division toxicology and cancer risk factors (c0200 / c010) · exploration of markers in large-scale...

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Research Program CCancer Risk Factors and Prevention

Division C0200Toxicology and Cancer Risk Factors

DKFZ 2003: Research Report 2001/2002

ScientistsDr. Matthias Bartelmann (-6/02) Dr. Barbara BertramDr. Heike Dally Dr. Norbert FrankDr. Clarissa Gerhäuser Dr. Reinhold KleinDr. Jagadeesan Nair Dr. Urmila NairProf. Dr. Hans Osswald Dr. Robert W. OwenPriv.-Doz. Dr. Odilia Popanda Dr. Angela RischDr. Margarita Rojas (-8/02) Dr. Hans-R. Scherf (-4/02)Dr. Peter Schmezer Dr. Bertold SpiegelhalderDr. Gisela Werle-Schneider

Visiting ScientistsDorota Butkiewicz (10/02-), Warschau, PolandDr. Roger Godschalk (-10/02), Maastricht, NetherlandsEmma Humphreys (-7/02), Cambridge, England, UKSomkid Sitthimonchai (-9/02), Saraburi, ThailandDr. Xin Sun, Bejing, China

PostgraduatesAmira Gamal Eldeen (-2/02) Elisabeth BertlChristopher Beynon Reinhard Ebbeler(-12/01)Kai Gassner (-7/02) Elke Heiss (-7/01)Marc Hoffmann (5/02-) Hui Huang (10/02-)Jörg Hümmerich Ulrike KnustLydia Pan (8/02-) Chi Tai PhongTorsten Schattenberg Inge SchönfeldSabine Stöckigt Isabel StreckYuttana Sudjaroen (12/02-) Changping Xie

UndergraduatesGerlinde Pappa (10/02-) Vivianne Raedts (-6/01)Anja Stroloke Andreas Vogt (-10/01)

Technical AssistantsDaniel Bodemer (-6/01) Ursula BollowChristel Ditrich Reinhard GliniorzRoswitha Haubner Michael Huber (5/01-9/01)Birgit Jäger Claudia Kalla (-3/01)Karin Klimo Jutta KnauftRegina Merkel (-2/02) Barbara Mostermann (9/02-)Regina Peichl (7/02-) Ulrike von Seydlitz-KurzbachPeter Waas Andreas Wölfelschneider (-7/02)Gerd Würtele Otto Zelezny

TraineesKai Doberstein (2/02-11/02) Karin Schüßler (8/01-2/02)SecretarySusanna Fuladdjusch

The overall goals of the Division involve: (i) identificationof exogenous and endogenous cancer risk factors andelucidation of their mechanisms of action, (ii) characteriza-tion of cancer-preventive agents and proof of their efficacyin preclinical and clinical studies, (iii) development, basedon advances in mechanistic knowledge, of new ultrasen-sitive detection methods for DNA-damage and biomarkersfor cancer susceptibility that are useful for molecular epi-demiology, studies on cancer etiology and prevention and(iv) initiation and participation in such studies by contribu-tion to methodology and design. This approach it is aimed

to provide data that are required to implement efficientcancer preventive measures either by elimination of riskfactors or by interruption of disease development (chemo-prevention).While there is diversity in the Division�s research activities,many studies fall under a common denominator, i.e. bio-marker development and application (Chart 1). This fol-lows a tier approach, before the markers are explored inlarge-scale epidemiological studies. Naturally, many of ourinvestigations have reached only the first phases of thisdevelopment, but recently larger-scale molecular epide-miological studies have been conducted [1].

Chart 1 Rationale for developing and validating biomarkers orintermediate endpoints relevant to human carcinogenesis for usein molecular epidemiological or clinical settings [2]

Epidemiological observations on risk and protectivefactors in human cancer

⇓⇓⇓⇓⇓Mechanistic studies in experimental systems to estab-lish biomarkers/intermediate endpoints as part of the

causal chain⇓⇓⇓⇓⇓

Development of (non-invasive) methods for exposure/risk markers

⇓⇓⇓⇓⇓Validation in animal/human pilot studies

⇓⇓⇓⇓⇓Exploration of markers in large-scale epidemiological

investigations⇓⇓⇓⇓⇓

Feedback: etiology, prevention, diagnosis, prognosisPrime objectives in the area of biomarker developmentand human applications involve (i) to identify new sourcesof carcinogen exposure [3], especially those arising fromendogenous sources [4,5], (ii) to quantify carcinogenDNA-damage in populations and measure the effect of ge-netic predisposition [6,7], (iii) to identify high-risk groups[8-10] and finally (iv) to verify the efficacy of preventivemeasures [1,11-14].Since 1996, more intense activities on secondary cancerprevention by chemopreventive (antidysplastic) agentshave been commenced in the Division. As chemopre-ventive agents are structurally heterogeneous and mecha-nistically diverse, ongoing work aims at the identificationand evaluation of new promising agents of natural origin[15,16] or synthetic analogues as lead compounds for thedevelopment of effective agents, the elucidation of theirmechanism of action and ultimately proof of their preven-tive efficacy in high-risk groups with dysplastic lesions orin the general population. A pan-European calcium fiberplacebo-controlled intervention study has been completedin patients with sporadic colorectal adenomas [1].The characterization of new chemopreventive agents withanti-inflammatory/antioxidant properties, but low long-termtoxicity call for a more intense interdisciplinary research

Division Toxicology and Cancer Risk Factors (C0200 / C010)Head: Prof. Dr. Helmut Bartsch

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network in secondary cancer prevention to include (i) con-duct of clinical trials, taking into account accessible dys-plasias for repeated direct dysplasia control followingtreatment with officinal and new antidysplastic drugs, (ii)development and validation of cancer-predictive biomark-ers for less accessible dysplasias, (iii) subsequent devel-opment of new antidysplastic agents with higher preven-tive efficacy for a broader spectrum of different dysplasias.

Publications (* = external co-author)[1] Bonithon-Kopp, C.*; Kronborg, O.*; Giacosa, A.*; Räth, U.*;Faivre, J.* [Experts:-Milan, C.*; Fenger, C.*; Piard, F.*; BelghitiC.*; Owen, R. W.; Pignatelli, M.*: Calcium and fibre supplementa-tion in the prevention of colorectal adenoma recurrence: a pla-cebo-controlled intervention trial from the European Cancer Pre-vention Organisation (ECP). Lancet 356 (2000) 1300-1306.[2] Bartsch, H. Studies on biomarkers in cancer etiology and pre-vention: a summary and challenge of interdisciplinary research.Mutation Research 462(2-3) (2000) 255-279.[3] Klein, R.G.; Schmezer, P.; Amelung, F.; Schroeder, H.-G.*;Woeste, W.*; Wolf, J.* Carcinogenicity assays of wood dust andwood activities in rats exposed by long-term inhalation. Int ArchOccup Environ Health 74 (2001) 109-118.[4] Bartsch, H.; Nair, J.; Owen, R. W. Exocyclic DNA adducts asoxidative stress markers in colon carcinogenesis: potential role oflipid peroxidation, dietary fat and antioxidants. Biological Chemis-try 383 (2002) 915-921.[5] Bartsch, H.; Nair, J. Potential role of lipid peroxidation derivedDNA damage in human colon carcinogenesis: studies on exocy-clic base adducts as stable oxidative stress markers. Cancer De-tection and Prevention 26 (2002) 308-312.[6] Godschalk, R.W.L.; Dallinga, J.W.*; Wikman, H.*; Risch, A.;,Kleinjans, J.C.S.*; Bartsch, H.; Van Schooten, F.-J.* Modulationof DNA and protein adducts in smokers by geneticpolymorphsims in GSTM1, GSTT1, NAT1 and NAT2. Pharmaco-genetics 11 (2001) 389-398.[7] Godschalk, R.; Nair, J.; Kliem, H.-C.; Wiessler, M.; Bouvier,G.*: Bartsch, H. Modified immuno-enriched 32P-HPLC assay forthe detection of 04-ethylthymidine in human biomonitoring studies.Chemical Research in Toxicology 15 (2002) 433-437.[8] Rajaee-Behbahani, N.*; Schmezer, P.; Ramroth, H.*; Bürkle,A.*; Bartsch, H.; Dietz, A.*; Becher, H.*: Reduced poly(ADP-ribosyl)ation in lymphocytes of laryngeal cancer patients: resultsof a case-control study. International Journal of Cancer 98 (2002)780-784.[9] Risch, A.; Wikman, H.*; Thiel, S.*; Schmezer, P.; Edler, L.;Drings, P.*; Dienemann, H.*; Kayser, K.*; Schulz, V.*;Spiegelhalder, B.; Bartsch, H. Glutathione-S-transferase M1, M3,T1 and P1 polymorphisms and susceptibility to non-small-celllung cancer subtypes and hamartomas. Pharmacogenetics 11(2001) 757-764.[10] Chang-Claude, J.; Kropp, S.; Jäger, B.; Bartsch, H.; Risch, A.Differential effect of NAT2 on the association between active andpassive smoke exposure and breast cancer risk. Cancer Epidemi-ology, Biomarkers and Prevention 11 (2002) 698-704.[11] Frank, N.; Knauft, J.; Amelung, F.; Nair, J.; Wesch, H.;Bartsch, H. No prevention of liver and kidney tumors in Long-Evans Cinnamon rats by dietary curcumin, but inhibition at othersites and of metastases. Mutation Research (2002)www.sciencedirect.com, 9466, 1-9[12] Nair, U; Bartsch, H. Metabolic polymorphisms as susceptibil-ity markers for lung and oral cavity cancer. In: Biomarkers in Can-cer Chemoprevention. Miller, A.B. et al. (eds.), IARC ScientificPublications N° 154 (IARC, Lyon, France) (2001) 271-290.

[13] Hagenlocher, T.; Nair, J.; Becker, N.; Bartsch, H. Influence ofDietary Fatty Acid, Vegetable and Vitamin Intake on Etheno-DNAAdducts in White Blood Cells of Healthy Female Volunteers: APilot Study. Cancer Epidemiology, Biomarkers & Prevention 10(2001) 1187-1191.[14] Hanaoka, T.; Nair, J.; Takahashi, Y.*; Sasaki, S.*; Bartsch, H.;Tsugane, S.*: Urinary excretion of 1, N6-ethenodeoxyadenosine, amarker of oxidative DNA damage in postmenopousal Japanesewomen participating in a dietary intervention trial in Northern Ja-pan. International Journal of Cancer 100 (2002) 71-75.[15] Gerhäuser, C.; Alt, A.*; Heiss, E.*; Gamal-Eldeen, A.; Klimo,K.; Knauft, J.; Neumann, I.*; Scherf, H.-R.; Frank, N.; Bartsch, H.;Becker, H. Cancer chemopreventive activity of Xanthohumol, anatural product derived from hop. Molecular Cancer Therapeutics1 (2002) 959-969.[16] Heiss, E.; Herhaus, C.*; Klimo, K.; Bartsch, H.; Gerhäuser,C. Nuclear factor kappa B is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms. J Biol Chem 276 (2001)32008-32015.

Chemoprevention (C0202)Group Leader: Dr. Clarissa Gerhäuser

1. Identification and evaluation of novel potentialcancer chemopreventive agents

Carcinogenesis can be regarded as an accumulation ofgenetic or biochemical cell damage, which offers a varietyof targets for chemopreventive agents to prevent or inhibitthe slow progression from early genetic lesions to tumordevelopment [1]. Well established molecular mechanismsof chemoprevention include modulation of drug metabo-lism, anti-oxidant, radical-scavenging, anti-inflammatory,anti-tumor promoting and anti-proliferative activities aswell as induction of terminal cell differentiation and apop-tosis. Knowledge of molecular mechanisms is of impor-tance for safe application of known, but also for further de-velopment of novel potential cancer preventive agents [2].Consequently, we have set up a battery of cell- and en-zyme-based in vitro marker systems relevant for inhibitionof carcinogenesis in vivo (Table 1) [3].

Table 1: Bioassay systems for the identification of potentialchemopreventive agents

Modulation of carcinogen-metabolizing enzymes- inhibition of Cyp1A activity in homogenates of β-naphtho-

flavone-induced H4IIE rat hepatoma cells (modified fromCrespi et al. 1997)

- induction of NAD(P)H:quinone oxidoreductase (QR) ac-tivity in cultured Hepa 1c1c7 cells (Prochaska andSantamaria 1988; Gerhauser et al. 1997)

Radical scavenging and anti-oxidant capacity- determination of free radical scavenging activity by reac-

tion with 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radi-cals (van Amsterdam et al. 1992)

- analysis of hydroxyl (OH*)- and peroxyl (ROO*)-radicalscavenging capacity in the oxygen radical absorbancecapacity (ORAC) assay (modified from Prior and Cao1999)

- scavenging of superoxide anion radicals, generated inthe xanthine oxidase system (X/XO) (Ukeda et al. 1997)

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- scavenging of superoxide anion radicals after stimulationof differentiated HL-60 cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (Pick and Mizel 1981)

- induction of glutathione levels in cellular systems(Gerhauser et al. 1997, Ridnour et al. 1999)

Anti-inflammatory mechanisms- inhibition of cyclooxygenase 1 (Cox-1) activity using

sheep seminal vesicle microsomes (Jang et al. 1997)- inhibition of human recombinant Cox-2 activity (Kuhl et

al. 1984)- inhibition of lipopolysaccharide (LPS)-mediated inducible

nitric oxide synthase (iNOS) induction in Raw 264.7 mu-rine macrophage cell culture (Heiss et al. 2001)

Anti-tumor promoting mechanisms- inhibition of TPA-mediated induction of ornithine decar-

boxylase in cultured 308 murine keratinocytes(Gerhauser et al. 1995)

- assessment of estrogenic and anti-estrogenic propertiesin Ishikawa human endometrium cancer cell culture viathe estrogen-dependent induction of alkaline phos-phatase (ALP) activity (Littlefield et al. 1990; Markiewiczet al. 1992)

- inhibition of human recombinant aromatase (Cyp19) ac-tivity (Stresser et al. 2000)

Anti-proliferative mechanisms relevant for the inhibition oftumor progression- induction of terminal cell differentiation in human (HL-60)

or murine (MELC) leukemia cell lines (Suh et al. 1995;Richon et al. 1996)

Taken together, these models allow fast (within days), sen-sitive and cost effective identification of promising leadcompounds and plant extracts and have been utilized foractivity-guided isolation of active principles. Further, thesemodels were utilized for detailed analyses and mechanis-tic investigations of promising compounds and series ofoptimized structures.

