] The ldquointerceptorrdquomode of CAF was described and analyzed for HCAs [15] andseveral other aromatic compounds ethidium bromide [2021] propidium iodide [19 20] quinacrine mustard ICR170ICR191 [22] proflavine [23] and several anticancer drugs [1724 25] Similarmechanismwas also described for interactionof chlorophyllin (CHL) [26] and other natural constituentsof human diet [27] with several aromatic compounds Itwas demonstrated that CHL forms stacking heterocomplexeswith HCAs and in consequence diminishes their mutagenicactivity and reduces their absorption from gastrointestinaltract [28 29] The second proposed mechanism of CAF and

other aromatic compounds protective action is defined asldquoprotectorrdquo mode of action [19] According to this mech-anism CAF may intercalate to DNA and compete withmutagen for free binding sites [21 23] However ldquoprotectorrdquomode of CAF or other aromatic compounds towards HCAsseems unlikely because biologically activated HCAs formcovalent adducts rather than intercalate to DNA [30 31]

3 Role of Chlorophyllin (CHL)and Other Porphyrins

Chlorophyllin (CHL) is a water-soluble salt of chlorophyllcontaining a chelated copper or sodium ion CHL is undoubt-edly one of the best-studied interceptor molecules which caneffectively reduce the activity of several aromatic mutagensand carcinogens such as PAHs aflatoxin B

1(AFB1) and

HCAs [32] Its protective activity is often attributed toits ability to form stacking heterocomplexes with planararomatic mutagens which can diminish their bioavailabilityand therefore protect against their biological effects

31 Direct Interactions between HCAs and CHL UV-visspectroscopy studies demonstrated that CHL forms molec-ular complexes with IQ with 2 1 stoichiometry and dis-sociation constant 119870

119878= 0141 plusmn 0028mM determined

from the Benesi-Hildebrand plot [33] Moreover CHL wasproven to inhibit mutagenic activity of a broad range ofIQ-type and non-IQ-type HCAs in short-term genotoxicity

4 ISRN Biophysics

assays [29] Direct interactions between CHL andHCAswereexamined spectrophotometrically with binding constants119870119887determined from Benesi-Hildebrand plot ranging from

307sdot103Mminus1 to 1274sdot103Mminus1 and 2 1 and 1 1 stoichiometriesof CHL-mutagen complexes formation for IQ-type and non-IQ-type HCAs respectively Observed protective effects ofCHL against HCAs mutagenic activity were markedly cor-related with strength of mutagen-CHL complex formationreflected by appropriate binding constant values These find-ings support the hypothesis that interception of aromaticmutagen molecules in noncovalent molecular complexeswith CHL can be considered as an effective mechanismfor CHL protective action especially for the systems whereinterceptor molecules can reach concentrations significantlyhigher than possiblemutagen concentrations Further studiesemploying molecular modeling analyses of IQ- and non-IQ-type HCA interactions with CHL and its structurallyrelated porphyrin derivatives revealed that two factors con-tribute to the molecular complex formation [34] Firstly anexocyclic amine group present in HCA can interact withacid groups on CHL and other porphyrins Replacement ofan amine group with a nitro group in NO

2IQ resulted in

its very weak interaction with CHL (binding constant 119870119887

estimated at lt 05sdot103Mminus1) and was accompanied with alack of antimutagenic activity of CHL towards nitro-IQ [29]Secondly stacking (120587-120587) interactions between overlappingaromatic rings is crucial for stable interactions betweenHCAsand porphyrin derivatives The presence of methyl groups inchlorophyll derivatives which can possibly introduce sterichindrance and thus interfere with 120587-120587 interactions wasshown to increase the minimal energies of the interactions[34] At the same time the authors noticed that many of theinterceptor molecules apart from heterocomplexation witharomatic mutagens are likely to interact with each otherforming dimers or longer homoaggregates This effect cancomplicate the quantitative analysis of the experimental sys-tem as the fraction of the freebound interceptor cannot bedirectly associated with the amount of added mutagen Thisproblem can be overcome by using thermodynamical modelsof mixed aggregation which allow for infinite homo- andheterocomplexation between interacting aromatic moleculesand enable one not only to calculate binding constantsthat is neighborhood association constants but also todetermine concentrations of each possible component ofevery analyzed mutagen-interceptor mixture Such approachwas successfully applied for interaction analyses of HCAs [15]and other aromatic molecules [22 35 36] with caffeine andother methylxanthines and will be discussed in detail in thenext section of this paper

32 Interception and Other Mechanisms of CHL ActionAlthough vast majority of studies concerning CHL as achemoprotective agent indicate that CHL acts as an ldquointer-ceptorrdquo molecule forming heterocomplexes with severalplanar mutagens there are some papers describing dif-ferent modes of CHL antimutagenic and anticarcinogenicactivity Yun et al [37] suggested that CHL can inactivatecytochrome P450 (CYP) involved in the bioactivation of

many environmental and food-borne mutagens whereasTachino et al [38] pointed out antioxidant and free rad-ical scavenging effects of CHL and increased mutagendegradation rates in the presence of CHL These alterna-tive mechanisms of CHL action can explain its protectiveeffects towards nonaromatic compounds such as alkylatingagents methyl methanesulfonate (MMS) methylnitrosourea(MNU) ethylnitrosourea (ENU) [39 40] or radiation-induced DNA damage [41 42] Moreover influence of CHLon phase II hepatic enzymes was proposed as a mode ofCHL protective action against low doses of PhIP in rats[43]

On the other hand CHL was demonstrated to protectagainst mutagenic activity of direct-acting N-hydroxy HCAsderivatives Trp-P-2[NHOH] [44] N-OH-IQ [45] and otherdirect-acting aromatic compounds benzo[a]pyrene-78-diol-910-epoxide (BPDE) [46] and aflatoxin B

1-89-epoxide [47]

In contrary no inhibitory effect of CHL was observedagainst small nonplanar alkylating agents N1015840-methyl-N1015840-nitrosourea (MNU) and ethyl methanesulfonate (EMS) [46]These findings strongly suggest that interception of aromaticmutagens in molecular complexes by CHL rather thanmodi-fication of xenobiotic metabolizing enzymes accounts for theobserved antimutagenic effects of CHL

In vitro protective effects of CHL towards DNA weredemonstrated in studies conducted by Pietrzak et al [26]Light absorption and fluorescence spectroscopy analysesof mutagen-CHL-DNA three-component mixtures revealedthat CHL induced a dose-dependent decrease in themutagencomplexed with DNA concentration Mutagens used in thestudy acridine orange quinacrinemustard and doxorubicinintercalate to DNA rather than form covalent adducts whichis typical of HCAs Nevertheless these in vitro findings sup-port the hypothesis that direct interactions between aromaticmutagen and CHLmolecules can influence its affinity to bindto DNA irrespectively of any other complex mechanisms ofaction which cannot be completely excluded when livingorganisms are used as experimental models

33 Adsorption of HCAs on Insoluble Carriers ContainingCHL The ldquointerceptorrdquo mode of CHL chemopreventiveaction was further investigated in studies involving adsorp-tion of aromatic mutagens on CHL bound to insoluble carri-ers Sepharose [48] and chitosan [49] It was demonstratedthat a broad range of polycyclic planar mutagens includ-ing non-IQ-type HCAs Trp-P-1 and Trp-P-2 are stronglyadsorbed on CHL covalently linked to Sepharose [48]Additionally the positive relationship between the strengthof adsorption of particular mutagens on CHL-Sepharose andthe efficacy of CHL in inhibiting their mutagenic activitywas revealed [48] The studies with CHL fixed on chitosandemonstrated that planar molecules having three or morefused rings (including IQ MeIQ MeIQx Glu-P-1 and Trp-P-2) efficiently adsorb on CHL-chitosan whereas for two-ring compounds tested only those with significant planarsurfaces such as PhIP noncovalently bind to CHL-chitosan[49] Overall the adsorption of aromatics to CHL-chitosanwas much stronger than to CHL-Sepharose and in contrast

ISRN Biophysics 5

to CHL-Sepharose mutagens adsorbed to CHL-chitosan aredifficult to elute even at very low pHs The adsorption ofHCAs on CHL-chitosan was proven to be relevant for invivo protective effects of CHL-chitosan coadministered witharomatic carcinogens The amount of Trp-P-2-DNA adductsin liver and other organs was significantly decreased in amice fed with Trp-P-2 and CHL-chitosan [50 51] which wasattributed to diminished absorption of CHL-chitosan boundmutagen from the gastrointestinal tract CHL-chitosan wasproven to be beneficial in humans promoting the excretionof dioxins in feces and sebum [52]

34 CHL Influence on Carcinogen Bioavailability Studiesusing Caco-2 monolayer transport model have shown thatCHL reduces in vitro carcinogen uptake from the intestine[53] In these studies the influence of CHL on absorptionrate of dibenzo[al]pyrene (DBP) AFB1 and PhIP throughintestinal epithelial cells was monitored CHL was shown toinhibit the transport of aromatic mutagens being the mosteffective towards DBP for which significant inhibition wasobserved for 1 10 ratio of mutagen CHL For AFB1 1 100ratio of mutagen CHL was sufficient to reduce transportby almost 50 whereas 1 1000 ratio of mutagen CHL wasneeded to inhibit PhIP absorption by 35 These differencesin CHL efficiency to inhibit mutagen transport throughCaco-2 monolayer can be explained by different affinities ofCHL for analyzed mutagens reflected by binding constantsof mutagen-CHL interactions the highest for BDP-CHLintermediate for AFB1-CHL and according to Dashwoodet al [34] much lower for PhIP-CHL These findings areconcordant with the report of Dashwood and Guo [33]who suggested basing on CHL-mutagen binding constantsthat interceptor CHL action can be effective only whenCHL concentrations are much higher than mutagen con-centrations for example IQ CHL weight ratio of 1 27 000ensures complete sequestration of IQ in complexeswithCHLNevertheless taking into consideration relatively high CHLdoses and low dietary exposure to most carcinogens it seemspossible that high CHL carcinogen ratio could be easilyachieved in the gastrointestinal tract Hence CHL mightact as a first line of defense forming noncovalent hetero-complexes with aromatic food-borne mutagenscarcinogensand lowering their absorption from the gastrointestinal tractEarlier in vivo studies with rat intestinal loop absorptionmodel gave similar results CHL administration to isolatedintestinal loops diminished the bioavailability and absorptionof IQ [28]

35 Animal Studies on CHL Protection towards HCAs andOther Dietary Carcinogens The antigenotoxic potential ofCHL against HCAs was observed in numerous studies withlaboratory animals Cotreatment of F344 rats with PhIPand CHL reduced mammary gland cancer incidences [54]Similar protection against PhIP-induced mammary carcino-genesis in F344 rats by CHL was observed by Hirose etal [55] Studies on the CHLrsquos influence on the carcinogenicactivity of IQ in rats revealed that CHL protected againstcolon liver and small intestine cancers [56] Furthermore

CHL was proven to protect against PhIP-induced aberrantcrypt foci (ACF) in F344 rats decreasing the uptake of amutagen from the gastrointestinal tract as concluded fromaugmented elimination of unmetabolized PhIP in feces inthe presence of CHL [57] CHL inhibited IQ-induced DNAadduct formation in rat liver by interacting with mutagendirectly and thus reducing its uptake from the intestine [28]In addition it was demonstrated that cotreatment of rats withIQ and CHL resulted in increased urinary and fecal excretionof unmetabolized mutagen confirming that CHL can act asinterceptor molecule interacting with planar mutagens anddecreasing their bioavailability in the gastrointestinal tractand tissues [58] Studies on Drosophila larvae revealed thatCHL significantly inhibited MeIQx-DNA adduct formationIt was concluded that large doses of CHL can interact withand effectively intercept MeIQx in feed as well as in thegastrointestinal tract of larvae inhibiting its bioavailabilityand absorption [59] Similar CHL protective effects werealso observed towards other aromatic mutagens Chlorophyllinhibited absorption of dioxins from the gastrointestinaltract [60] Direct interactions between chlorophyll CHLand dibenzo[al]pyrene (DBP) contributed to the reduceduptake of the mutagen to the liver thus diminishing stomachand liver cancer incidents in rainbow trout [61] Chemo-preventive properties of natural chlorophyll and CHL werealso described for another aromatic mutagencarcinogen-AFB1 and AFB1-induced carcinogenesis in rats [62] Theprotection resulted from decreased mutagen uptake fromthe intestines which was concluded from the increasedexcretion of AFB1 in feces of CHL- and chlorophyll-fedanimals Moreover molecular complexes formation betweentwo anticarcinogens and AFB1 were confirmed by fluores-cence quenching experiments with 1 1 stoichiometries anddissociation constants 119870

119889= 122 120583M and 305 120583M for AFB1-

chlorophyll and AFB1-CHL interactions respectively [62]CHL administered together with AFB1 in the diet inhibitedAFB1-DNA binding in trout Interestingly lower protectiveeffects were observed when AFB1 was given to animalsby intraperitoneal injection thus significantly reducing thepossibility of direct interactions between CHL and mutagenin the intestines [47] Similar observations weremade by Guoand Dashwood [63] They demonstrated that simultaneousadministration of IQ and CHL to rats is crucial to exert themost effective inhibition of IQ-DNA binding These findingsserve as additional evidence for interceptor mode of CHLprotective action

36 Chemoprotective Effects of CHL in Human StudiesCHL was proven to act as HCAs protective agent in humanstudies It was demonstrated that CHL tablets administered tohumans exert inhibitory effects againstDNAdamage inducedby HCA-containing fried meat in target colorectal cells [64]Clinical chemoprevention trial on a human population withhigh AFB1 exposure revealed that 100mg of dietary CHLthree times per day significantly reduces the level of aflatoxin-N7-guanine hepatic DNA adduct biomarker in urine [65]This study clearly demonstrated that CHL can be an effectiveagent suitable for use in reducing human cancer risk from

6 ISRN Biophysics

dietary AFB1 Detailed pharmacokinetics studies on humanvolunteers treated withmicrodoses (30 ng) of AFB1 indicatedthat chlorophyll as well as CHL administration significantlyinfluenced various pharmacokinetic parameters and reducedsystemic AFB1 absorption [66] These findings directly sug-gest that CHL and chlorophyll exert protective effects inhumans by decreasing aromatic mutagens bioavailability

4 Role of Caffeine and Other Methylxanthines

Caffeine (CAF) a component of several popular beverages(coffee tea and energy drinks) is the most widely consumedalkaloid worldwide Statistical data from the USA revealedthat the average daily intake of CAF can reach 193mgdayper person [67] Many epidemiological studies suggest thatcaffeine can reduce the risk of cancer [68ndash71] Since theinfluence of caffeine on human body is exceedingly complexits overall mechanism of action as a chemopreventive agentis still not fully understood It is commonly known thatCAF can modulate the metabolism of xenobiotics mainly bythe induction of cytochrome P450 enzymes Therefore it isbelieved that CAF may exert its protective action throughthe enhancement of detoxication pathways of numerouscarcinogenic compounds On the other hand CAF is inten-sively metabolized through cytochrome P450 isoenzyme 1A2(CYP1A2) [72] Thus competitive inhibition of CYP1A2can be mentioned as one of the possible mechanisms ofCAF protective action towards several procarcinogens forexample HCAs which are biologically activated by CYP1A2

41 Inhibition of HCAs Activity by CAF It was demonstratedthat caffeine-rich beverages such as different types of tea [73ndash76] as well as coffee [77] can inhibit HCAs biological activityMoreover decaffeinated tea was shown to be less effective inreducing PhIP mutagenic activity than regular tea [75]