2. Establishment of novel test systems for thedetection of chemopreventive activity

2.1. Inhibition of lipoxygenase activityIt is estimated that 10% of all cancer cases are related tochronic inflammatory processes. Chronic inflammationand infections stimulate the inducible form of nitric oxidesynthase and Cox-2 expression and result in elevated lev-els of pro-inflammatory mediators like NO and prostaglan-dins in tumor tissue. In addition, a role of lipoxygenase(Lox) products, i.e. leucotrienes and hydroxy eicosatetra-enoic acids (HETEs) in carcinogenesis has been estab-lished in recent years. Lipoxygenases constitute an en-zyme family; in humans, four different forms were identi-fied so far and were designated according to their primaryproducts as 5-, 8-, 12-, and 15-Lox. Leukotriene B4, aproduct of 5-Lox, has been found to promote proliferationof colon and prostate cancer cell lines, whereas 8- and 12-HETE overproduction in mouse skin led to an increase inmutagenic DNA-adducts (Nair et al. 2000). In contrast, ex-pression of the anti-carcinogenic isoenzymes 15-Lox-1

and -2 seems to be downregulated in colon and esoph-ageal cancer tissue (Shureiqi and Lippman, 2001).Based on these findings, we decided to include lipoxy-genase as a target for anti-inflammatory activity in ourscreening program. As a first approach, we used soybeanlipoxygenase 1 (SBL-1), which catalyses the conversion ofarachidonioc acid mainly to 12-HETE. The enzymatic ac-tivity was determined photometrically, monitoring an in-creased absorption at 234 nm (modified from Ben-Aziz etal. 1970 in 96-well plate format) or more selectively byHPLC. Based on the results obtained with four referencecompounds (nordihydroguaiaretic acid, quercetin, myrice-tin and esculetin), the correlation of IC50 values obtainedby both methods was high (r2=0.92). Consequently, a pri-mary screening with 200 pure compounds derived from 18structural classes was performed by the faster photometricmethod. Triterpenoids, flavonols and hydroxychalconesprovided most inhibitors, and phenolic compounds witho-dihydroxy-substitution were identified as the most prom-ising structures. This system now provides a means to effi-ciently identify Lox-inhibitors, which can then be further in-vestigated for human isoenzymes selectivity and chemo-preventive efficacy (publication in preparation).2.2. A novel human in vitro screening method for

anti-angiogenic substancesAngiogenesis - the formation of new blood vessels fromalready established microvasculature - plays a pivotal rolein the growth of solid tumors and the spreading of me-tastases. According to Judah Folkman (Folkman, 1974) amicrotumor requires an intact blood vessel system in orderto be supplied with oxygen, nutrients and the possibility toshed its metabolites to grow beyond a critical size of 1-2mm². This blood vessel formation is the result of a com-plex cascade of molecular events and can lead to fast tu-mor growth. Therefore, the inhibition of angiogenesis witha wide range of substances is an alternative approach tochemoprevention. Especially natural compounds mightplay an important role in this field.The aim of our project was to establish different test sys-tems to investigate anti-angiogenic properties of knownand novel cancer chemopreventive agents. Consequently,we have established a human in vitro anti-angiogenic as-say based on work of Brown (Brown et al., 1996) andWatson (Watson et al., 1997). This test is premised on theprinciple of wound healing where angiogenesis occursnaturally and therefore no additional stimulation is re-quired. Placentas from Caesarian births were obtainedfrom a local hospital and proceeded within 8 hours. Super-ficial vessels of venous or arterial origin were dissecti-oned, cut into 2-3mm fragments and embedded into a fi-brin gel. The vessels were cultivated for three weeks; themedium mix containing MCBD 131 and Medium 199 waschanged twice a week. After one week, sprouts can be de-tected, then a constant growth phase is observed until day19 and leads to a growth plateau around day 21. Afterthree weeks, vessel growth is monitored microscopicallyand pictures are acquired using a digital camera. Thequantitative analysis of vessel density (area covered bynewly formed vessels) is carried out using Adobe Photo-

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shop®. Four known inhibitors of angiogenesis, i.e. resver-atrol, indomethacin, hydrocortisone, and surmamin wereused to validate the model. Fragments treated with the dif-ferent substances showed less capillary growth comparedto the solvent control (0.1% DMSO). Indomethacin at aconcentration of 1µM had the strongest effect (65.2 ±18.6% inhibition) on capillary growth, whereas 0.1µM hy-drocortisone revealed only 31 ± 8.5% inhibition. Resver-atrol (1 µM) demonstrated the most reproducible effects(41 ± 6.4% inhibition) and was selected as a positive con-trol for screening of substances for which anti-angiogenicactivity is not known yet. Suramin did not significantly in-hibit vessel growth. Additional testing of potential chemo-reventive agents is currently in progress. Overall, our hu-man anti-angiogenic model is suitable to screen the ef-fects of a wide variety of anti-angiogenic substances oncapillary growth in vitro. To further elucidate the mechanis-tic details of inhibition, the influence of inhibitors on endo-thelial cell growth and properties will be analyzed in addi-tional assays using HMEC-1 cell culture (publication inpreparation).

3. Identification of novel lead structures3.1. Screening of cancer chemopreventive

potential of Thai medicinal plant extractsIn cooperation with Chulabhorn Mahidol, M. Ruchirawat and S.Ruchirawat (Chulabhorn Research Institute, Bangkok, Thailand)

In a screening program to investigate the cancer chemo-preventive potential of Thai medicinal plants, 118 crudeextracts derived from various parts of 37 Thai plants be-longing to 24 families were analyzed for their potential toscavenge DPPH-, superoxide anion-, and peroxyl-radicalsand to inhibit Cox-1, soybean lipoxygenase 1 (SBL-1) andaromatase. Of these, 18 samples demonstrated multipleactivities indicative of chemopreventive effects. They werederived from Artabotrys spinosus Craig. (Annonaceae),Calamus acanthophyllus Becc. (Arecaceae), Euonymuscochinchinensis Pierre (Celastraceae), Lophopetalumduperreanum (Celastraceae), Fimbristylis insignis (Cyper-aceae), Dendrophthoe varians (Loranthaceae), Dendro-phthoe falcata (Loranthaceae), Helixanthera parasitica(Loranthaceae), Psychotria monticola (Rubiaceae),Madhuca kerri (Sapotaceae), Pradongleuad and Tumerin.Interestingly, mostly aqueous extracts displayed the high-est activities in comparison with more lipophilic extracts.Extracts from three species, Dendrophthoe falcata(stems), Euonymus cochinchinensis (bark) and Helix-anthera parasitica (whole plant) were selected for activity-guided fractionation. Fifteen fractions with potent radical-scavenging activity were obtained. The isolation and struc-ture elucidation of pure active constituents is ongoing.

3.2. Cancer chemopreventive in vitro activities ofisoflavones isolated from Iris germanica

In cooperation with E. Wollenweber (Technische Universität,Darmstadt), and F. Stevens (Oregon State University, Corvallis,OR, USA)

For centuries, the use of underground parts (RhizomaIridis) of several species of Iris (Iridaceae) has been wellestablished in traditional European folk medicine due to

their emetic, cathartic, diuretic, stimulant, expectorant anderrhine properties, but so far, Iris has not been investi-gated for chemopreventive effects. Activity-guided fraction-ation of a methanolic extract of fresh rhizomes of I. germa-nica led to the isolation of six isoflavones known as irisol-idone (1), irisolidone-7-O-α-D-glucoside (1a), irigenin (2),irilone (3), iriflogenin (4), and iriskashmirianin (5). Thecompounds were tested in a selection of the bioassays de-scribed above, and the activities were compared withthose of the well-known cancer chemopreventive isoflav-one genistein. The Iris isoflavones (2), (3) and (4) wereshown to be potent inhibitors of cytochrome P450 1Awhich is involved in the metabolic conversion of procarci-nogens into carcinogens. Cyp1A inhibitory activities werecomparable with that of genistein, with IC50 values of1.2 µM, 0.3 µM, and 1.4 µM, respectively. (2), (3) and (5)displayed moderate activity as inducers of NAD(P)H:quin-one reductase (QR), a carcinogen detoxifying enzyme,with CD values (concentration required to double the spe-cific activity of QR) of 3.5-16.7 µM, whereas weak activitywas observed with compounds (4) and (5) in the radical(DPPH) scavenging bioassay (IC50 values 89.6 and120.3 µM, respectively). With respect to anti-tumor pro-moting potential based on anti-inflammatory mechanisms(Cox-1 inhibition, inhibition of LPS-mediated NO inductionin murine macrophages), none of the compounds demon-strated significant activity in the concentration rangetested. Importantly, the activity profile observed with irilone(3) was very similar to that of genistein (6), which is inagreement with their structural similarity [4].

O

OOHOH

O

O

Irilone (3) Genistein

3.3. Biological activity of tyrolobibenzyls fromScorzonera humilis

In cooperation with C. Zidorn, R. Spitaler, H. Stuppner, E.-P.Ellmerer-Müller (Institut für Pharmazie und Organische Chemie,Universität Insbruck, Austria), and N.B. Perry (Department ofChemistry, University of Otago, Dunedin, New Zealand)

Recently, a new class of naturally occurring bibenzyl de-rivatives was discovered in the Asteraceae Scorzonerahumilis L., and a novel tyrolobibenzyl derivative, 1→6-β-D-apiosyl-β-D-glucopyranosyl 4-[2-(4-hydroxyphenyl)ethyl]-benzofuran-2-carboxylate (tyrolobibenzyl D) was isolated.Given the unique structure of this new class of compoundsand the fact that Scorzonera hispanica L., a related spe-cies, is used as a vegetable (black salsify), we investi-gated the potential chemopreventive activity of some re-lated tyrolobibenzyls, i.e. tyrolobibenzyls A and C (1, 4)and a semi-synthetic peracetyl derivative (1a). Com-pounds 1 (37 %) and 4 (15 %) demonstrated moderateand weak radical scavenger activity, respectively, in theDPPH assay at concentrations of 250 µM, whereas theacetyl derivative 1a was inactive at this concentration. Allthree tested compounds showed no measurable inhibitory(anti-oxidative) activity in the TPA-induced superoxide for-mation assay in concentrations of 100 µM. In contrast to

O

OOH

HO

OH

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other bibenzyls investigated earlier, compounds 1, 1a and4 showed only negligible inducing activity on QR with CDvalues above 50 µM. The influence on the enzyme Cyp1Awas weak (13 and 18 % inhibition, respectively) for com-pounds 1 and 4 and moderate for compound 1a (38 % in-hibition) in concentrations of 5 µM. Inhibitory activity ofcompounds 1, 1a, and 4 on the LPS-induced expressionof iNOS in Raw macrophages was also negligible with IC50

values above 50 µM. Cox-1 inhibitory activity of the testedtyrolobibenzyls was weak at a concentration of 100 µMwith inhibition values of 10 % (1), 15 % (1a) and 18 % (4),respectively. These data demonstrate that the substitutionof the bibenzyl moiety strongly influences biological activi-ties [5].

3.4. Isolation and potential cancer chemopre-ventive activities of phenolic compounds ofbeer

In cooperation with Alt, A. and Becker, H., Universität desSaarlandes, Saarbrücken

Beer contains a variety of phenolic compounds. During thebrewing process, some of these compounds are removedby polyvinylpolypyrrolidon (PVPP) to prevent haze forma-tion. We have analyzed the phytochemical composition ofa PVPP residue as well as of unstabilized beer and iso-lated a total of 51 compounds, belonging to various struc-tural classes, i.e. benzoic and cinnamic acid derivatives,acetophenones, flavonoids (chalcones, flavanones, fla-vones, flavan-3-ols, and proanthocyanidins) and somemiscellaneous compounds. Eight structures were identi-fied as novel, i.e. 2-(4�-hydroxyphenyl)-3,5-dihydroxyben-zoic acid, 2�-(4�-hydroxyphenyl)isoferulic acid ester,1,2,5,7-tetrahydroxyanthraquinon and 4,7-dihydroxy-5-(2�,4�,6�-trihydroxyphenyl)-indan-1,2-dione from the PVPPresidue, and catechin-7-O-β-(6�-O-nicotinoyl)-β-D-gluco-pyranosid, ent-epigallo-catechin-(4α→8, 2α→O→7) cat-echin, ent-epigallocatechin (4α→6, 2α→O→7)catechinand 2,3-cis-3,4-trans-2-[2,3-trans-3,3�,4�,5,7-pentahydroxy-flavan-8-yl]-4-(3,4-dihydroxyphenyl)3,5,7-trihydroxybenzo-pyran from the unstabilized beer. Most of the compoundswere tested for potential cancer chemopreventive activitiesin in vitro test systems detecting a modulation of carcino-gen metabolism (inhibition of Cyp1A activity, induction ofQR activity) and anti-inflammatory mechanisms (inhibitionof LPS-mediated iNOS induction), inhibition of Cox-1 activ-ity).

O

O

OHOH

OH

HO

1,2,5,7-Tetrahydroxyanthraquinon

1,2,5,7-Tetrahydroxyanthraquinone was identified as oneof the most interesting compounds. It inhibited Cyp1A ac-tivity with an IC50 of 0.07µM and induced QR activity with aCD value of 10.3µM. With respect to anti-inflammatorymechanisms, it reduced iNOS induction with an IC50 of33.8 µM, and also inhibited Cox-1 activity with an IC50 of7.2 µM. Importantly, this agent inhibited chemically-in-duced preneoplastic lesions in an ex vivo mouse mam-mary gland organ culture model (MMOC) with an IC50 of

0.1 µM, respectively.Our results demonstrate that beer is an interesting sourceof potential cancer chemopreventive agents and should befurther investigated with this respect [6].

3.5. Xanthohumol from hop (Humulus lupulus) asa novel potential cancer chemopreventiveagent

In cooperation with Alt, A. and Becker, H., Universität desSaarlandes, Saarbrücken.

Characterization and use of effective cancer chemopre-ventive agents has become an important issue in publichealth-related research. To this end, xanthohumol (XN), aprenylated chalcone from hop (Humulus lupulus) and aseries of natural and semi-synthetic analogs and relatedhop constituent were tested in a broad-spectrum of in vitrobioassays.

OOCH3

HO

OH

OH

Xanthohumol

Of all hop constituents and analogs tested, XN was identi-fied as the most promising agent with multiple hitherto un-known activities indicative of cancer preventive potential.XN has been reported previously to modulate carcinogenmetabolism and to act by cytotoxic/-static mechanisms;however, a conclusive characterization of its chemopre-ventive potential was missing. XN was able to scavenge avariety of physiological relevant radicals including peroxyl-,hydroxyl-, and superoxide anion-radicals more effectivelythan the known antioxidant Trolox. Anti-initiating mecha-nisms by modulation of enzymes involved in carcinogenmetabolism and detoxification were confirmed. For the firsttime, XN was characterized as an effective anti-inflamma-tory agent. It was found to inhibit both the constitutive formof cyclooxygenase Cox-1 and, more importantly, the induc-ible Cox-2 which is linked to carcinogenesis. In culturedRaw 264.7 murine macrophages, XN was shown to de-crease LPS-mediated iNOS induction. Another novel as-pect of chemopreventive potential of XN can be seen in itsmultiple anti-proliferative mechanisms. XN was found toinhibit human DNA polymerase α, the only eukaryotic poly-merase that can initiate DNA synthesis de novo. This ef-fect might be responsible for the previously described inhi-bition of cell growth. Using alkaline phosphatase inductionin the Ishikawa cell line, XN was identified as an anti-es-trogen without possessing estrogenic potential. Addition-ally, XN was found to induce terminal cell differentiation incultured HL-60 cells. Differentiation markers and a de-crease in cellular proliferation were detected at a concen-tration range of 0.8- 6.25 µM. Most importantly, XN atnanomolar concentrations prevented carcinogen-inducedpreneoplastic lesions in mouse mammary gland organ cul-ture (MMOC), providing a first direct proof for its chemo-preventive potential. Investigations on bio-availability, effi-cacy and safety in animal models are ongoing. Together,

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our data provide promising evidence for novel preventiveapplications of XN and hop products [7,8].

4. Mechanistic investigations of potential cancerchemopreventive agents

4.1. Ellagic acid induces antioxidant mechanismsin cultured human hepatocellular carcinomacells HUH-7

Dietary factors play an important role in modulating the de-velopment of certain types of human cancers. Chemopre-ventive agents are found in all foods, especially fruits andvegetables. Ellagic acid is a dietary polyphenol present infruits and nuts including raspberries, strawberries and wal-nuts. It possesses both anti-mutagenic and anti-carcino-genic activities, thus, anti-carcinogenic effects were dem-onstrated in various rodent chemoprevention models. El-lagic acid is known to modulate the activation of carcino-gens by inhibition of phase 1 cytochrome P450 enzymesand by induction of phase 2 enzymes which are involvedin carcinogen detoxification.The present study aimed to investigate whether antioxi-dant mechanisms contribute to its anti-carcinogenic activ-ity. In this context it was interesting to analyze whether el-lagic acid acted mainly as a direct antioxidant, based onits polyphenol structure, or might activate additional intra-cellular antioxidant mechanisms.

O

O

HO

HO

OH

OH

O

O

Ellagic acid

In the ORAC assay, ellagic acid exhibited a high scaven-ging capacity against different physiologically relevant re-active species, including ROO*, OH*, and Cu2+, and en-hanced significantly the antioxidant activities of both theprotein and nonprotein fractions of human hepatocellularcarcinoma cells HUH-7 cells against ROO* and OH* radi-cals. It significantly increased total intracellular thiol levelsand moderately elevated the GSH/GSSG ratio. The in-crease in total thiols was not only due to elevated GSHlevels, but mainly due to thiol-containing proteins, whichmight include enzymes like catalase, glutathione peroxi-dase and thioredoxin reductase, or cysteine-rich proteinslike metallothioneins (MT).Human metallothioneins (hMTs) are a conserved family ofheavy metal-binding proteins that participate in detoxifica-tion of transition metals such as Cd and Zn and protectagainst oxidative stress. Since MT isoforms have been re-ported to belong to the antioxidant responsive element(ARE)-regulated family of genes and MT was inducible bygenistein-treatment of cultured Caco-2 human colon carci-noma cells, the influence of ellagic acid on MT expressionwas further investigated. Using ELISA and RT-PCR tech-niques, EA was shown to moderately induce MT proteinlevels and to fivefold upregulate MT-1a mRNA expressionin HUH-7 cells, whereas MT-2a mRNA levels were about50% reduced. Transcriptional regulation of MT expressioninvolves the activation of metal response elements (MRE),

which are present in multiple copies in the proximal pro-moters of MT genes, by the MRE-binding transcription fac-tor-1 (MTF-1). In electrophoretic mobility shift assay(EMSA) analyses we could demonstrate that ellagic aciddifferentially up-and downregulated DNA binding of MTF-1and of transcription factor Sp1 to MREs (a-d), whereastranscription factor AP-1 DNA binding was not influenced.In addition, ellagic acid treatment time- and dose-depen-dently enhanced the activities of essential cellular antioxi-dant enzymes including catalase, glutathione peroxidaseand thioredoxin reductase. As a consequence, chemicallyinduced lipid peroxidation in HUH-7 cells, determined asmalondialdehyde levels, was effectively prevented by el-lagic acid treatment. Taken together, our studies supportthe role of ellagic acid as a cancer chemopreventive agentacting by multiple antioxidant mechanisms (publication inpreparation).

4.2. NF-κκκκκB as a molecular target of sulforaphane-mediated anti-inflammatory mechanisms

Sulforaphane is an aliphatic isothiocyanate found as aglucosinolate-precursor in cruciferous vegetables likebroccoli. Its chemopreventive activity, shown by inhibitionof chemically-induced rat mammary carcinogenesis, hasmainly been attributed to the modulation of carcinogenmetabolism.

SO

NCS Sulforaphane

As an additional mechanism of action, we demonstratedthat sulforaphane potently inhibits LPS-mediated nitric ox-ide (NO), PGE2 and TNF-α production in Raw 264.7 mac-rophages with IC50s of 0.7, 1.4 and 7.8 µM, respectively.Excessive production of NO during infection and chronicinflammation is considered as a causative factor of cellularinjury and cancer e.g. via nitrosative deamination and lipidperoxidation. Sulforaphane did not directly interact withNO, nor inhibited the enzymatic activity of iNOS or Cox-1.Rather, western blot analyses revealed a dose- and time-dependent decrease of LPS-stimulated iNOS as well asCox-2 protein expression (Gerhäuser et al., 1999).To further elucidate the mechanism of sulforaphane-medi-ated iNOS induction, RT-PCR analyses confirmed thatsulforaphane regulated iNOS expression at the transcrip-tional level. A pivotal transcription factor in LPS-mediatediNOS and Cox-2 induction is NF-κB. In unstimulated mac-rophages, it is located in the cytosol as a complex with IkB(inhibitor of NF-κB), which is phosphorylated and de-graded upon LPS-stimulation, thus releasing NF-κB andallowing nuclear translocation and initiation of transcrip-tion. In EMSA experiments sulforaphane was shown to im-pair DNA-binding of NF-κB without affecting the DNA-bind-ing of transcription factors AP-1 and C/EPBβ. Using west-ern blotting and immunofluorescence detection of I-κB andNF-κB subunits p65 and p50, respectively, we could dem-onstrate that sulforaphane does neither interfere with in-duced degradation of IκB nor with nuclear translocation ofNF-κB. Interestingly, treatment of Raw macrophages withsulforaphane caused a rapid decrease in intracellular GSH

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levels, which might influence the redox-sensitive activa-tion, translocation and transactivation of NF-κB. Taken to-gether, our data indicate that sulforaphane mediates itschemopreventive effects additionally by anti-inflammatorymechanisms [9].

4.3. Induction of HL-60 cell differentiation bysesquiterpene lactones

In cooperation with C.A. Klaas and I. Merfort (Institute of Pharma-ceutical Biology, Albert-Ludwigs-University, Freiburg) and V.Castro (Escuela de Quimica, Universidad de Costa Rica, SanJose, Costa Rica).

Cancer can be regarded as an imbalance between cellproliferation and cell differentiation, i.e. cell maturation anddevelopment to a defined cell type. Consequently, induc-tion of cell differentiation to a normal, not cancerous phe-notype is regarded as a valid mechanism of cancerchemoprevention and chemotherapy. Naturally occurringsesquiterpene lactones (SLs) have been shown previouslyto possess anti-inflammatory and anti-tumoral potential.Anti-inflammatory activity was linked to the inactivation ofNF-κB by alkylation of its p65 subunit. To investigate apossible correlation between NF-κB inhibition and induc-tion of cell differentiation, we utilized the human promyelo-cytic leukemia cell line HL-60. Cellular differentiation tomorphological and functional mature granulocytes, mono-cytes or macrophages was determined by monitoring cel-lular properties, i.e. reduction of nitroblue tetrazolium(NBT) after TPA-challenge, appearance of nonspecific(NSE)/specific acid esterase (SE) and a decrease in cellu-lar proliferation.

OO

O

Parthenolid Dihydrohelenalin acetate

For SLs possessing a reactive α-methylene-γ-lactone moi-ety, e.g. Parthenolid, we observed good correlation be-tween inhibition of NF-κB and of cell proliferation (r2=0.96),but no correlation with induction of cell differentiation.Rather, weak NF-κB inhibitors, including α-methylene-butyrolactone used as a reference compound and SLswithout the α-methylene-γ-lactone moiety, e.g. dihydro-helenalin acetate (DH-Ac), were identified as potent differ-entiation inducers by NSE straining, indicating cell matura-tion along the monocytic lineage. Taken together, we coulddemonstrate that induction of HL-60 cell differentiation bySLs is independent of NF-κB inhibition (Gerhäuser et al.2001).To obtain further information on molecular targets of SLsinvolved in the induction of cell differentiation, we appliedan array-based gene expression screening (SuperArray@)using DH-Ac as a model compound. In general, after DH-Ac treatment, expression levels of factors promoting cellcycle progression, such as cyclin-dependent kinase 1 and4, and the universal transcription factor E2F, were de-creased. mRNA levels of cell cycle inhibitors such as p21and p57 were elevated, which is consistent with their pro-posed role in cell cycle regulation. Further Western blot

data confirmed these findings on the protein level. Theseresults implicate that the induction of differentiation in HL-60 cells by SLs is tightly associated with cell cycle regula-tion by an intricate network of various cell cycle-relatedgenes (publication in preparation).

4.4. Differential effects of sodium butyrate andtrichostatin A on differentiation induction incolonic cancer cell lines

In cooperation with M. Jung (University of Münster).

Histone deacetylase (HDAC) inhibitors have been shownto induce cell differentiation in various cancer cell types invitro. The aim of the present work was to investigate themolecular basis of differentiation induction in colonic can-cer cell lines treated with two types of HDAC inhibitors,namely sodium butyrate (SB) and trichostatin A (TSA) [10,11].We measured induction of the brush border glycoproteinalkaline phosphatase (ALP) as a marker of cell differentia-tion in the wild-type (wt) HCT 116 adenocarcinoma cell lineand a p53 (-/-) and p21(-/-) derivative thereof (generouslyprovided by Dr. B. Vogelstein). ALP activity was deter-mined by fluorimetric detection of the dephosphorylation of8-methyl-umbelliferylphosphate. Histone acetylation wasassessed by AUT-PAGE. Further, expression ofp21waf-1¶Cip-1, p27, p57, E2F, and phosphorylation of pRbwere determined by Western blot analyses.By comparing the results of ALP induction obtained withthe wt HCT 116 cell line and the p53 (-/-) and p21 (-/-) de-rivatives thereof, we could demonstrate that SB-mediatedinduction of cell differentiation was dependent on p21, butindependent of p53. In HCT 116 cells, SB-treatment led toa significant induction of p21 levels 12 and 24 h post-treat-ment. On the other hand, TSA treatment of wt HCT 116cells resulted in growth inhibition, but caused no inductionof ALP activity as a differentiation marker. Since TSA treat-ment still induced hyperacetylation of histone H4 and p21expression, these parameters seemed to be not sufficientfor differentiation induction. Importantly, SB and TSA dif-fered with respect to their effect on Rb phosphorylation:Upon TSA treatment, pRb was mainly hypo-phosphory-lated in HCT 116 (consistent with enhanced expression ofp21), but hyper-phosphorylated in the p21 (-/-) derivative.The effect of SB was weaker in the HCT 116 cell line. Inthe p21 (-/-) derivative, SB treatment still resulted ingrowth arrest and hypo-phosphorylation of pRb, indicatingan effect of SB on Cdk-inhibitors other than p21. Conse-quently, we analyzed the expression levels of additionalcell cycle regulating proteins. Upon treatment with SB, thecdk inhibitors p27 and p57 were also induced in a time-dependent manner. The level of underphosphorylated Rbslightly increased after 24 h treatment, whereas the ex-pression of E2F was significantly reduced. By immunopre-cipitation analyses using Rb antibody, we could demon-strate reduced E2F binding to Rb. This is most likely dueto reduced E2F protein levels and not a result of the modi-fications in the phosphorylation status of Rb.Despite the differences on Rb phosphorylation, experi-ments using DNA array technology (SuperArray@) dem-onstrated an overall similar pattern of mRNA expression in

O

H

OOAc

O

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SB- and TSA-treated HCT 116 cells. Egr-1 and p19 mRNAexpression were significantly induced by both compounds.Selected genes (cdk6, p53 and skp1) were differentiallyexpressed. Overall, we conclude that hyperacetylation ofhistone H4 and induction of p21 seem to be essential, butnot sufficient for differentiation induction by HDAC inhibi-tors in HCT 116 cells (publication in preparation).