CAF was proven to attenuate mutagenic effects of MeIQTrp-P-2 MeIQx [78 79] and PhIP [75] This protectiveCAF potential was attributed to the inhibition of microsomalenzymes (eg CYP1A2) involved in HCAs bioactivationExperiments with laboratory animals revealed that admin-istration of CAF-containing beverages not only protectedagainst IQ-induced genotoxicity but also caused about 20ndash30 induction of CYP1A2 and CYP1A1 isoenzymes [76]Thelatter should augment rather than diminish HCAs genotoxi-city by increased HCAs bioactivation ratio to highly reactivearylnitrenium intermediates suggesting that mechanismsother than themodulation of phase I enzymes can be involvedin observed protective action The inhibitory effects of teaextracts towards promutagens (IQ Glu-P-1) as well as direct-acting mutagens (9-aminoacridine and N-methyl-N-nitro-N-nitrosoguanidine) further support the hypothesis thatprotective effects of CAF cannot be fully explained by itsinfluence on xenobiotic activation system [80] The presenceof mechanisms additional to the inhibition of cytochromeP450 enzymatic activity was also suggested by Chen andYen [73] Furthermore Weisburger et al specified alternativemechanisms of CAF action against HCAs among which the

possibility of direct interactions between planar CAF andHCAs molecules was proposed [75]

42 Molecular Complexes Formation between HCAs CAFand Other Methylxanthines The idea of heterocomplexationbetween CAF and HCAs was further investigated for IQ-type HCAs and CAF as well as other methylxanthines(MTX) pentoxifylline (PTX) and theophylline (TH) [15]In order to examine direct interactions between IQ-typeHCAs and MTX light absorption spectra of mutagenstitrated with MTX were measured and analyzed for eachHCA-MTX pair Distinctive spectral changes bathochromicand hypochromic shift were observed indicating stacking(120587-120587) complexes formation between analyzed compoundsProvided CAF and other MTX form homoaggregates inaqueous solution the determination of association constantfor mutagen-MTX interaction 119870

119887from Benesi-Hildebrand

plot is not a very accurate approach MTX homocomplexa-tion can be significant especially for high (milimolar range)MTX concentrations hence freeboundMTX ratio cannot bedirectly related to the amount of added mutagen Thereforestatistical-thermodynamical model of mixed aggregation[81] which assumes infinite self-aggregation of one of theinteracting ligands was applied in this study in order tocalculate neighborhood association constants 119870AC of HCA-MTX interactions The obtained 119870AC values of HCA-MTXinteractions were within 102Mminus1 range Moreover the modelwas used to estimate the concentrations of each possiblecomponent of every analyzed HCA-MTX mixture (ie con-centration of MTX monomers and MTX homoaggregatesand concentration of HCA monomers and HCA-MTX com-plexes) It was demonstrated that 100 1 ratio of MTX HCAwas enough to sequester more than 80 mutagen moleculesin complexes with MTX [15] Thus CAF and other MTXseem to be much more effective in intercepting HCAs thanCHL for which 27 000 1 CHL mutagen ratio was proposedfor efficient HCA binding [33] IQ-MTX binding efficiencydetermined in vitro was confirmed in mutagenicity studies[15] Ames test experiments revealed that CAF PTX and THcaused a significant dose-dependent reduction of IQ muta-genic activity A strong linear correlation between mutagenicactivity of IQ-MTX mixtures and free form of IQ suggeststhat CAF and other MTX antimutagenic activities can beexplained by sequestration of HCAs molecules in hetero-complexes with MTX Moreover these findings indicate thatthermodynamical models can be a useful tool to estimate thebiological effects of biologically-active aromatic compoundsheterocomplexation and to predict to what extent interceptorpotential may contribute to overall protective action ofseveral aromatic compounds

Hernaez et al reported that CAF forms molecularcomplexes with direct-acting IQ derivative N-OH-IQ withassociation constant 119870

119887lt 05sdot103Mminus1 determined by spec-

trophotometric titration experiments andBenesi-Hildebrandplot [45] However in contrast to the above-mentionedstudies [15] no protective effect of CAF against mutagenicactivity of N-OH-IQ was observed up to 250 nmole CAF perplate

ISRN Biophysics 7

43 Heterocomplexation of MTX with Other Aromatic Lig-ands Although there are only few reports describing studieson interception role of CAF and other MTX towards HCAsheterocomplexation ofMTXwith other aromatic compoundshas been broadly investigated It has been reported previ-ously that CAF and other MTX form stacking aggregateswith aromatic dyes acridine orange [82ndash84] ethidium bro-mide [20 81 85] 410158406-diamidino-2-phenylindole (DAPI)[81] methylene blue [86] anticancer drugs doxorubicinmitoxantrone and daunomycin [17 24 81 87] antibacte-rial agent norfloxacin [88] vitamin B

2derivative flavin-

mononucleotide [87] neurotoxins [36 89] and aromaticmutagens [22 35] Some of these reports reveal in vivo effectsof observed heterocomplexation indicating that molecularcomplex formation between aromatic compounds can berelevant in biological systems [22 35 36 89]

It was demonstrated that CAF and otherMTX can inhibitDNA binding or even induce deintercalation of aromaticligands from DNA [20 21 84 88] It is still discussedwhether this effect results barely from the decrease in ligandavailability due to its interception by MTX or can be alsocontributed to the reduction of binding sites on DNA avail-able for ligands caused by MTX intercalation Neverthelessthese findings clearly demonstrate that direct interactionsbetween aromatic compounds can significantly modify theirbiological properties

5 Other BACs

51 Role of Polyphenols Polyphenols are naturally occurringaromatic compounds containing at least two phenolic ringsThey are extensively studied for their antimicrobial anticar-cinogenic and antiinflammatory properties [90] In the fieldof chemoprevention they show potential to both scavengereactive oxygen species and form heterocomplexes with aro-matic mutagens [91 92] The latter mechanism as describedbefore reduces mutagenic potential of aromatic mutagensby formation of molecular complexes with aromatic muta-gens thus reducing their bioavailability Such effects weredescribed for one of the model mutagens acridine orangeand resveratrol by Osowski et al [27]

Numerous studies concerning chemopreventive mech-anisms of polyphenolic compounds against heterocyclicaromatic amines were conducted Research carried out byHernaez et al reveals thatN-OH-IQ (direct-acting IQderiva-tive) binds to (minus)-epigallocatechin gallate (EGCG) (minus)-epigallocatechin (EGC) (+)-catechin and (minus)-epicatechingallate (ECG) directly The binding constants determinedfrom Benesi-Hildebrand plot for these polyphenolic com-pounds are in the range of 1sdot103Mminus1 [45] Neverthelessauthors of this work suggest that determined interactionconstants are too low to contribute to observed inhibitionof HCAs mutagenic activity [45] However two works ofDashwood and Guo demonstrate IQ-CHL interaction con-stant determined from Benesi-Hildebrand plot in the rangeof 7sdot103Mminus1 Nonetheless authors consider the interactionsprobable and important CHL chemopreventive mechanismespecially for high CHL IQ ratios (the ratio of CHL IQ

required to bind 999 is greater than 27 000 1) [29 33]Results for IQ-MTX interactions published by Woziwodzkaet al demonstrate that MTX reduce mutagenic activity ofHCAs despite mixed association constants in the range of1sdot102Mminus1 [15] Considering these data it can be speculatedthat binding constants in the range of 1sdot103Mminus1 indicatethe mechanism of HCAs sequestration by polyphenoliccompounds as plausible and suggest its role in chemopre-ventive action of these compounds against HCAs apart fromactivation of phase II enzymes and free radicals scaveng-ing

Additionally Artemisia argyi analysis performed byNakasugi et al revealed that flavones reduce mutagenicactivity of Trp-P-2 [93] Comparison of Ames mutagenicitytest results for Trp-P-2 and its active form Trp-P-2[NHOH]indicate that apart from other mechanisms also hetero-complexes formation should be considered as importantchemopreventive mechanism of different flavones namelyeupatilin jaceosidin apigenin and chrysoeriol Presentedresults demonstrate that mechanism of heterocomplexesformation is responsible for up to 30 of overall reductionof HCAs mutagenic activity in the Ames test Moreover pre-sented data demonstrate Trp-P-2 fluorescence quenching ina dose-dependent manner by mentioned flavones indicatingheterocomplexes formation between Trp-P-2 and flavones[93]

Furthermore Arimoto-Kobayashi et al findings suggestthat polyphenolic compounds present in beer can act asHCAs interceptors [94] Beer and other phenol-containingbeverages (ie red and white wine sake and brandy)exhibited strong antimutagenic effects against Trp-P-2 andTrp-P-2[NHOH] in the Ames test The protective effect ofbeer polyphenols was observed only when mutagen wasadministered simultaneously with beer Neither pre- norposttreatment with beer caused any protective effects towardsbacteria in the Ames test [94] In contrast bacteria pretreat-ment with wine polyphenolic compounds (eg ellagic acid)resulted in decrease in Trp-P-2[NHOH] mutagenic activityprobably due to their antioxidative properties [95]

52 Role of Plant Dyes Plant dyes of aromatic structure thatcannot be classified as porphyrins or phenolic compoundsalso show promising chemopreventive activity Izawa etal analyzed Monacus dyes chemopreventive action againstdirect-acting Trp-P-2 derivative Trp-P-2[NHOH] [96]Theirfindings suggest that aromatic dyes present in Monacusplants especially red and yellow dye significantly reduceHCAs mutagenic activity with observed inhibitory effect forred and yellow dyes reaching 55 What is important is thatspectrophotometric study of Trp-P-2[NHOH] interactionswith yellow dye showed significant spectral changes suggest-ing strong interactions between dye and mutagen Moreoverthe authors suggested that dye-HCAheterocomplexes forma-tion can induce observed degradation of Trp-P-2[NHOH][96] These results indicate that apart from chlorophyllinother natural dyes can be also involved in protection towardsHCAs by reduction ofmutagen bioavailability and enhancingits degradation through heterocomplexes formation

8 ISRN Biophysics

53 Role of Dietary Fiber Several reports describe bindingof HCAs including MeIQx Trp-P-1 Trp-P-2 and Glu-P-1 to the dietary fibers [77 97] Formation of MeIQx-fiber complexes reduces mutagen uptake in rats increasingexcreted amount of mutagen and reducing time of HCAstransition through gastrointestinal tract [98 99] The affinityof such binding is dependent on both fiber and MeIQxconcentrations [97 100] with both environment pH andcompounds hydrophobicity being other factors to consider[100 101] Additionally Ryden and Robertson observed noinfluence of bile salts on MeIQx binding to fiber Thisremark allowed the authors to suggest that planar structureof molecule is required to bind to the fiber [100] These find-ings indicate fiber potential to form heterocomplexes witharomatic mutagens and its possible role in chemoprevention

6 Conclusions

According to the World Health Organization cancer isresponsible for as much as 13 of all deaths being secondmost frequent cause of death worldwide The number ofcancer incidents increases every year and it is expected toexceed 11 million in 2030 It is estimated that up to 40 ofall cancer deaths result from the exposure to behavioral andenvironmental risk factors Diet accounting for even 30 ofcancers in industrialized countries is one of the risk factorswhich can be easily modified Thus the research on the roleof diet in cancer appears to be a matter of great interestThere are many food constituents with carcinogenic or anti-carcinogenic potential Among carcinogens great attentionis paid to HCAs present in thermal processed meat On theother hand it was demonstrated that consumption of severalBACs can protect against several types of cancer Since theinfluence of BACs on human body can be very diverse theirpossible mechanisms of chemoprevention are still discussedOne of the possible explanations of BACs protective actionis their direct noncovalent interactions with HCAs whichmay lead to the sequestration of mutagens blocking theirbioavailability and thus reducing their genotoxic potentialEven though numerous works describing action of dietaryBACs are published there is still a lot to be done to evaluatethe mechanism of such compounds action and their role incancer prevention

Acknowledgment

One of the authors (A Woziwodzka) was financed fromEuropean Social Fund as a part of the project ldquoEducatorsfor the elite-integrated training program for PhD studentspost-docs and professors as academic teachers at the Uni-versity of Gdanskrdquo within the framework of Human CapitalOperational Programme Action 411 improving the qualityof educational offer of tertiary education institutions

References

[1] E M PWidmark ldquoPresence of cancer-producing substances inroasted foodrdquo Nature vol 143 no 3632 p 984 1939

[2] M Nagao M Honda Y Seino T Yahagi and T SugimuraldquoMutagenicities of smoke condensates and the charred surfaceof fish and meatrdquo Cancer Letters vol 2 no 4-5 pp 221ndash2261977

[3] K Wakabayashi M Nagao H Esumi and T Sugimura ldquoFood-derived mutagens and carcinogensrdquo Cancer Research vol 52no 7 pp 2092sndash2098s 1992

[4] H Kataoka ldquoMethods for the determination of mutagenicheterocyclic amines and their applications in environmentalanalysisrdquo Journal of Chromatography A vol 774 no 1-2 pp 121ndash142 1997

[5] T Sugimura K Wakabayashi H Nakagama and M NagaoldquoHeterocyclic amines mutagenscarcinogens produced duringcooking ofmeet and fishrdquoCancer Science vol 95 no 4 pp 290ndash299 2004

[6] Q Dai X O Shu F Jin Y T Gao Z X Ruan and W ZhengldquoConsumption of animal foods cooking methods and risk ofbreast cancerrdquoCancer Epidemiology Biomarkers and Preventionvol 11 no 9 pp 801ndash808 2002

[7] M Gerhardsson De Verdier U Hagman R K Peters GSteineck and E Overvik ldquoMeat cooking methods and colorec-tal cancer a case-referent study in Stockholmrdquo InternationalJournal of Cancer vol 49 no 4 pp 520ndash525 1991

[8] S E Norell A Ahlbom R Erwald et al ldquoDiet and pancreaticcancer a case-control studyrdquoAmerican Journal of Epidemiologyvol 124 no 6 pp 894ndash902 1986

[9] G Steineck U Hagman M Gerhardsson and S E NorellldquoVitamin a supplements fried foods fat and urothelial cancerA case-referent study in Stockholm in 1985-87rdquo InternationalJournal of Cancer vol 45 no 6 pp 1006ndash1011 1990

[10] E G Snyderwine H A J Schut R H Adamson U PThorgeirsson and S SThorgeirsson ldquoMetabolic activation andgenotoxicity of heterocyclic arylaminesrdquo Cancer Research vol52 no 7 pp 2099ndash2102 1992

[11] IARC Some Naturally Occurring and Synthetic Food Compo-nents Furocoumarins andUltraviolet Radiation vol 40 of IARCMonographs on the Evaluation of Carcinogenic Risks to HumansIARC Lyon France 1986

[12] IARC Some Naturally Occuring Substances Food Items andConstituents Heterocyclic Aromatic Amines andMycotoxins vol56 of IARCMonographs on the Evaluation of Carcinogenic Risksto Humans IARC Lyon France 1993

[13] C E Schwab W W Huber W Parzefall et al ldquoSearch forcompounds that inhibit the genotoxic and carcinogenic effectsof heterocyclic aromatic aminesrdquoCritical Reviews in Toxicologyvol 30 no 1 pp 1ndash69 2000

[14] R H Dashwood ldquoModulation of heterocyclic amine-inducedmutagenicity and carcinogenicity an ldquoA-to-Zrdquo guide to chemo-preventive agents promoters and transgenicmodelsrdquoMutationResearch vol 511 no 2 pp 89ndash112 2002