5. In vivo investigations5.1 Influence of curcumin on tumor development

in the Long Evans Cinnamon (LEC) rat modelIn cooperation with F. Amelung (Division of Cellular and MolecularPathology, DKFZ) and Horst Wesch (Division of Oncological Diag-nostic and Therapy, DKFZ)

Long-Evans Cinnamon (LEC) rats, an inbred mutant strainwhich accumulates copper due to an aberrant copper-transporting ATPase gene, develop acute hepatitis,chronic liver injury and liver tumors as a result of copper-induced oxidative stress, lipid peroxidation and DNA-dam-age. Curcumin, an antioxidant and anti-inflammatorychemopreventive agent, has shown anticancer propertiesin many rodent models. We investigated the modulatingrole of curcumin on liver and kidney carcinogenesis inLEC-rats. Two groups of 4 wks-old LEC-rats (n=60 each)were fed either a standard diet (control) or received 0.5%curcumin in the diet for life. In untreated LEC-rats, the rateof acute liver failure, the incidence of liver tumors and ofkidney tumors were 32%, 100% and 10% respectively,which was not altered by curcumin treatment. However,curcumin reduced tumor incidence at other organ sites(15% vs 0%; p = 0.025) and suppressed formation of me-tastases (18% vs 0%; p = 0.01). Median survival time wasdecreased from 88.7 to 78.1 wks in curcumin treated rats(p = 0.002). The lack of chemoprevention of liver and kid-ney tumors in LEC-rats by curcumin may be caused by en-hanced toxicity and oxidative stress due to excess copper.We conclude that curcumin should be contra-indicated forpatients suffering from inherited and acquired metal stor-age diseases that include patients with hepatitis C virusinfection [12].

5.2. Prevention of ischemic stress in rat muscleby proline dithiocarbamate

In cooperation with S.T. Lille (Dept. of Chemistry and Biochemis-try, Arizona State University, Tempe, AZ, USA).

NF-κB is thought to play an important role in the expres-sion of genes expressed in response to ischemia/reperfusion (I/R) injury. In this study, the activation of NF-κB in rat skeletal muscle during reperfusion following afour hour ischemic period was investigated. NF-κB activa-tion displayed a biphasic pattern, showing peak activitiesfrom 30 min to 3 h postperfusion and 6 h to 16 h post-perfusion with a decline to baseline activity levels between3 and 6 h. Inhibition of NF-κB activation was investigatedusing the chemopreventive agent proline dithiocarbamate(Pro-DTC). NF-κB activity during reperfusion was signifi-cantly reduced by iv administration of Pro-DTC. Addition-ally, Pro-DTC resulted in decreased muscle edema andneutrophil activity with increased percentage of musclesurvival compared to vehicle controls. These results dem-

onstrated that NF-κB is activated during reperfusion in abiphasic manner and that the regulation of the initial phaseof NF-κB activation by Pro-DTC afforded physiologic pro-tection against a severe ischemic stress [13].

Publications (* = external co-author)[1] Gerhäuser, C. Mechanismen der Krebsentstehung -Ansatzpunkte für die Krebs-Chemoprävention. Ernährungs-Umschau 48 (2001) 48 - 51.[2] Gerhäuser, C. Flavonoide und andere pflanzliche Wirkstoffe.Was hat praktische Relevanz? Sollen wir unser Essverhaltenändern? Akt. Ernähr. Med. 26 (2001) 1-7.[3] Gerhäuser, C.; Klimo, K.; Heiss, E.; Neumann, I.; Gamal-Eldeen, A.; Knauft, J.; Liu, G.; Sitthimonchai, S.; Frank, N.Mechanism-based in vitro screening of potential cancerchemopreventive agents. Mutation Research, in press.[4] Wollenweber, E.*; Stevens, J.F.*; Klimo, K.; Knauft, J.;Frank, F.; Gerhäuser, C. Cancer Chemopreventive in vitro Activi-ties of Isoflavones Isolated from Iris germanica. Planta Medica 69(2003) 15-20.[5] Zidorn, C.*; Spitaler, R.*; Ellmerer-Müller, E.P.*.: Perry, N.B.*;Gerhäuser, C.; Stuppner, H.*: Structure of Tyrolobibenzyl D andbiological activity of tyrolobibenzyls from Scorzonera humilis. Z.Naturforsch. 57 (2002) 614-619.[6] Gerhäuser, C.; Alt, A.P.*; Klimo, K.; Knauft, J.; Frank, N.;Becker, H.*: Isolation and potential cancer chemopreventive ac-tivities of phenolic compounds of beer. Phytochemistry Reviews,in press.[7] Gerhäuser, C.; Alt, A.*; Heiss, E.; Gamal-Eldeen, A.; Klimo, K.;Knauft, J.; Neumann, I.; Scherf, H.-R.; Frank, N.; Bartsch, H.;Becker, H.*: Cancer chemopreventive activity of Xanthohumol, anatural product derived from hop. Molecular Cancer Therapy 1(2002) 959-969.[8] Gerhäuser, C.; Alt, A.*; Heiss, E.; Gamal-Eldeen, A.; Klimo, K.;Knauft, J.; Neumann, I.; Nookandeh, A.*; Scherf, H.; Frank, N.;Bartsch, H.; Becker, H.*: Identification and cancerchemopreventive potential of Xanthohumol, a prenylated chal-cone from hop (Humulus lupulus L.) Hopfenrundschau Interna-tional 2002/2003 (2002) 50-55.[9] Heiss, E.; Herhaus, C.; Klimo, K.; Bartsch, H.; Gerhäuser, C.Nuclear factor-κB is a molecular target for sulforaphane-mediatedanti-inflammatory mechanisms. Journal of Biological Chemistry276 (2001) 32008-32016.[10] Wittich, S.*; Scherf, H.R.; Xie, C.; Brosch, G.*; Loidl, P.*;Gerhäuser, C.; Jung, M.*: Structure-activity relationships on phe-nylalanine containing inhibitors of histone deacetylase - In-vitroenzyme inhibition, induction of differentiation and inhibition of pro-liferation in erythroleukemic cells. Journal of Medical Chemistry45 (2002) 3296-3309.[11] Wittich, S.*; Scherf, H.; Xie, C.P.; Heltweg, B.*; Dequiedt, F.*;Verdin, E.*; Gerhäuser, C.; Jung, M.*: A structure-activity study onbiphenylalanine inhibitors of histone deacetylase, submitted.[12] Frank, N.; Knauft, J.; Amelung, F.; Nair, J.; Wesch, H.;Bartsch, H. No prevention of liver and kidney tumors in Long-Evans Cinnamon rats by dietary curcumin, but inhibition at othersites and of metastases. Mutation Research (2002),www.sciencedirect.com, 9466, 1-9.[13] Lille, S.T.*; Lefler, S.R.*; Mowlavi, A.*; Suchy, H.*; Boyle, Jr.E.M.*; Farr, A.L.*; Su, C.-Y.*; Frank, N.; Mulligan, D.C.*: Muscleand Nerve 24 (2001) 534-541.[14] Gerhäuser, C. and Frank, N. New Promising Chemopreven-tive Agents and Mechanisms. In: Handbook of Experimental Phar-macology. Harri Vainio: Springer Verlag, Heidelberg (in press).

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[15] Patents: Nr. 100 15 525.1. Synthetische Derivate vonLunularsäure, Arzneimittel enthaltend diese Verbindungen,Verfahren zur Herstellung der Lunularsäurederivate sowie derenVerwendung. Erf. Gerhäuser, Eicher*, Pick*: Patentanmeldungeingereicht beim Deutschen Patentamt am 30.3.2000,Internationale Patentanmeldung PCT/DE01/01264 am 30.3.2001.External funding:Wissenschaftsförderung der Deutschen Brauereiwirtschaft e.V.1.11.1999 - 31.12.2001 DM 280.500,- Dr. C. Gerhäuser/Prof. H.Becker (Uni Saarbrücken), 1.1.2002 - 31.7.2003, � 129.011,- Dr.C. Gerhäuser/Prof. H. Becker (Uni Saarbrücken); BMBF:1.7.2002-30.6.2005, � 314.232,- Dr. C. Gerhäuser/Dr. N. Frankplant constituents, NADP(H):quinone oxidoreductase, nuclear fac-tor NF-κB, Thai medical plants, dehydrohelenalin acetate, coppertoxicity.

Genetic Toxicology and DNA Repair (C0203)Group Leader : Dr. Peter Schmezer

P. Schmezer and O. Popanda

Cellular DNA is constantly attacked by both reactive spe-cies inside the cell and environmental agents. About 130human DNA repair or repair-related genes are currentlyknown which continuously monitor the cellular DNA in or-der to minimize toxic and mutagenic consequences result-ing from this endogenously and exogenously induced DNAdamage. Deficiencies in DNA repair systems may lead tothe development of cancer. It is therefore our aim to de-velop and apply sensitive methods to detect different typesof DNA damage and to study the repair of these lesions.We have focused our work on the identification of high riskindividuals in human population studies: In cooperationwith epidemiological and clinical partners, an optimisedmicrogel electrophoresis technique (alkaline comet assay)is used to monitor cellular mutagen sensitivity (induced bychemicals or radiation) and DNA repair capacity in periph-eral blood lymphocytes [1, 2]. Additionally, PCR tech-niques are applied to identify individuals carrying specificrepair and metabolising enzyme polymorphisms [9,10,15].Furthermore, gene expression of DNA repair enzymes isstudied using DNA array technology and quantitative real-time RT-PCR [12]. Using these methods, impaired DNArepair capacity and enhanced mutagen sensitivity arestudied as potential markers for the identification of indi-viduals with (i) a high risk for cancer, and (ii) an enhancedrisk to develop clinical side effects to radiation therapy.The identification of these high risk individuals has sub-stantial preventive implications or leads to therapeuticconsequences as in the case of radiotherapy patients.These high risk individuals could be targeted for intensivecancer screening, and they could be enrolled into chemo-preventive trials. A new activity of the group consists in thesearch and evaluation of compounds capable to inducecellular repair mechanisms [18].Genotoxicity is a property of most human and many rodentcarcinogens, but often a (tissue)-specific metabolism hasto occur to form the ultimate DNA reactive species. Wehave therefore used metabolically active mammalian cells(human and rodent) freshly isolated from various organs/tissues to study the genotoxic activity of compounds. Thisgoal can effectively be reached only when assays are

used which allow the detection of genotoxicity on thesingle cell level. In this case cells isolated from small hu-man biopsies can be analyzed. The alkaline comet assayis an appropriate method for this purpose. Our researchactivities included investigations on airborne genotoxic/carcinogenic compounds, mainly in the respiratory tract[4, 5,6,8, 13]. To identify possible genotoxic carcinogens,mutagenicity was also studied in transgenic rodent muta-tion assays (BigBlue®, Muta®Mouse)[7].

1. DNA repair capacity and mutagen sensitivity asrisk markers for non-small cell lung cancer

P. Schmezer, N. Rajaee-Behbahani, A. Risch,R. GliniorzIn cooperation with: W. Rittgen, Biostatistics Unit, DKFZ;P. Drings, H. Dienemann, K.W. Kayser, V. Schulz, ThoraxklinikHeidelberg-Rohrbach

Individual susceptibility to carcinogens is an important de-terminant of disease risk. It is influenced by host factorssuch as the ability to repair DNA lesions. We have devel-oped a microgel electrophoresis assay for use in molecu-lar epidemiological studies in order to identify subjectswho are at high risk [1]. The assay was validated in acase-control study on non-small cell lung cancer [2]. Pe-ripheral blood lymphocytes were collected from 160 can-cer patients and 180 control patients without cancer andfrom the same hospital, and stored at -80°C. After thaw-ing, the phytohaemagglutinin-stimulated cells were treatedwith bleomycin at 20 µg/ml for 30 min and the extent ofDNA damage and DNA repair capacity were determined:Bleomycin sensitivity was significantly higher in lung can-cer patients than in tumor-free hospital controls(p<0.0001). DNA repair capacity, after 15 min repair time,in lymphocytes of non-small cell lung cancer patients andcontrols was 67% and 79.3%, respectively (p<0.0004).There was no correlation, in either patient or control group,between the bleomycin sensitivity and DNA repair capacitywith age or gender. The median values of DNA repair ca-pacity and sensitivity in controls were used as cut-offpoints for calculating odds ratios (OR). After adjustment forage, gender and smoking status, the cases vs. controlshad reduced DNA repair capacity (OR = 2.1; 95% confi-dence limit: 1.1-4) and increased bleomycin sensitivity(OR = 4; 95% confidence limit: 2.2-7.4). Both endpointswere independent risk factors for smoking-related cancer.Repeated analysis of peripheral lymphocytes from thesame individual demonstrated good reproducibility of theassay. Cryopreservation of the lymphocytes for more than12 months did not significantly affect their sensitivity.These results show that our standardised microgel electro-phoresis assay is suitable for determining individual sensi-tivity to mutagens and DNA repair capacity: It is sensitiveand faster than cytogenetic assays, and it can be appliedto native and cryopreserved peripheral blood lymphocytes.Validation of this assay in large prospective studies for theidentification of subjects at high cancer risk is now war-ranted.