[15] A Woziwodzka A Gwizdek-Wisniewska and J Piosik ldquoCaf-feine pentoxifylline and theophylline form stacking complexeswith IQ-type heterocyclic aromatic aminesrdquo Bioorganic Chem-istry vol 39 no 1 pp 10ndash17 2011

[16] D D Andrejuk A A Hernandez Santiago V V KhomichV K Voronov D B Davies and M P Evstigneev ldquoStructuraland thermodynamic analysis of the hetero-association of theo-phylline with aromatic drug moleculesrdquo Journal of MolecularStructure vol 889 no 1ndash3 pp 229ndash236 2008

[17] J Piosik M Zdunek and J Kapuscinski ldquoThe modulation byxanthines of the DNA-damaging effect of polycyclic aromatic

ISRN Biophysics 9

agents part IIThe stacking complexes of caffeine with doxoru-bicin andmitoxantronerdquoBiochemical Pharmacology vol 63 no4 pp 635ndash646 2002

[18] M A Semenov I Blyzniuk T V Bolbukh A V ShestopalovaM P Evstigneev and V Y Maleev ldquoIntermolecular hydrogenbonds in hetero-complexes of biologically active aromaticmolecules probed by the methods of vibrational spectroscopyrdquoSpectrochimica Acta A vol 95 pp 224ndash229 2012

[19] E Bedner L Du F Traganos and Z Darzynkiewicz ldquoCaffeinedissociates complexes between DNA and intercalating dyesapplication for bleaching fluorochrome-stained cells for theirsubsequent restaining and analysis by laser scanning cytome-tryrdquo Cytometry vol 43 no 1 pp 38ndash45 2001

[20] J Piosik K Wasielewski A Woziwodzka W Sledz and AGwizdek-Wisniewska ldquoDe-intercalation of ethidium bromideand propidium iodine from DNA in the presence of caffeinerdquoCentral European Journal of Biology vol 5 no 1 pp 59ndash66 2010

[21] S F Baranovsky P A Bolotin M P Evstigneev and D NChernyshev ldquoInteraction of ethidium bromide and caffeinewith DNA in aqueous solutionrdquo Journal of Applied Spectroscopyvol 76 no 1 pp 132ndash139 2009

[22] J Piosik K Ulanowska A Gwizdek-Wisniewska A Czyz JKapuscinski and GWegrzyn ldquoAlleviation of mutagenic effectsof polycyclic aromatic agents (quinacrine mustard ICR-191 andICR-170) by caffeine and pentoxifyllinerdquoMutation Research vol530 no 1-2 pp 47ndash57 2003

[23] A S Buchelnikov A A Hernandez Santiago FM Gonzalez RRVazquezD BDavies andMP Evstigneev ldquoGeneral analysisof competitive binding in drug-interceptor-DNA systemsrdquoEuropean Biophysics Journal vol 41 no 3 pp 273ndash283 2012

[24] J Piosik A Gwizdek-Wisniewska KUlanowska J OchocinskiA Czyz and G Wegrzyn ldquoMethylxanthines (caffeine pen-toxifylline and theophylline) decrease the mutagenic effect ofdaunomycin doxorubicin and mitoxantronerdquo Acta BiochimicaPolonica vol 52 no 4 pp 923ndash926 2005

[25] G M Hill D M Moriarity and W N Setzer ldquoAttenuation ofcytotoxic natural productDNA intercalating agents by caffeinerdquoScientia Pharmaceutica vol 79 pp 729ndash747 2011

[26] M Pietrzak ZWieczorek J Wieczorek and Z DarzynkiewiczldquoThe ldquointerceptorrdquo properties of chlorophyllin measured withinthe three-component system intercalator-DNA-chlorophyllinrdquoBiophysical Chemistry vol 123 no 1 pp 11ndash19 2006

[27] A Osowski M Pietrzak Z Wieczorek and J WieczorekldquoNatural compounds in the human diet and their ability tobind mutagens prevents DNA-mutagen intercalationrdquo Journalof Toxicology and Environmental Health A vol 73 no 17-18 pp1141ndash1149 2010

[28] R H Dashwood ldquoProtection by chlorophyllin against thecovalent binding of 2-amino-3-methylimidazo[45-f]quinoline(IQ) to rat liver DNArdquo Carcinogenesis vol 13 no 1 pp 113ndash1181992

[29] R Dashwood and D Guo ldquoAntimutagenic potency of chloro-phyllin in the Salmonella assay and its correlation with bindingconstants of mutagen-inhibitor complexesrdquo Environmental andMolecular Mutagenesis vol 22 no 3 pp 164ndash171 1993

[30] H A J Schut and E G Snyderwine ldquoDNA adducts of hetero-cyclic amine food mutagens implications for mutagenesis andcarcinogenesisrdquoCarcinogenesis vol 20 no 3 pp 353ndash368 1999

[31] R J Turesky and P Vouros ldquoFormation and analysis ofheterocyclic aromatic amine-DNA adducts in vitro and in vivordquoJournal of Chromatography B vol 802 no 1 pp 155ndash166 2004

[32] M D Waters H F Stack M A Jackson H E Brockman andS De Flora ldquoActivity profiles of antimutagens in vitro and invivo datardquoMutation Research vol 350 no 1 pp 109ndash129 1996

[33] R Dashwood and D Guo ldquoInhibition of 2-amino-3-methy-limidazo[45-f]quinoline (IQ)-DNA binding by chlorophyllinstudies of enzyme inhibition and molecular complex forma-tionrdquo Carcinogenesis vol 13 no 7 pp 1121ndash1126 1992

[34] R Dashwood S Yamane and R Larsen ldquoStudy of the forcesof stabilizing complexes between chlorophylls and heterocyclicamine mutagensrdquo Environmental and Molecular Mutagenesisvol 27 no 3 pp 211ndash218 1996

[35] J Kapuscinski B Ardelt J Piosik M Zdunek and ZDarzynkiewicz ldquoThe modulation of the DNA-damaging effectof polycyclic aromatic agents by xanthines part I Reduction ofcytostatic effects of quinacrine mustard by caffeinerdquo Biochemi-cal Pharmacology vol 63 no 4 pp 625ndash634 2002

[36] K Ulanowska J Piosik A Gwizdek-Wisniewska and GWegrzyn ldquoImpaired mutagenic activities of MPDP+ (1-methyl-4-phenyl-23-dihydropyridinium) and MPP+ (1-methyl-4-phenylpyridinium) due to their interactions withmethylxanthinesrdquo Bioorganic and Medicinal Chemistry vol 15no 15 pp 5150ndash5157 2007

[37] C H Yun H G Jeong JW Jhoun and F P Guengerich ldquoNon-specific inhibition of cytochrome P450 activities by chloro-phyllin in human and rat livermicrosomesrdquoCarcinogenesis vol16 no 6 pp 1437ndash1440 1995

[38] N TachinoDGuoWMDashwood S Yamane R Larsen andRDashwood ldquoMechanisms of the in vitro antimutagenic actionof chlorophyllin against benzo[a]pyrene studies of enzymeinhibition molecular complex formation and degradation ofthe ultimate carcinogenrdquoMutation Research vol 308 no 2 pp191ndash203 1994

[39] G C Bez B Q Jordao V E P Vicentini and M S Manto-vani ldquoInvestigation of genotoxic and antigenotoxic activities ofchlorophylls and chlorophyllin in cultured V79 cellsrdquoMutationResearch vol 497 no 1-2 pp 139ndash145 2001

[40] O Olvera C Arceo and S Zimmering ldquoChlorophyllin[CHLN] and the mutagenicity of monofunctional alkylatingagents in Drosophila the action of CHLN need not include aninfluence on metabolic activationrdquoMutation Research vol 467no 2 pp 113ndash117 2000

[41] S S Kumar R C Chaubey T P A Devasagayam K IPriyadarsini and P S Chauhan ldquoInhibition of radiation-induced DNA damage in plasmid pBR322 by chlorophyllin andpossible mechanism(s) of actionrdquo Mutation Research vol 425no 1 pp 71ndash79 1999

[42] S S Kumar B Shankar andK B Sainis ldquoEffect of chlorophyllinagainst oxidative stress in splenic lymphocytes in vitro and invivordquo Biochimica et Biophysica Acta vol 1672 no 2 pp 100ndash1112004

[43] Y K Bae A Brown E Garrett et al ldquoHypermethylation inhistologically distinct classes of breast cancerrdquo Clinical CancerResearch vol 10 no 18 pp 5998ndash6005 2004

[44] T Negishi S Arimoto C Nishizaki and H HayatsuldquoInhibitory effect of chlorophyll on the genotoxicity of 3-amino-1-methyl-5H-pyrido[43-b]indole (Trp-P-2)rdquo Carcinogenesisvol 10 no 1 pp 145ndash149 1989

[45] J Hernaez M Xu and R Dashwood ldquoEffects of tea andchlorophyllin on the mutagenicity of N-hydroxy-IQ studies ofenzyme inhibitionmolecular complex formation and degrada-tionscavenging of the active metabolitesrdquo Environmental andMolecular Mutagenesis vol 30 no 4 pp 468ndash474 1997

10 ISRN Biophysics

[46] L Romert M Curvall and D Jenssen ldquoChlorophyllin is botha positive and negative modifier of mutagenicityrdquo Mutagenesisvol 7 no 5 pp 349ndash355 1992

[47] R H Dashwood V Breinholt and G S Bailey ldquoChemo-preventive properties of chlorophyllin inhibition of aflatoxinB1 (AFB1)-DNA binding in vivo and anti-mutagenic activityagainst AFB1 and two heterocyclic amines in the Salmonellamutagenicity assayrdquo Carcinogenesis vol 12 no 5 pp 939ndash9421991

[48] S Arimoto S Fukuoka C Itome H Nakano H Rai andH Hayatsu ldquoBinding of polycyclic planar mutagenesis tochlorophyllin resulting in inhibition of the activityrdquo MutationResearch vol 287 no 2 pp 293ndash305 1993

[49] S Arimoto-Kobayashi N Harada R Tokunaga J I Odo andHHayatsu ldquoAdsorption ofmutagens to chlorophyllin-chitosanan insoluble form of chlorophyllinrdquoMutation Research vol 381no 2 pp 243ndash249 1997

[50] N Anzai T Taniyama N Nakandakari et al ldquoInhibition ofDNA adduct formation and mutagenic action of 3-amino-1-methyl-5H-pyrido[43-b]indole by chlorophyllin-chitosan inrpsL transgenic micerdquo Japanese Journal of Cancer Research vol92 no 8 pp 848ndash853 2001

[51] C Sugiyama N Nakandakari H Hayatsu and S Arimoto-Kobayashi ldquoPreventive effects of chlorophyllin fixed on chi-tosan towards DNA adduct formation of 3-amino-1-methyl-5H-pyrido[43-b]indole in CDF1 micerdquo Biological and Pharma-ceutical Bulletin vol 25 no 4 pp 520ndash522 2002

[52] K Kitamura M Nagao H Hayatsu and M Morita ldquoEffectof chlorophyllin-chitosan on excretion of dioxins in a healthymanrdquo Environmental Science and Technology vol 39 no 4 pp1084ndash1091 2005

[53] J E Mata Z Yu J E Gray D E Williams and RRodriguez-Proteau ldquoEffects of chlorophyllin on transport ofdibenzo(al)pyrene 2-amino-1-methyl-6-phenylimidazo-[45-b]pyridine and aflatoxin B1 across Caco-2 cell monolayersrdquoToxicology vol 196 no 1-2 pp 117ndash125 2004

[54] R Hasegawa M Hirose T Kato et al ldquoInhibitory effect ofchlorophyllin on PhIP-induced mammary carcinogenesis infemale F344 ratsrdquo Carcinogenesis vol 16 no 9 pp 2243ndash22461995

[55] M Hirose A Nishikawa M Shibutani T Imai and T ShiraildquoChemoprevention of heterocyclic amine-induced mammarycarcinogenesis in ratsrdquo Environmental and Molecular Mutage-nesis vol 39 no 2-3 pp 271ndash278 2002

[56] D Guo D T Horio J S Grove and R H DashwoodldquoInhibition by chlorophyllin of 2-amino-3-methylimidazo[45-f]quinoline-induced tumorigenesis in the male F344 ratrdquo Can-cer Letters vol 95 no 1-2 pp 161ndash165 1995

[57] D Guo H A J Schut C D Davis E G SnyderwineG S Bailey and R H Dashwood ldquoProtection by chloro-phyllin and indole-3-carbinol against 2-amino-1-methyl-6-phenylimidazo[45-b]pyridine (PhIP)-induced DNA adductsand colonic aberrant crypts in the F344 ratrdquoCarcinogenesis vol16 no 12 pp 2931ndash2937 1995

[58] R Dashwood and C Liew ldquoChlorophyllin-enhanced excre-tion of urinary and fecal mutagens in rats given 2-amino-3-methylimidazo[45-f]quinolinerdquo Environmental and MolecularMutagenesis vol 20 no 3 pp 199ndash205 1992

[59] C Sugiyama A Shinoda H Hayatsu and T NegishildquoInhibition of 2-Amino-38-dimethylimidazo[45-f]quinoxal-ine-mediated DNA-adduct formation by chlorophyllin in

Drosophilardquo Japanese Journal of Cancer Research vol 87 no 4pp 325ndash328 1996

[60] K Morita M Ogata and T Hasegawa ldquoChlorophyll derivedfrom Chlorella inhibits dioxin adsorption from the gastroin-testinal tract and accelerates dioxin excretion in ratsrdquo Environ-mental Health Perspectives vol 109 no 3 pp 289ndash294 2001

[61] M T Simonich T McQuistan C Jubert et al ldquoLow-dosedietary chlorophyll inhibits multi-organ carcinogenesis in therainbow troutrdquo Food and Chemical Toxicology vol 46 no 3 pp1014ndash1024 2008

[62] M T Simonich P A Egner B D Roebuck et al ldquoNaturalchlorophyll inhibits aflatoxin B1-induced multi-organ carcino-genesis in the ratrdquo Carcinogenesis vol 28 no 6 pp 1294ndash13022007

[63] D Guo and R Dashwood ldquoInhibition of 2-amino-3-methy-limidazo[45-f] quinoline (IQ)-DNA binding in rats givenchlorophyllin dose-response and time-course studies in theliver and colonrdquoCarcinogenesis vol 15 no 4 pp 763ndash766 1994

[64] D T Shaughnessy L M Gangarosa B Schliebe et al ldquoInhibi-tion of fried meat-induced colorectal dna damage and alteredsystemic genotoxicity in humans by crucifera chlorophyllinand yogurtrdquo PLoS ONE vol 6 no 4 Article ID e18707 2011

[65] P A Egner A Munoz and T W Kensler ldquoChemopreventionwith chlorophyllin in individuals exposed to dietary aflatoxinrdquoMutation Research vol 523-524 pp 209ndash216 2003

[66] C Jubert J Mata G Bench et al ldquoEffects of chlorophyll andchlorophyllin on low-dose aflatoxin B 1 pharmacokinetics inhuman volunteersrdquo Cancer Prevention Research vol 2 no 12pp 1015ndash1022 2009

[67] C D Frary R K Johnson andM Q Wang ldquoFood sources andintakes of caffeine in the diets of persons in the United StatesrdquoJournal of the American Dietetic Association vol 105 no 1 pp110ndash113 2005

[68] D GanmaaW CWillett T Y Li et al ldquoCoffee tea caffeine andrisk of breast cancer a 22-year follow-uprdquo International Journalof Cancer vol 122 no 9 pp 2071ndash2076 2008

[69] A Nkondjock ldquoCoffee consumption and the risk of cancer anoverviewrdquo Cancer Letters vol 277 no 2 pp 121ndash125 2009