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2. Expression profiles of DNA repair genesP. Schmezer, C. Mayer, J. Hümmerich,G. Werle-Schneider, O. Zelezny, P. Waas,O. PopandaIn cooperation with: W. Rittgen, Biostatistics Unit, DKFZ; A. Bach,M.C. von Brevern, Axaron Bioscience AG, Heidelberg

For characterisation and identification of DNA repair geneswhich might be responsible for an impaired cellular DNArepair capacity, we developed cDNA arrays for expressionanalysis of human DNA repair genes: PCR fragmentswere amplified from IMAGE cDNA clones of genes whichare known to be directly or indirectly involved in DNA re-pair. Following verification by sequence analysis, the frag-ments were spotted onto arrays. These cDNA arrays arenow used to produce transcriptional profiles from individu-als with high and low cellular DNA repair capacity. We andothers are using this individual capacity to repair DNAdamage in peripheral blood lymphocytes (PBLs) as a bio-marker for cancer risk. As the cell�s ability to remove DNAdamage may be correlated with proliferative activity, it isan important question whether quiescent or dividing cellsshould be used in such studies. It was therefore the aim ofa pilot study to compare DNA repair capacity and expres-sion profiles of 70 known DNA repair genes, both in rest-ing and phytohemagglutinine (PHA) stimulated humanPBLs [12]. Using the alkaline comet assay γ-radiation-in-duced DNA damage and repair in lymphocytes was ana-lysed. No difference, neither in the rate of radiation-in-duced DNA damage nor in DNA repair capacity betweenPHA-stimulated and non-stimulated PBLs was observed.Stimulated cells, however, showed significantly elevatedvalues for background damage. Transcriptional profiles ofrepair genes were analysed using our newly developedcDNA arrays. Hybridisation experiments were performedwith mRNA isolated from both unstimulated and PHA-stimulated PBLs. More than 70 % of all evaluated geneshad constant expression levels. Twelve genes respondedwith a more than 2-fold (max.: >18-fold) increase of tran-scripts to the mitogenic stimulus. Most of the up-regulatedrepair enzymes are also known to play a role in DNA repli-cation. We concluded from our data that all repair proteinsneeded for the repair of γ-irradiation induced DNA-dam-age, that can be detected by the alkaline comet assay, arealready present in G0 cells at sufficient amounts and donot need to be induced once lymphocytes are stimulatedto start cycling. Our results thus do not support a generalincrease in DNA repair activity of PBLs by PHA stimula-tion, and the use of stimulated PBLs in molecular epide-miological studies on DNA repair of γ-irradiation inducedDNA damage seems not to be mandatory. Further studiesare currently performed to optimise our expression profil-ing technology. The cDNA arrays have been extended tocarry sequences which represent now more than 100 hu-man DNA repair or repair-related genes.

3. Radiation-induced DNA damage and repair inlymphocytes from cancer patients and theircorrelation with side effects to radiotherapy

O. Popanda, R. Ebbeler, P. Waas, O. Zelezny,J. Hümmerich, P. SchmezerIn cooperation with: J. Chang-Claude, D. Twardella, I. Helmbold,Clinical Epidemiology; J. Debus, Radiation Oncology;H.W. Thielmann, Interactions of Carcinogens with Biological Mac-romolecules, all DKFZ; D. von Fournier, Department of Gyneco-logical Radiology, Heidelberg University Hospital, Heidelberg; W.Haase, Clinic for Radiotherapy and Radiooncology, St.Vincentius-Hospital Karlsruhe; M.L. Sautter-Bihl, Clinic for Radio-therapy, Karlsruhe Hospital GmbH, Karlsruhe; F. Wenz, Depart-ment of Radiation Oncology, Universitätsklinikum Mannheim,Mannheim

Repair of radiation-induced DNA damage plays a criticalrole for both a patient�s susceptibility to side effects afterradiotherapy and his subsequent cancer risk. Study objec-tive is to evaluate whether DNA repair data determined invitro are correlated with the occurrence of acute side ef-fects during radiotherapy. For this purpose, breast cancerpatients receiving radiation therapy after a breast-conserv-ing surgery were investigated in a prospective epidemio-logical study [21]. As an indicator for clinical radiosensitiv-ity, adverse reactions of the skin irradiated during radio-therapy were recorded. For the determination of DNA re-pair capacity, cryo-preserved lymphocytes from 113 studyparticipants were γ-irradiated with 5 Gy in vitro and ana-lysed using the alkaline comet assay. Reproducibility ofthe assay was determined by repeated analysis (n=26) ofcells from a healthy donor. A coefficient of variation of 0.3was calculated. The various parameters determined tocharacterize the individual DNA repair capacity (back-ground DNA damage, DNA damage induced by radiationand DNA repair capacity after 15 and 30 min) showedlarge differences between patients. 11 patients were identi-fied with considerably enhanced DNA damage inductionand 7 patients exhibited severely reduced DNA repair ca-pacity after 15 and 30 min. Six patients were consideredas clinically radiosensitive indicated by moist desquama-tion of the skin after a radiation dose of about 50 Gy. Us-ing the alkaline comet assay as described here, breastcancer patients were identified which showed abnormalcellular radiation effects. However, the comparison of ex-perimental and clinical results revealed that repair defi-ciency of lymphocytes occurred only at a very limited ex-tent in patients exhibiting acute radiation sensitivity of theskin. Thus, development of acute side effects of the skincannot be explained by the measured defects in DNA re-pair. In some lymphocyte samples, a considerable amountof radiation-induced DNA damage remained after an incu-bation time of 30 min. Therefore, future studies should in-clude the measurement of slowly repaired DNA lesions. Asimpaired DNA repair could be involved in the developmentof late irradiation effects, individuals exhibiting severely re-duced DNA repair capacity should be followed for the de-velopment of late clinical symptoms. To identify a furtherpossible marker for radiation sensitivity, we are now com-paring DNA repair parameters and expression profiles ofDNA repair genes in prostate cancer patients (see chapter2).

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4. The role of DNA polymorphisms in DNA repairgenes

O. Popanda, T. Schattenberg, C.T. Phong, P. Waas,A. Risch, P. SchmezerIn cooperation with: L. Edler, Biostatistics Unit, DKFZ; P. Drings,H. Dienemann, K.W. Kayser, V. Schulz, Thoraxklinik Heidelberg-Rohrbach

Polymorphisms in DNA repair genes have been suggestedto modulate the individual risk for lung cancer. As reducedDNA repair capacity is known to be associated with elev-ated lung cancer risk, we are investigating the influence ofDNA repair gene variants on cellular DNA repair capacityin an ongoing lung cancer case-control study (see chapter1). Individual DNA repair capacity was determined in pe-ripheral blood lymphocytes using the alkaline single-cellmicrogel electrophoresis (comet) assay and bleomycin asa DNA-damaging agent [2]. Impaired DNA repair capacitywas significantly associated with increased lung cancerrisk (OR=2.1fold, p<0.001). Four DNA polymorphismswere analysed for which a correlation with increased lungcancer risk has been reported: XRCC1 (Arg399Gln),XRCC3 (Thr241Met), and XPD (Lys751Gln andAsp312Asn). These variants are localised in three genesof different DNA repair pathways. The allele distributionbetween cases (172 individuals) and controls (146 indi-viduals) was very similar for the XRCC1 and XRCC3genotypes. The XPD-variants Gln/Gln and Asn/Asn weredetected with a higher frequency in cases; this differencedid not reach significance, possibly due to the limited num-ber of individuals screened so far. Furthermore, 53 indi-viduals were homozygous for variant bases at 2 or 3 of the4 sites investigated. These combinations were found to bemore frequent among cancer cases than controls (12% incontrols vs 21% in cases; p=0.03). In fact, these 53 indi-viduals showed an enhanced risk for lung cancer(OR=1.9; p=0.05). 155 lung cancer cases and 129 tumor-free control subjects were investigated by the comet as-say. DNA repair capacity was not affected by the genevariants analysed. Our interim results indicate that lungcancer risk is increased with impaired DNA repair capacityand a combination of sequence variations in DNA repairgenes. For a more comprehensive characterization of howgene variants affect DNA repair capacity and cancer risk,we are investigating the effects of additional DNA repairgene variants in a larger number of individuals in furtherstudies.

5. Genetic variants in replicative DNApolymerases from tumor cells

O. Popanda, P. WaasIn cooperation with: H.W. Thielmann, J. Dai, A. Hunziker (DKFZ)

Mutated constituents of the DNA replication complex mightcontribute to the mutational load of the genome during tu-mor development by impairing DNA synthesis as well ascell cycle-related control of DNA replication. For example,mutations in genes coding for DNA polymerases might im-pair their catalytic functions, particularly their copying fidel-ity. In consequence, a large number of mutations would

accumulate in replicated genes, contributing to the step-wise development of cancer. To prove or disprove this hy-pothesis, we looked for mutations in the cDNA sequencesof the four subunits of the DNA polymerase α-primasecomplex from both highly malignant Novikoff hepatomacells and regenerating normal rat liver, and ii) comparedphysicochemical and catalytic properties of the DNA poly-merase α-primase complexes purified from both sources[3]. i) Sequence analysis revealed two mutations insubunit B from Novikoff cells: one in nucleotide position855 (CCG→CCA) that did not result in an amino acid ex-change, and one in position 862 (GTG→ATG) that causeda change of Valine to Methionine in codon 288. In thethree other subunits, no mutation was found. The wild typeand the mutated sequence of subunit B were cloned andexpressed in vitro. Sedimentation analysis of both poly-peptides revealed different sedimentation constants indi-cating that the amino acid exchange affected the proper-ties of subunit B. ii) The biochemical characterization ofthe purified enzymes revealed a sedimentation valuewhich was significantly higher for the enzyme complexfrom normal liver than for that from Novikoff cells. In addi-tion, DNA polymerase α-primase complexes from Novikoffcells showed higher sensitivity to camptothecin, topotecan,and structurally related compounds (such as 7-ethyl-10-hydroxycamptothecin (SN-38), 9-aminocamptothecin and10-hydroxycamptothecin) than the enzyme from normal ratliver. In conclusion, the change in subunit B caused bythe amino acid variation probably imparts altered confor-mational strain on its binding partners in the DNA poly-merase α-primase complex from Novikoff hepatoma cells.It thus may influence the overall catalytic properties of thecomplex. Whether this mutation influences genetic insta-bility or tumor development needs to be explored. To fur-ther analyse the effect of genetic variants, such variants inDNA replication and repair genes including DNA poly-merases will be studied in cooperation with clinical andepidemiological partners (see chapter 4).

6. Quantitative assessment of poly(ADP-ribosyl)ation

P. Schmezer, N. Rajaee-Behbahani, R. GliniorzIn cooperation with: A. Bürkle, University of Konstanz; H. Becher,Department of Tropical Hygiene and Public Health, University ofHeidelberg; H. Ramroth, Division of Clinical Epidemiology, DKFZ;A. Dietz, Department of Otolaryngology, Head and Neck Surgery,University of Heidelberg

Poly(ADP-ribose) polymerase (PARP), a nuclear enzymethat is catalytically activated by DNA strand breaks, playsa complex role in DNA repair. Using NAD+ as precursor, itcatalyses the formation of ADP-ribose polymers, which areattached to various proteins including DNA repair en-zymes. As defects in DNA repair pathways have been as-sociated with increased risks for cancer in humans, we in-vestigated whether differences in the activity of PARP areassociated with the risk for laryngeal cancer. In a case-control study on genetic, lifestyle and occupational riskfactors for laryngeal cancer, the PARP activity was as-sessed as DNA damage-induced poly(ADP-ribose) forma-

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tion in human peripheral blood lymphocytes by quantita-tive immunofluorescence analysis [14]. Polymer formationwas determined as the cellular response to bleomycin, awell-known inducer of DNA strand breaks, in lymphocytesfrom 69 laryngeal cancer patients and 125 healthy popula-tion controls. The frequency of bleomycin-induced polymerformation, measured as mean pixel intensity, was signifi-cantly lower in cases (74.6; standard error [SE] = 3.7) thanin controls (94.5; SE = 3.5) and was not influenced bysmoking, age or sex. There was no significant differencebetween cases (59.1; SE = 5.2) and controls (50.5; SE =3.7) in basal polymer formation (in cells not treated withbleomycin). When the lowest tertile of polymer formationwas used as the reference, the odds ratio (OR) for the low-est tertile of bleomycin-induced polymer formation was3.79 (95% confidence interval [CI], 1.37-10.47, p = 0.01).Peripheral blood lymphocytes from laryngeal cancer pa-tients thus showed significantly less bleomycin-inducedpoly(ADP-ribose) formation. Our results suggest that a re-duced capacity of somatic cells to synthesize poly(ADP-ribose) might be associated with an increased risk for la-ryngeal cancer. The underlying mechanism remains to beinvestigated.

7. Influence of natural compounds on poly(ADP-ribosyl)ation

B. Bertram, U. Bollow, P. SchmezerIn cooperation with: A. Bürkle, University of Konstanz

With regard to a future use of natural compounds capableof inducing cellular repair mechanisms in intervention trialswith individuals at high cancer risk, we performed experi-ments with (-)-epigallocatechin gallate (EGCG), the mainconstituent of green tea. Tea does not belong to the largegroup of botanicals with mutagenic and carcinogenic po-tential, but shows remarkable preventive effects againstthe development of cancer and cardiovascular disease[11,16,17,19,20]. We studied effects of EGCG onpoly(ADP-ribose) (PAR) levels and on DNA-damage in hu-man peripheral blood lymphocytes [18]. A dose and timedependent increase of both PAR formation as assessedby quantitative immunofluorescence analysis and DNA-damage as assessed by the alkaline comet assay wereobserved after treatment with EGCG at 20, 40 and 80µMfor 10 to 240 min. Maximum levels of PAR formation andof DNA damage were observed after 10 minutes at all con-centrations tested. Increased PAR levels were still detect-able by 240 min in the 40 and 80µM groups. At the lowestconcentration which is near the physiological values foundafter tea ingestion, PAR formation was not correlated withDNA damage. Here, EGCG led to pronounced PAR levels,whereas the comet assay was almost negative. In con-trast, such marked differences in time course and extent ofboth genotoxicity and PAR formation following EGCGtreatment were not detected after γ-irradiation. Our resultssuggest that the known chemopreventive effects of EGCG,representing the main constituent of tea, may be partly at-tributed to an induction of PAR formation. The capacity ofEGCG to restore repair processes will be further investi-gated in repair deficient cells.