[70] L Rosenberg ldquoCoffee and tea consumption in relation to therisk of large bowel cancer a review of epidemiologic studiesrdquoCancer Letters vol 52 no 3 pp 163ndash171 1990

[71] L J Vatten K Solvoll and E B Loken ldquoCoffee consumptionand the risk of breast cancer A prospective study of 14593Norwegian womenrdquo British Journal of Cancer vol 62 no 2 pp267ndash270 1990

[72] H R Ha J Chen S Krahenbuhl and F Follath ldquoBiotransfor-mation of caffeine by cDNA-expressed human cytochromes P-450rdquo European Journal of Clinical Pharmacology vol 49 no 4pp 309ndash315 1996

[73] H Y Chen and G C Yen ldquoPossible mechanisms of antimu-tagens by various teas as judged by their effects on mutagene-sis by 2-amino-3-methylimidazo[45-f]quinoline and benzo[a]pyrenerdquoMutation Research vol 393 no 1-2 pp 115ndash122 1997

[74] B Stavric T I Matula R Klassen and R H Downie ldquoTheeffect of teas on the in vitro mutagenic potential of heterocyclicaromatic aminesrdquo Food and Chemical Toxicology vol 34 no 6pp 515ndash523 1996

[75] J H Weisburger L Dolan and B Pittman ldquoInhibition of PhIPmutagenicity by caffeine lycopene daidzein and genisteinrdquoMutation Research vol 416 no 1-2 pp 125ndash128 1998

ISRN Biophysics 11

[76] M Xu A C Bailey J F Hernaez C R Taoka H A J Schut andRHDashwood ldquoProtection by green tea black tea and indole-3-carbinol against 2-amino-3-methylimidazo[45-f]quinoline-induced DNA adducts and colonic aberrant crypts in the F344ratrdquo Carcinogenesis vol 17 no 7 pp 1429ndash1434 1996

[77] T Kato S Takahashi and K Kikugawa ldquoLoss of heterocyclicamine mutagens by insoluble hemicellulose fiber and high-molecular-weight soluble polyphenolics of coffeerdquo MutationResearch vol 246 no 1 pp 169ndash178 1991

[78] A J Alldrick and I R Rowland ldquoCaffeine inhibits hepatic-microsomal activation of some dietary genotoxinsrdquo Mutagen-esis vol 3 no 5 pp 423ndash427 1988

[79] A J Alldrick W E Brennan-Craddock and I R RowlandldquoDietary caffeine reduces the genotoxicity ofMeIQx in the host-mediated assay inmicerdquoNutrition and Cancer vol 24 no 2 pp143ndash150 1995

[80] A Bu-Abbas M N Clifford D Walker and C IoannidesldquoMarked antimutagenic potential of aqueous green tea extractsmechanism of actionrdquo Mutagenesis vol 9 no 4 pp 325ndash3311994

[81] M Zdunek J Piosik and J Kapuscinski ldquoThermodynamicalmodel of mixed aggregation of ligands with caffeine in aqueoussolution Part IIrdquo Biophysical Chemistry vol 84 no 1 pp 77ndash852000

[82] J Kapuscinski and M Kimmel ldquoThermodynamical model ofmixed aggregation of intercalators with caffeine in aqueoussolutionrdquoBiophysical Chemistry vol 46 no 2 pp 153ndash163 1993

[83] M B Lyles and I L Cameron ldquoInteractions of the DNAintercalator acridine orange with itself with caffeine and withdouble stranded DNArdquo Biophysical Chemistry vol 96 no 1 pp53ndash76 2002

[84] M B Lyles and I L Cameron ldquoCaffeine and other xanthinesas cytochemical blockers and removers of heterocyclic DNAintercalators from chromatinrdquo Cell Biology International vol26 no 2 pp 145ndash154 2002

[85] H Kimura and T Aoyama ldquoDecrease in sensitivity to ethidiumbromide by caffeine dimethylsulfoxide or 3-aminobenzamidedue to reduced permeabilityrdquo Journal of Pharmacobio-Dynamics vol 12 no 10 pp 589ndash595 1989

[86] P A Bolotin S F Baranovsky and M P Evstigneev ldquoSpec-trophotometric investigation of the hetero-association of Caf-feine and thiazine dye in aqueous solutionrdquo Spectrochimica ActaA vol 64 no 3 pp 693ndash697 2006

[87] M P Evstigneev V P Evstigneev and D B Davies ldquoNMRinvestigation of the effect of caffeine on the hetero-associationof an anticancer drug with a vitaminrdquo Chemical Physics Lettersvol 432 no 1ndash3 pp 248ndash251 2006

[88] M P Evstigneev K A Rybakova and D B Davies ldquoCom-plexation of norfloxacin with DNA in the presence of caffeinerdquoBiophysical Chemistry vol 121 no 2 pp 84ndash95 2006

[89] K Ulanowska J Piosik A Gwizdek-Wisniewska and GWegrzyn ldquoFormation of stacking complexes between caf-feine (123-trimethylxanthine) and 1-methyl-4-phenyl-1236-tetrahydropyridine may attenuate biological effects of thisneurotoxinrdquo Bioorganic Chemistry vol 33 no 5 pp 402ndash4132005

[90] L Bravo ldquoPolyphenols chemistry dietary sources metabolismand nutritional significancerdquo Nutrition Reviews vol 56 no 11pp 317ndash333 1998

[91] D M Shankel S Kuo C Haines and L A Mitscher ldquoExtra-cellular interception of mutagensrdquo Basic life sciences vol 61 pp65ndash74 1993

[92] M M Manson ldquoCancer preventionmdashthe potential for diet tomodulate molecular signallingrdquo Trends in Molecular Medicinevol 9 no 1 pp 11ndash18 2003

[93] T Nakasugi M Nakashima and K Komai ldquoAntimutagens ingaiyou (Artemisia argyi Levl et Vant)rdquo Journal of Agriculturaland Food Chemistry vol 48 no 8 pp 3256ndash3266 2000

[94] S Arimoto-Kobayashi C Sugiyama N Harada M TakeuchiM Takemura and H Hayatsu ldquoInhibitory effects of beer andother alcoholic beverages onmutagenesis and DNA adduct for-mation induced by several carcinogensrdquo Journal of Agriculturaland Food Chemistry vol 47 no 1 pp 221ndash230 1999

[95] G J Soleas G Tomlinson E PDiamandis andDMGoldbergldquoRelative contributions of polyphenolic constituents to theantioxidant status of wines development of a predictivemodelrdquoJournal of Agricultural and Food Chemistry vol 45 no 10 pp3995ndash4003 1997

[96] S Izawa N Harada T Watanabe et al ldquoInhibitory effects offood-coloring agents derived frommonascus on themutagenic-ity of heterocyclic aminesrdquo Journal of Agricultural and FoodChemistry vol 45 no 10 pp 3980ndash3984 1997

[97] R Vikse B Balsrud Mjelva and L Klungsoyr ldquoReversiblebinding of the cooked food mutagen MeIQx to lignin-enrichedpreparations from wheat branrdquo Food and Chemical Toxicologyvol 30 no 3 pp 239ndash246 1992

[98] P Sjodin M Nyman L L Nielsen HWallin andM JagerstadldquoEffect of dietary fiber on the disposition and excretion of afood carcinogen (2-14C-labeled MeIQx) in ratsrdquo Nutrition andCancer vol 17 no 2 pp 139ndash151 1992

[99] L R Ferguson AM RobertonM EWatson CM Triggs andP J Harris ldquoThe effects of a soluble-fibre polysaccharide on theadsorption of carcinogens to insoluble dietary fibresrdquo Chemico-Biological Interactions vol 95 no 3 pp 245ndash255 1995

[100] P Ryden and J A Robertson ldquoThe effects of pH and bile salts onthe binding of MeIQx to wheat bran fibrerdquo Mutation Researchvol 351 no 1 pp 45ndash52 1996