8. Transcription profiling in predictive toxicologyG. Werle-Schneider, W. Beerheide , H. Bartsch, M. BartelmannIn collaboration with C.K. Behrens, M.C. v. Brevern, J. Scheel, T.Storck, A. Bach, AXARON Bioscience AG, Heidelberg; D. Müller,R. Glöckner, Institute of Pharmacology and Toxicology, Friedrich-Schiller-University, Jena; J. Hengstler, M.Ringel, Institute of Toxi-cology; Johannes Gutenberg-University, MainzSupported in part by the Bio-Regio-Project 0311942

Classical toxicology testing using animals as model sys-tems like the rodent cancer bioassay are expensive, time-consuming and require large numbers of animals. There-fore more efficient and alternative testing methodologieshave to be developed. In vitro techniques have beenevolved to measure toxicity, many of which measure toxi-cant-induced DNA damage, but fail to identify non-DNA re-active carcinogens, like tumor promoters. Based on thefact that toxicity is often preceded by, and results in alter-ations of gene expression, transcription profiling technol-ogy is promoted as a alternative to traditional rodent bio-assays to identify and assess the safety of chemicals anddrug candidates in drug development. The emerging newsubdiscipline, termed toxicogenomics, is based on the ex-pectation, that gene expression profiles will be unique fora particular class of compounds. By using hierarchicalclustering of transcription profiles which are focused onselected marker genes, the hypothesis has to be testedthat gene expression profiles of compounds with similartoxicity cluster together.In this project �toxicology-focused� DNA micro arrays[Storck T, et al., Curr Opin Drug Discov Devel 5 (2002) 90]containing 1600 selected genes of the rat genome wereused to investigate the transcription profiles of differentknown tumor promoters from three distinct hepatotoxicclasses: Enzyme inducers [phenobarbital (PB), α-hexa-chlorocyclohexane (HCH) and cyproterone acetate (CP)],peroxisome proliferators [WY-14643, dehydroepiandro-sterone (DHEA), ciprofibrate (CF) and nafenopine], andfinally the hormone ethinylestradiol (EE). Generating com-pound specific transcription profiles, marker genes shouldbe identified which are predictive and characteristic for theaction of tumor promoters.Our collaboration partner Axaron Bioscience AG couldshow that different classes of promoting agents in the livercan be classified according to their effect on gene expres-sion profiles in rat liver after in vivo treatment.For drug screening, there is however a need for high-throughput in vitro assays which should reflect the in vivoconditions.Development of an in vitro test system to analyse gene ex-pression patterns which are predictive for the action ofnon-genotoxic carcinogens.To develop an in vitro tool for gene expression studies, dif-ferent systems derived from rat liver were analysed: A cellline established from rat liver (C2I [Mayer D and SchäferB. Exp Cell Res 138(1982)1]), primary hepatocytes[Hengstler et al. Drug Metab Rev 32(2000)81] and liverslices [Kuhn et al. Exp Toxicol Pathol 50(1998)491]. PB,

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which changes the expression of many xenobiotic metabo-lizing enzymes was selected as the first model compound.(I) Cell linesEstablished cell lines would be, in terms of handling, themost convenient system for toxicogenomic studies. But wecould not observe alterations in transcription profiles of thecell line C2I, typical for phenobarbital. This could be due tothe high proliferation rate and subsequent dedifferentiationof cell lines, which therefore do not display metabolic ca-pabilities typical for hepatocytes. We concluded that thevalue of cell lines as a model for physiology of hepato-cytes is questionable. Hence, cell lines are unsuitable ingene expression studies.(II)Primary cultured hepatocytesPrimary cultured hepatocytes are well suited for toxicologi-cal studies because they display a certain level of meta-bolic activity. Using specific culture conditions, e.g. Matri-gel (extracellular matrix) or dexamethasone, phenobarbitalspecific changes of the activity for several cytochromeP450 enzymes were obtained similar to those in vivo.Different culture conditions were analysed: (a) two-dimen-sional culture, first as monoculture with collagen- orMatrigel-coated culture dishes or collagen-sandwich; sec-ond as co-culture with rat liver epithel cells; and three-di-mensional culture using calcium alginate beads; (b) differ-ent media: FCS, with or without dexamethasone andchemically defined medium. The culture conditions af-fected gene expression. As a result transcription profilesdetermined in vitro differed from those generated in vivo inrat liver. Best results (34 % of the regulated genes weredetermined in vivo and in vitro) were obtained for mono-culture and chemically defined medium.(III) Liver slicesThe use of precision-cut liver slices is a relatively new invitro test system with increasing application in toxicology.Liver slices provide decided advantages because of main-taining the functional acinar architecture of the liver. Aftertreatment of rat liver slices with PB [22], regulated genescorrespond to 28 % with in vivo treated rat liver. Strongagreement between rat liver slices and in vivo treated ratliver was obtained for the total number of expressed genes(60 %), in comparison to 14 % with cryopreserved rat liverslices.(IV) Transcription profiles of different tumor promotersDifferent tumor promoters were investigated in two in vitrotest systems: primary rat hepatocytes in monoculture andchemically defined medium and in rat liver slices. The invitro responses observed in drug metabolising genes, in-cluding cytochrome P-450 genes (CYPs), epoxide hydro-lases, UDP-glucuronosyl transferases (UGTs), glutathionetransferases (GSTs) and peroxisomal genes correspondedwell with published data. Expression of UGTs, GSTs andseveral CYP genes, like members of the CYP2 and CYP3families were increased in rat liver slices and primaryhepatocytes treated with enzyme inducers like PB andHCH. After treatment with the peroxisome proliferatorsDHEA, WY-14,643 and CF the induced genes were thosepredominantly involved in lipid metabolism and ß-oxida-tion; genes of the CYP4A subfamily were also upregul-

ated. After exposure of primary hepatocytes or rat liverslices to EE, the expression of only a few genes, like ste-roid 3-alpha-dehydrogenase, which is involved in the ste-roid metabolism and the Bcl-2 apoptosis related gene waschanged.To establish gene expression profiles, a two-dimensionalhierarchical clustering analysis was performed. Transcrip-tion profiles of enzyme inducers such as PB and HCH, toa lesser extend CP, either generated in the liver of treatedrats and in exposed rat liver slices or primary hepatocytesclustered together. The expression profiles of DHEA-, WY-14,643 and CF-treated rat liver slices or hepatocytes andthose obtained in rat liver after in vivo treatment withDHEA, WY-14,643 or Nafenopin were similar, according totheir similar mechanism of toxicity. However the expres-sion profiles observed in liver after in vitro and in vivo ex-posure by EE was distinctly different from those producedby the other chemicals.In conclusion, we could demonstrate that gene expressionprofiles of compounds that act via similar mechanismsshow common effects on transcription in vivo and in vitroboth in rat liver slices and in primary hepatocytes, sug-gesting that in vitro studies are predictive for the in vivotoxicity. Thus, our results support the use of in vitro testsystems like rat liver slices and primary hepatocytes astool in predictive toxicology, i.e. for the development andscreening of new drugs.

Publications (* = external co-author)[1] Schmezer, P.; Rajaee-Behbahani, N.; Risch, A.; Thiel, S.*;Rittgen, W.; Drings, P.*; Dienemann, H.*; Kayser, K.W.*; Schulz,V.*; Bartsch, H. Rapid screening assay for mutagen sensitivityand DNA repair capacity in human peripheral blood lymphocytes.Mutagenesis 16 (2001) 25-30.[2] Rajaee-Behbahani, N.; Schmezer, P.; Risch, A.; Rittgen, W.;Kayser, K.W.*; Dienemann, H.*; Schulz, V.*; Drings, P.*; Thiel,S.*; Bartsch, H. Altered DNA repair capacity and bleomycin sensi-tivity as risk markers for non-small cell lung cancer. InternationalJournal of Cancer 95 (2001) 86-91.[3] Popanda, O.; Flohr, C.; Dai, J.-C.; Hunzicker, A.; Thielmann,H.W. A mutation in subunit B of DNA polymerase alpha fromNovikoff hepatoma cells is concomitant with conformationalchanges and abnormal catalytic properties of the DNA poly-merase alpha-primase complex. Molecular Carcinogenesis 31(2001) 171-183.[4] Klein, R.G.; Schmezer, P.; Amelung, F.; Schroeder, H.-G.*;Woeste, W.*; Wolf, J.*: Carcinogenicity assays of wood dust andwood additives in rats exposed by long-term inhalation. Interna-tional Archives of Occupational and Environmental Health 74(2001) 109-118.[5] Frei, E.; Kuchenmeister, F.*; Gliniorz, R.; Breuer, A.;Schmezer, P. N-nitrosodimethylamine is activated in microsomesfrom hepatocytes to reactive metabolites which damage DNA ofnon-parenchymal cells in rat liver. Toxicology Letters 123 (2001)227-234.[6] Tisch, M.*; Lohmeier, A.*; Schmezer, P.; Bartsch, H.; Maier,H.*: Genotoxic effect of the insecticides pentachlorophenol andlindane on human nasal mucosal epithelium. DeutscheMedizinische Wochenschrift 126 (2001) 840-844.[7] Turner, S.D.*; Tinwell, H.*; Piegorsch, W.*; Schmezer, P.;Ashby, J.*: The male rat carcinogens limonene and sodium sac-charin are not mutagenic to male Big Blue rats. Mutagenesis 16(2001) 329-332.

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[8] Warbrick, E.V.*; Dearman, R.J.*; Ashby, J.*; Schmezer, P;Kimber, I.*: Preliminary assessment of the skin sensitizing activityof selected rodent carcinogens using the local lymph node assay.Toxicology 163 (2001) 63-69.[9] Wikman, H.; Thiel, S.*; Jäger, B.; Schmezer, P.; Spiegelhalder,B.; Edler, L.; Dienemann, H.*; Kayser, K.W.*; Schulz, V.*; Drings,P.*; Bartsch, H.; Risch, A. Relevance of N-acetyltransferase 1 and2 (NAT1, NAT2) genetic polymorphisms in non-small cell lungcancer susceptibility. Pharmacogenetics 11 (2001) 157-168.[10] Risch, A.; Wikman, H.; Thiel, S.*; Schmezer, P.; Edler, L.;Drings, P.*; Dienemann, H.*; Kayser, K.W.*; Schulz, V.*;Spiegelhalder, B.; Bartsch, H. Glutathione-S-transferase M1, M3,T1 and P1 polymorphisms and susceptibility to non-small-celllung cancer subtypes and hamartomas. Pharmacogenetics 11 (2001) 757-764.[11] Bertram, B.; Hemm, I.*; Tang, W.*: Mutagenic and carcino-genic constituents of medicinal herbs used in Europe and in theUSA. Die Pharmazie 56: (2001) 99-120.[12] Mayer, C.; Popanda, O.; Zelezny, O.; von Brevern, M.-C.*;Bach, A.*; Bartsch, H.; Schmezer, P. DNA repair capacity aftergamma-irradiation and expression profiles of DNA repair genes inresting and proliferating human peripheral blood lymphocytes.DNA Repair 1 (2002) 237-250.[13] Tisch, M.*; Schmezer, P.; Faulde, M.*; Groh, A.*; Maier, H.*:Genotoxicity studies on permethrin, DEET and diazinon in pri-mary human nasal mucosal cells. Eur Arch Otorhinolaryngol. 259(2002) 150-153.[14] Rajaee-Behbahani, N.; Schmezer, P.; Ramroth,H.; Bürkle, A.*; Bartsch, H.; Dietz, A.*; Becher, H.*:Reduced poly(ADP-ribosyl)ation in lymphocytes oflaryngeal cancer patients: results of a case-controlstudy. International Jounral of Cancer 98 (2002) 780-784.[15] Dally, H.; Gassner, K.; Jäger, B.; Schmezer, P.;Spiegelhalder, B.; Edler, L.; Drings, P.*; Dienemann,H.*; Schulz, V.*; Kayser, K.*; Bartsch, H.; Risch, A.Myeloperoxidase (MPO) genotype and lung cancerhistologic types: The MPO -463 A allele is associ-ated with reduced risk for small cell lung cancer insmokers. International Jounral of Cancer 102 (2002)530-535.[16] Bertram, B.; Bartsch, H. Krebsprävention durchgrünen Tee: Wirklichkeit und Wunschdenken.Wiener Medizinische Wochenschrift 5/6 (2002) 153-158.[17] Becker, R.*; Ritter, A.*; Eichhorn, U.*; Lips, J.*;Bertram, B.; Wiessler, M.; Zdzienicka, M.Z.*; Kaina,B.*: Induction of DNA breaks and apoptosis incrosslink-hypersensitive V79 cells by the cytostatic drug beta-D-glucosyl-ifosfamide mustard. British Journal of Cancer 86 (2002)130-135.[18] Bertram, B.; Bollow, U.; Rajaee-Behbahani, N.; Bürkle, A.*;Schmezer, P. Induction of poly(ADP-ribosyl)ation and DNA dam-age in human peripheral lymphocytes after treatment with (-)-epigallocatechin-gallate. Mutation Research 534 (2003) 77-84.[19] Tang, W.*; Hemm, I.*; Bertram, B. Recent Development ofantitumor agents from Chinese Herbal Medicines. Part I. Low mo-lecular compounds. Planta Medica 69 (2003) 1-12.[20] Tang, W.*; Hemm, I.*; Bertram, B. Recent Development ofanti-tumor agents from Chinese Herbal Medicines. Part II. Highmolecular compounds. Planta Medica, in press.[21] Popanda, O.; Ebbeler, R.; Twardella, D.; Helmbold, I.;Gotzes, F.; Schmezer, P.; Thielmann, H.W.; von Fournier, D.*;Haase, W.*; Sautter-Bihl, M.L.*; Wenz, F.*; Bartsch, H.; Chang-Claude, J. Radiation-induced DNA damage and repair in lympho-cytes from breast cancer patients and their correlation with acuteskin reactions to radiotherapy. International Journal of RadiationOncology Biology Physics, in press.

[22] Werle-Schneider, G.; Kalla, C.; Hollstein, M.; Bollow, U.;Behrens, C.K.*; v. Brevern; M.V.*; Storck; T.*; Müller, D.*;Steinmetzer, P.*; Bach, A.*; Beerheide, W. Comparison of gene inrat liver slices and primary hepatocytes after treatment with phe-nobarbital. Journal of Cancer Research and Clinical Oncology127 (2001) 30.

Biomarkers (C0206)Group Leader: Dr. Jagadeesan NairCooperations: A. B. Miller et al., G. Fürstenberger DKFZ; K.H.Adzersen, G. Bastert, Frauenklinik Heidelberg; P. Galle, UniversityHospital, Mainz; G. Winde, Klinikum Kreis Herford, Herford; E.W.Vogel, Leiden University, Leiden, The Netherlands; JacquesLaval, Institute Gustave Roussy, Villejuif, France; A. Barbin,IARC, Lyon, France; B. Tudek, Instytut Biochemii i Biofizyki, War-saw, Poland; R.H. Elder, Paterson Institute for Cancer Research,Manchester, UK; J.F. van Schooten, University Maastricht, theNetherlands; R. Srám, Technical Academy of Science, Prague;Czech Republic; K.H. Beger, University Ulm; P. Dolara, UniversityFlorence, Italy.

External Fundings: Marie Curie Fellowship Program from EU; EUContract MCFI-2000-01241. EU-Contract QOL-2000-4.2.1 Oxida-tive Stress and Chronic Diseases: Exocyclic DNA Adducts asMarkers for Disrupted Genomic Integrity and Risk (awarded inMarch 2001).

The group continued and extended the application the ex-pertise gained on the development and validation of meth-ods for analysing DNA-adducts as biomarkers for under-standing the mechanism of their formation from exo-genous and endogenous reactive species and to applythese markers in human biomonitoring and chemopre-vention trials. The formation of adducts from a carcinogenor the proximate metabolite of carcinogen (exogenous orendogenous) is one of the earliest damage to the genomein the cells. If not repaired, the adduct formation in a sur-viving cell, will lead to a mutation upon cell division andthe accumulated mutations that disrupt genomic integrityleads to cancer (see Fig. 1).1. Etheno-DNA adductsThe studies concluded so far established the validity ofmeasurement of etheno-DNA adducts to assess endog-enous DNA damage due to oxidative stress and lipidperoxidation. This included assessment of DNA damage

PLA2

DNA-reactive aldehydes

(HNE, MDA)

Exocyclic-DNAadducts

ROSRNS

Linoleic acid (diet)

Arachidonic acid

Lipid peroxidation

Impaired DNA repairLoss of apoptotic response

Increased DNA damage, cell proliferation and mutations

Hyperplasia�� Adenoma �� Carcinoma

Normal colon

epithelium

COX2,LOX,iNOS,Cytokines

Inflammatoryprocesses Membrane

phospholipids

Oxidativestress

Fig. 1

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caused by chronic infection/inflammation and dietary fac-tors such as intake of polyunsaturated fatty acids (ω-6PUFA) [Bartsch et al. Carcinogenesis 20 (1999) 2209-2218] and oxidative stress caused by metal storage dis-eases. The most common method for evaluating oxidativeDNA damage is the measurement of oxidized DNA base8-oxo-deoxyguanosine, however, uncertainties exist on theuse of this biomarker due to artefact formation. The mea-surement of more stable secondary DNA damage such asetheno-DNA adducts proved to be more reliable to assessthe DNA damage caused by increased oxidative stressand lipid peroxidation (LPO). The following investigationsare accomplished in order to link increased etheno adductformation as a result of enhanced lipid peroxidation due toinflammation and intake dietary ω-6 PUFA [reviewed in 2],[10,12].1. Formation of etheno adducts 1,N6-ethenodeoxyadeno-sine (εdA) and N3,4-ethenodeoxycytidine (εdC) were in-vestigated in diseases, prone to develop into colon cancersuch as familial adenomatous polyposis (FAP), ulcerativecolitis (UC) and Crohn�s disease (CD). εdA and εdC wereelevated in both FAP and CD whereas in UC εdC alonewas elevated. The increased etheno-DNA adducts in FAPmay resulting from increased arachidonic acid metabolismdue to overexpression of phospholipase A2 and cyclo-oxy-genase 2. In case of CD and UC the increased adduct for-mation may be due to increased oxidative stress/lipidperoxidation as a consequence of chronic inflammationand overproduction of nitric oxide [5,13].2. Formation of etheno adducts were found to be higher inDNA of pancreatic tissues obtained from chronic pancre-atitis patients compared to normal pancreas. Ethanol andiron-overload enhanced the adduct level in the liver [8, 9].3. Detection of 1,N6-ethenodeoxyadenosine (εdA) in hu-man urine by immunoaffinity-HPLC-fluorecsence. In orderto investigate the mechanism of formation and repair ofthese lipid peroxidation induced ε-base adducts in human,which appear to be a complex one due to the influence ofdietary factors, hormonal metabolism, inflammatory pro-cess etc., a non-invasive assay is desirable. For this pur-pose we have developed a specific and sensitive urinaly-sis assay for εdA using immunoaffinity-HPLC-fluores-cence. Using urinary εdA as biomarker it is now possibleto study the DNA damage due to oxidative stress and lipidperoxidation in human caused by different etiology suchas high dietary fatty acid and low antioxidant intake, in-flammatory process etc. using this non-invasive and easilyavailable biological samples [Nair et al. IARC Publications150 (1999) 55-61]. The method is successfully used toevaluate an intervention study in Japan. Excretion of(εdA), was analyzed in urine of non-smoking postmeno-pausal women participanting in a dietary intervention trialin Northern Japan. Hereby the efficacy of dietary counsel-ing in reducing salt and increasing vitamin C and carot-enes during one year was estimated. Thirty postmeno-pausal women, aged 60 - 69 years, from the interventiongroup, and 30 age-matched women from the control groupwere randomly selected. At the pre-intervention, εdA ex-cretion was positively associated with urinary salt excre-

tion (R = 0.33, P = 0.01) and ω-6 polyunsaturated fattyacid intake (% energy value, R = 0.28, P = 0.03) in the 59women). Results from this pilot study suggest urinary εdAas a potential biomarker of DNA damage possibly derivedfrom salt-induced inflammation and LPO; further explora-tion of edA in human biomonitoring studies is warranted[6]. (In collaboration with Dr. T. Hanaoka, National CancerResearch Institute, Chiba, Tokyo, Japan.)4. An immunohistochemical method has been developedfor the measurement of εdA at the cellular level and suc-cessfully applied in rat liver exposed to vinyl chloride andiron overload [14].5. Investigation of the role of COX-2 and LOX on the for-mation of etheno DNA adducts. Studies on DMBA-TPAmouse skin carcinoma revealed a close correlation ofetheno DNA adducts and 8 and 12, HETE; the products ofLOX path ways of arachidonic acid metabolism (Nair et al.2000). Increased etheno DNA adducts were detected inthe polyp epithelia of FAP patients (Shimid et al. 2000). Itis further planned to investigate the relative roles of COX-2and LOX using cell lines expressing high levels of theseenzymes [11]. (In collaboration with Dr. G.Fürstenberger,DKFZ.)6. Relationship between dietary fatty acid intake andetheno adducts in WBC-DNA of premenopausal women[7]. Isolated serum and buffy coat from available samplescollected from women (EPIC- Heidelberg study) were se-lected. The samples were randomised in two groups i.e.dietary intake of >15g (A) or <5g (B) linoleic acid per dayaccording to the dietary questionnaire. Serum sampleswere analysed for free fatty acids (OA and LA) by gaschromatography mass-spectrometry. and the DNA wasisolated from buffy coat was analysed for etheno-DNA ad-ducts by 32P-postlabelling. On a group level serum LA lev-els were significantly higher in group A than group B. However the OA levels were also higher in group in A com-pared to group B. The ratio between LA and RA levelswere similar in both groups. Both adduct levels were notsignificantly different in the two groups, however a third ofthe individuals had more than double the adduct levelsthan the rest. Oxidative stress in humans is partly counter-acted by dietary intake of anti-oxidants. We tried to corre-late the amount of different anti-oxidants taken by indi-vidual subjects determined by detailed questionnaire andthe etheno-DNA adducts in their white blood cells. Amongthe parameter analysed vegetable intake and vitamin Esignificantly reduced the εdA in these subjects. εdC wasalso reduced considerably however, did not reach the sig-nificance due to large variation in the values. This obser-vations is in accordance with the fact that vitamin E andvegetables reduce oxidative stress and lipid peroxidation.(In collaboration with Dr. N. Becker, Division of ClinicalEpidemiology, DKFZ.)7. Etheno DNA adducts were investigated in G6PD-defi-cient mice. Brains of G6PD-deficient males exhibited asignificant distortion of redox control (~3-fold decrease inthe ratio of reduced glutathione to oxidized glutathione), aconsiderable accumulation of promutagenic etheno DNAadducts (~13-fold increase in ethenodeoxyadenosine and

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~5-fold increase in ethenodeoxycytidine), and a substan-tial elevation of somatic mutation rates (~3-fold increase inmutant frequencies in lacZ, the target and reporter gene ofmutagenesis in the shuttle vector, pUR288) [4]. (In col-laboration with Dr. K. Felix and Dr. J. Siegfried, NationalCancer Institute, NIH, Bethesda, USA.)

2. O4-ethyl-dT.Besides etheno-DNA adducts a new method has beendeveloped to detect O4-ethylthymidine [3]. Among about adozen different alkylation products induced in genomicDNA by N-nitroso carcinogens, O4-ethyl-thymidine(O4-etdT) can be considered as a major premutational le-sion causing AT→GC transition mutations. Although O4-etdT is initially formed at far lower amounts than otheralkylation products in cellular DNA, it accumulates be-cause of its inefficient repair and may thus play an impor-tant role in carcinogenesis. Increased excretion ofethylated DNA bases has been reported in the urine ofcigarette smokers. To study DNA ethylation in the targetorgans of smokers, an immunoenriched 32 P-postlabelingassay for O4-ethyl-thymidine (O4-etdT) was developed. O4-etdT-3-monophosphate (O4-etdT-3P) was synthesized, pu-rified, and characterized by LC-MS, ESI-MS, and NMR. Tovalidate the method, O4-etdT levels were determined incalf thymus DNA treated with N-ethyl-N-nitrosourea, and adose-dependent formation of O4-etdT was observed. Fur-thermore, O4-etdT was found to be present in the cells ob-tained from the lower respiratory tract by sputum inductionof two out of four smokers but not in three nonsmokers.O4-etdT is a poorly repaired promutagenic DNA lesion;thus, it could be of potential use for biomonitoring smok-ing-related DNA damage. Our improved assay was foundto be sufficiently sensitive and specific to detect O4-etdT insurrogate cells from cigarette smoke exposed humans.Lung DNA samples obtained from patients undergoingsurgery revealed in creased formation of O4-etdT in smok-ers lungs [1]. (In collaboration with Dr. M. Wiessler and Dr.C. Kliem, DKFZ.)

Publications (* = external co-author)[1] Godschalk, R.; Nair, J.; van Schooten, F.-J.*; Risch, A.;Drings, P.*; Kayser, K.*; Dienemann, H.*; Bartsch H. Comparisonof multiple DNA adduct types in tumor adjacent lung tissue: ef-fect of cigarette smoking. Carcinogenesis 23 (2002) 2081-2086.[2] Bartsch, H.; Nair, J.; Owen, R.W. Exocyclic DNA adducts asoxidative stress markers in colon carcinogenesis: potential role oflipid peroxidation, dietary fat and antioxidants. Biological Chemis-try 383 (2002) 915-921.[3] Godschalk, R.; Nair, J.; Kliem, H.C.; Wiessler, M.; Bouvier,G.*; Bartsch, H. Modified immunoenriched (32)P-HPLC assay forthe detection of O(4)-ethylthymidine in human biomonitoring stud-ies. Chemical Research in Toxicology 15 (2002) 433-437.[4] Felix, K.*; Rockwood, L.D.*; Pretsch, W.*; Nair, J.; Bartsch,H.; Bornkamm, G.-W.*; Janz, S.*: Moderate g6pd deficiency in-creases mutation rates in the brain of mice. Free Radical Biology& Medicine 32 (2002) 663-673.[5] Bartsch, H.; Nair, J. Potential role of lipid peroxidation derivedDNA damage in human colon carcinogenesis: studies on exocy-clic base adducts as stable oxidative stress markers. Cancer De-tection and Prevention 26 (2002) 308-312.