[101] P J Harris C M Triggs A M Roberton M E Watsonand L R Ferguson ldquoThe adsorption of heterocyclic aromaticamines by model dietary fibres with contrasting compositionsrdquoChemico-Biological Interactions vol 100 no 1 pp 13ndash25 1996

Submit your manuscripts athttpwwwhindawicom

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Enzyme Research



Microbiology

2 ISRN Biophysics

N

NN

N

NN

N

NN

N N

N

NH

NN

N

N

IQ MeIQ MeIQx

PhIP Trp-P-2 Glu-P-1

CH3

NH2NH2

CH3

CH3

H3C

NH2

CH3

NH2

CH3

NH2

CH3

CH3

NH2

Figure 1 Examples of heterocyclic aromatic amines (HCAs) chemical structures IQ 2-amino-3-methylimidazo[45-f ]quinolineMeIQ 2-amino-34-dimethylomidazo[45-f ]quinoline MeIQx 2-amino-38-dimethylimidazo[45-f ]quinoxaline PhIP 2-amino-1-methyl-6-phenylimidazo[45-b]pyridine Trp-P-2 3-amino-1-methyl-5H-pyrido[43-b]indole Glu-P-1 2-amino-6-methyldipyrido[12-a 3101584021015840-d]imidazole

2AB carcinogens [11 12] therefore being extensively inves-tigated worldwide with a particular interest in findingmethods to diminish their genotoxic potential As a resultnumerous studies concerning protective effects of severaldietary components against HCAs mutagenic and carcino-genic activity have been performed It is estimated thatapproximately 600 different individual compounds as well ascomplex mixtures have been described as antimutagenic oranticarcinogenic towards HCAs representing diverse modesof action [13] Proposed mechanisms of protection involveinhibition of mutagen formation inhibition of HCAs acti-vating enzymes induction of HCAs detoxifying enzymeselectrophile-scavenging enhanced DNA repair alteration ofsignaling pathways involved in apoptosis and proliferationand direct binding of HCAs and their metabolites by inter-ceptor molecules [14]

In the studies concerning the molecular mechanisms ofchemoprevention against HCAs activity observed for severalfood components the main focus is placed on the interactionof these compounds with different metabolic pathways ofHCAs with protective effects resulting from reduction inHCAs bioactivation andor enhancement of their detoxi-cation Nevertheless taking into consideration that manynatural protective compounds have aromatic moieties intheir structure it seems possible that noncovalent directinteractions especially stacking (120587-120587) complexes formationbetween protective molecules and HCAs can at least in partcontribute to the observed chemopreventive effects Suchsequestration of mutagenmolecules in heterocomplexes withbiologically active aromatic compounds (BACs) can reducetheir bioavailability and in consequence effectively preventfrom their genotoxic effects (Figure 2)

This paper summarizes the current state of knowledgeon the importance of heterocomplexation in diminishingvarious biological effects of HCAs

2 Mechanism of Heterocomplexation

Mechanism of small aromatic molecules protective actionagainst HCAs activity is often attributed to their ability toformheterocomplexes between these compounds Formationof such complexes is dependent on hydrophobic interactionsbetween 120587-120587 electron systems from overlapping aromaticrings present in both molecules The ability of stackingheterocomplexes formation can be described by severalbiophysical parameters such as enthalpy changes (ΔH) orassociation constants (eg neighborhood association con-stant 119870AC) For example calculated values of neighborhoodassociation constants (119870AC) for HCAs-theophylline complexformation are greater than 119870AC values of HCAs-caffeine andHCAs-pentoxifylline heterocomplexes [15] Andrejuk et alreported similar relationship between association constantvalues of heterocomplexes formation for several aromaticcompounds and theophylline and other methylxanthines[16] This difference was interpreted as a consequence ofa 7-Me group absence in theophylline molecules whichis present in caffeine and can introduce steric hindrancereducing heterocomplexation possibility [16] Possibility ofhigh-order complex formation can be considered as anotherreason for differences between association constants val-ues of stacking complexes formation Formation of suchhigh-order complexes was proposed previously for theinteraction between caffeine (CAF) and anticancer drugmitoxantrone (MIT) [17] The authors suggested that theformation of MIT-(CAF)

2complex is more probable and

more thermodynamically favorable than 1 1 heterocomplexas concluded from lower values of enthalpy changes for2 1 heterocomplexes [17] Apart from stacking complexesformation direct binding of mutagen to inhibitor moleculescan involve electrostatic and H-bond interactions whichcan act independently or additionally to heterocomplexes

ISRN Biophysics 3

Cu

N

N N

N

O

ONa

ONa

ONa

O

O

N

NN

N

O

O

O

O

OOH

OH

OH

OH

OH

OH

OH

HO

O

OOH

HO

OHO

O

HO

OH

O

CHO

CHL EGCG


CH3

CH3

CH3

H3C

H3C

H2C

CH3

C5H11

H3C

CH3

CH3







1(AFB1) and





4 ISRN Biophysics



2IQ resulted in






1-89-epoxide [47]




ISRN Biophysics 5





119889= 122 120583M and 305 120583M for AFB1-



6 ISRN Biophysics













ISRN Biophysics 7


2derivative flavin-



5 Other BACs







8 ISRN Biophysics


6 Conclusions


Acknowledgment


References


















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10 ISRN Biophysics
































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Volume 2014

Zoology





Volume 2014


















Advances in

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Volume 2014




Enzyme Research



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ISRN Biophysics 3

Cu

N

N N

N

O

ONa

ONa

ONa

O

O

N

NN

N

O

O

O

O

OOH

OH

OH

OH

OH

OH

OH

HO

O

OOH

HO

OHO

O

HO

OH

O

CHO

CHL EGCG


CH3

CH3

CH3

H3C

H3C

H2C

CH3

C5H11

H3C

CH3

CH3







1(AFB1) and





4 ISRN Biophysics



2IQ resulted in






1-89-epoxide [47]




ISRN Biophysics 5





119889= 122 120583M and 305 120583M for AFB1-



6 ISRN Biophysics













ISRN Biophysics 7


2derivative flavin-



5 Other BACs







8 ISRN Biophysics


6 Conclusions


Acknowledgment


References


















ISRN Biophysics 9






























10 ISRN Biophysics
































ISRN Biophysics 11


































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 ISRN Biophysics



2IQ resulted in






1-89-epoxide [47]




ISRN Biophysics 5





119889= 122 120583M and 305 120583M for AFB1-



6 ISRN Biophysics













ISRN Biophysics 7


2derivative flavin-



5 Other BACs







8 ISRN Biophysics


6 Conclusions


Acknowledgment


References


















ISRN Biophysics 9






























10 ISRN Biophysics
































ISRN Biophysics 11


































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biophysics 5





119889= 122 120583M and 305 120583M for AFB1-



6 ISRN Biophysics













ISRN Biophysics 7


2derivative flavin-



5 Other BACs







8 ISRN Biophysics


6 Conclusions


Acknowledgment


References


















ISRN Biophysics 9






























10 ISRN Biophysics
































ISRN Biophysics 11


































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 ISRN Biophysics













ISRN Biophysics 7


2derivative flavin-



5 Other BACs







8 ISRN Biophysics


6 Conclusions


Acknowledgment


References


















ISRN Biophysics 9






























10 ISRN Biophysics
































ISRN Biophysics 11


































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biophysics 7


2derivative flavin-



5 Other BACs







8 ISRN Biophysics


6 Conclusions


Acknowledgment


References


















ISRN Biophysics 9






























10 ISRN Biophysics
































ISRN Biophysics 11


































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 ISRN Biophysics


6 Conclusions


Acknowledgment


References


















ISRN Biophysics 9






























10 ISRN Biophysics
































ISRN Biophysics 11


































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biophysics 9






























10 ISRN Biophysics
































ISRN Biophysics 11


































Volume 2014

Zoology





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Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

10 ISRN Biophysics
































ISRN Biophysics 11


































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biophysics 11


































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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