[6] Hanaoka, T.*; Nair, J.; Takahashi, Y.*; Sasaki, S.*; Bartsch,H.; Tsugane, S.*: Urinary excretion of 1, N6-ethenodeoxyadeno-sine, a marker of oxidative DNA damage in postmenopousalJapanese women participating in a dietary intervention trial inNothern Japan. International Journal of Cancer 100 (2002) 71-75.[7] Hagenlocher, T.; Nair, J.; Becker, N.; Korfmann, A.; Bartsch, H.Influence of Dietary Fatty Acid, Vegetable, and Vitamin Intake onEtheno-DNA Adducts in White Blood Cells of Healthy Female Vol-unteers: A Pilot Study. Cancer Epidemiology, Biomarkers andPrevention 10 (2001) 1187-1191.[8] Marrogi. A.J.*; Khan, M.A.*; van Gijssel, H.E.*; Welsh, J.A.*;Rahim, H.*; Demetris, A.J.*; Kowdley, K.V.*; Hussain, S.P.*; Nair,J.; Bartsch, H.; Okby, N.*; Poirier, M.C.*; Ishak, K.G.*; Harris,C.C.*: Oxidative Stress and p53 Mutations in the Carcinogenesisof Iron Overload-Associated Hepatocellular Carcinoma. Journal ofNational Cancer Institute 93 (2001) 1652-1655.[9] Navasumrit, P.*; Ward, T.H.*; O�Connor, P.J.*; Nair, J.; Frank,N.; Bartsch, H. Ethanol enhances the formation of endogenouslyand exogenously derived adducts in rat hepatic DNA. MutationResearch 479 (2001) 81-94.[10] Bartsch, H.; Nair, J. Ultrasensitive and specific detectionmethods for exocylic DNA adducts: markers for lipid peroxidationand oxidative stress. Toxicology 153 (2000) 105-114.[11] Nair, J.; Fürstenberger, G.; Burger, F.; Marks, F.; Bartsch, H.Promutagenic etheno-DNA adducts in multistage mouse skin car-cinogenesis: correlation with lipoxygenase-catalyzed arachidonicacid metabolism. Chemical Research in Toxicology 13 (2000)703-709.[12] Bartsch, H.; Nair, J. New DNA-based biomarkers for oxidativestress and cancer chemoprevention studies. European Journal ofCancer 36 (2000) 1229-1234.[13] Schmid, K.; Nair, J.; Winde, G.*; Velic, I.*; Bartsch, H. In-creased levels of promutagenic etheno-DNA adducts in colonicpolyps of FAP patients. International Journal of Cancer 87 (2000)1-4.[14] Yang, Y.; Nair, J.; Barbin, A.*; Bartsch, H. Immunohistochemi-cal detection of 1,N(6)-ethenodeoxyadenosine, a promutagenicDNA adduct, in liver of rats exposed to vinyl chloride or an ironoverload. Carcinogenesis 21 (2000) 777-781.

A) Intervention Studies and Characterizationof Cancer Protective Food Components(C0207)Group Leader: Dr. Bertold Spiegelhalder incollaboration with Dr. Robert W. Owen

1.1. Intervention studies and colorectal cancerR.W. Owen, G. Würtele, B. Spiegelhalder, H. Bartsch,J. Wahrendorf*, B. Hofstad**, M. Vatn**, C. Bonithon-Kopp*** and J. Faivre****Environmental Epidemiology, DKFZ; **Rikshospitalet, The Na-tional Hospital, Medical Department A, Oslo, Norway; ***ECP Co-lon Cancer Working Group, Registre des Tumeurs Digestives dela Cote-d�Or, Faculte de Medicine, Dijon, France.

A high content of calcium [Owen, Recent Results in Can-cer Research 146 (1998) 195-213]. and/or fibre [Hill et al.European Journal of Cancer Prevention 6 (1997) 512-514]in the diet is regarded to be protective against colorectalcancer. The major mechanisms of action are thought to bechelation (calcium) and dilution (fibre) of potentially co-car-cinogenic intestinal lipids. To test this hypothesis a numberof intervention studies in adenoma patients have been em-barked upon. In a pilot placebo-controlled calcium inter-vention trial (35 patients) intervention had no greater effect

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than placebo on reduction of intestinal cell proliferation[Weisgerber et al. Gut 38 (1996) 396-402]. In anotherslightly larger study (110 patients) intervention with cal-cium and antioxidants had a small but insignificant effecton the repression of growth of adenomas left in-situ but asignificant effect on the formation of new adenomas[Hofstad et al. Digestion 59 (1998) 14]. Publication of thedata pertaining to these studies is now complete.Finally in collaboration [Faivre et al. European Journal ofCancer Prevention 6 (1997) 132-138] with the EuropeanAgency for Cancer Prevention (ECP) a pan-European (in-volving 10 countries) long-term (3 year) calcium/fibre(fybogel) placebo-controlled intervention study in patients(665) with sporadic adenoma has recently been com-pleted. At the DKFZ (during 1997 and 1998) intestinal lipidand mineral content of the stools was completed in 1003Inclusion and Intervention stool specimens.The relation between diet, cell proliferation, adenoma re-currence, intestinal lipids and minerals, blood DNA andantioxidant status and the effect of intervention on theseparameters is being evaluated and manuscripts preparedfor publication.The clinical data [8] shows that overall, calcium interven-tion had a small but non-significant preventive effect onthe recurrence of adenomas and this attained statisticalsignificance in those patients whose habitual intake of cal-cium was very low. On the other hand intervention with fi-bre had a significant enhancing effect on the recurrence ofadenomas. This effect was particularly enhanced in North-ern European men. Furthermore intervention with fibrehad a clear dose dependent interactive effect with habitualdietary calcium intake.The hypothesis that bile acids [4] are involved in the aden-oma-carcinoma sequence was not upheld by any of theabove studies but the data from the ECP study is currentlyundergoing exhaustive statistical analyses.

1.2. Phenolic and lipid components of seasoningoils and their precursors in relation to cancerof the colorectum and breast

R.W. Owen, R. Haubner, B. Spiegelhalder,H. Bartsch, W.E. Hull*, A. Giacosa** andP Srivatanakul****Central Spectroscopy, DKFZ; **Istituto Nazionale per la ricercasul cancro, Istituto scientifico per lo studio e la cura del tumori,Genova, Italy; ***National Cancer Institute, Bangkok, Thailand.

1.2.1. Olive drupes and olive oilThe Mediterranean diet is associated with decreased inci-dence of a range of diseases, especially cancer of thecolorectum and breast. A major reason for this is the highconsumption of olive oil which contains over 70% of its lip-ids as the monounsaturated long chain fatty acid oleic acid(n9) and in addition a range of phenolic substances. Be-cause high intakes of polyunsaturated long chain fatty ac-ids (n6) have recently been implicated in the aetiology ofbreast cancer [Nair et al. Cancer Epidemiology, Biomar-kers and Prevention 6 (1997) 597-601] and to better deter-mine the mechanisms [3] by which olive oil is superior toother oils in its health protecting properties the phenolic

(antioxidant) and lipid components was evaluated in arange of olive and seed oils (n = 30) currently on the Ital-ian market.Extraction, GLC and HPLC protocols have been devel-oped to maximise detection and separation of the lipid andphenolic components. A range of major antioxidants havebeen identified by mass spectrometry and nuclear mag-netic resonance spectroscopy including squalene, terpe-noids, simple phenols, secoiridoids, lignans and flavon-oids [7]. The detection and characterisation of lignans andflavonoids is a novel phenomenon in olive oil.These studies have been extended to an evaluation of thephenolic antioxidant content of olive drupes. Both blackand green olives contain very high concentrations (10-20times higher than in extravirgin olive oil) of these sub-stances and therefore may represent an even greater con-tribution to the health promoting properties of the Mediter-ranean diet.All of the individual phenolic compounds extracted andisolated from olive oils have potent antioxidant propertiesand compare favourably with the classic antioxidant, vita-min E. These data may have a bearing not only on chemo-preventive strategies but also on future epidemiologicstudies which are recommended to take into account notonly the types and grades of oils but also the intake of ol-ive drupes.These studies are have been extended to olive oils andolive drupes [2] from a range of different countries withinthe Mediterranean basin and to a range of different prod-ucts derived from olives currently on the Italian market.

1.2.2. Palm, Sesame, Soy oils in comparison toother seasoning oils in Thailand

Along with the diet of the Mediterranean basin of Europe,food consumption within countries of Eastern civilisationsare also likely to provide a rich source of antioxidant sub-stances which are not present in typical Western diets. Agood example of this is Thailand which has an extremelylow incidence of colon and breast cancer. To test this hy-pothesis a collaborative study has been established be-tween the Division of Toxicology and the National CancerInstitute, Bangkok, Thailand. A range (n = 20) of season-ing oils currently on the Thai market were studied by thesame analytical techniques applied in the olive oil studies.Crude palm oil was found to contain substantial amountsof antioxidant phenolic acids such as 3,4-dihydroxy ben-zoic acid, vanillic acid, p-coumaric acid. In contrast refinedpalm oils (n = 6) were devoid of these phenolic acids buttwo were found to contain considerable amounts of whatappears to be a synthetic antioxidant. In comparison to bu-tylated hydroxyanisole and butylated hydroxytoluene(BHT) it was found to contain two hydroxyl moeities on thephenol ring. This was confirmed by both GC/MS and NMRand has been assigned the structure butylateddihydroxyanisole (BDHA). Considerable variation in theconcentration of this substance in refined palm oils wasevident and raises the question, is it a synthetic addition ora natural product? Probably the natural antioxidants incrude palm oil are destroyed in the refining process and itis possible that BDHA is formed artificially during the refin-

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ing process from BHT which may be added to preservethe keeping quality of palm oil. Studies are in progress toelucidate how industrial refining of palm oil is conducted inThailand so that the presence of this new substance canbe rationalised. Of the other oils studied, sesame oil wasfound to be particularly rich in two non-polar antioxidantphenolics i.e. sesamin and sesamolin. Levels of up to 2g/kg were detected. Precursor sesame seeds however con-tained a more complex profile and contained in additionthe mono-, di- and tri-glucosides of sesaminol andpinoresinol glucoside along with free sesaminol,sesamolinol, pinoresinol and samin. Also the presence ofsubstantial quantities of lariciresinol, iso-lariciresinol,matairesinol, syringaresinol and vanillic acid have beendetected for the first time. In contrast soy along with sun-flower, corn and peanut oils are devoid of antioxidantsustances belonging to these classes. The structures ofthe compounds described in this study were identified byESI-MS, GC/MS and NMR. The data is currently being col-lated for publication Work is also in progress to isolate suf-ficient of these antioxidants for a compreshensive screenof their anticancer potential in collaboration with DrClarissa Gerhäuser and Dr Peter Schmezer.1.2.3. Linseeds and linseed oil in relation to

faecal mammalian lignansMammalian lignans are formed in the large intestine by mi-crobial transformation of dietary precursors and they areconsidered to be protective against breast cancer espe-cially because of their similar structure to Tamoxifen. Thelignans formed are termed enterodiol (ENND) and entero-lactone (ENNL) and the major precursor is deemed to besecoisolariciresinol diglucoside (SDG) which is a majorcomponent of a complex phenolic polymer (CPP) in lin-seeds. Linseed oil in contrast to e.g. olive and sesame oilscontains very low levels of SDG because the CPP is in-soluble in oil matrices. Although SDG has been isolatedand identified as a major component of the CPP we haveconducted further investigations into the nature of thepolymer. After extraction and methanolysis of the polymerwe have demonstrated that SDG is bound within an arrayof phenolic acids such as p-coumaric, ferulic and caffeicvia glucose intermediate linkages. Therefore high intakesof linseeds in the diet will not only afford a rich source ofsubstrate for the formation of mammalian lignans in thelarge intestine where they can exert local protective effectsagainst colon cancer and via absorbtion, at other sites, butalso provides considerable amounts of antioxidant phe-nolic acids after digestion of the polymer in the large bowelwhich should enhance the protective effects of the lignans.The exact nature of all the polymeric fractions are cur-rently being subjected to a range of spectroscopic meth-ods so that their definitive structures can be delineatedand work is in progress to provide sufficient quantities forcomprehensive screening of their chemopreventive poten-tial.

1.3. Faecal phenolsR.W. Owen, B. Spiegelhalder, H. Bartsch

Many of the compounds isolated from various seasoningoils and their precursors are glycosides and therefore atool used along with spectroscopic techniques to deter-mine their structures is deglycosylation. Commercial en-zymes are very ineffective at deglycosylating phenolic gly-cosides i.e. under prescribed conditions only 50% effec-tiveness after one weeks incubation. Therefore weadopted a different approach. The faecal matrix is rich in avariety of glycosidases and deglycosylation can be com-pleted in hours in phosphate buffer with very smallamounts of faecal matrix. In studies with SDG for example,complete conversion via the monoglucoside to secoiso-lariciresinol is evident after 3 h. It was noted however inthese experiments that phenolic antioxidants not derivedfrom SDG were evident in the HPLC and GC/MS chroma-tograms. These were identified as metabolites of phenolicantioxidants present in olive drupes and olive oil andtherefore were �contaminants´ in the faecal matrix. Pilotstudies with the faecal matrix alone confirmed this. Sur-prisingly phenolic antioxidants within the faecal matrix areresistant to extraction by organic solvents but under theconditions of the deglycosylation experiments are releasedinto the aqueous medium which then allows extraction withthese fluids.A study was therefore conducted with faecal samplesdrawn from the ECP calcium/fibre intervention study andthe phenolic antioxidants were identified by both GC/MSand ESI-LC/MS. This allowed their quantitation usingsingle ion monitoring techniques and over thirty phenolicantioxidant compounds can be detected in the faecal ma-trix. The new methodology has been applied to as yetsamples from three countries in the ECP study i.e. Den-mark, Germany and Italy representing a north-south gradi-ent. In this pilot study a remarkable correlation betweenthe content of the phenolic antioxidant substances in thefaecal matrix and the incidence of both breast and coloncancer has been demonstrated. A full scale study is cur-rently being designed.

1.4. Fermentation studiesR.W. Owen, B. Spiegelhalder, H. Bartsch

In previous work published in the 1980´s a limited pathwayof mammalian lignan formation from linseed meal wasproposed but the exact mechanism was not elucidated. Inour studies on linseeds (1.2.3) gram quantities of SDGwere isolated and purified and therefore we endeavouredto shed further light on the nature of this pathway. Toachieve this, anaerobic fermentation experiments wereconducted. Briefly, Brain Heart Infusion broth supple-mented with reducing agents and the substrate SDG (0.5mg/ml) were inoculated with faecal matrix (1%) and incu-bated at 37°C for 72 h in an anaerobic chamber. The fer-mentation broths were extracted on extrelute and duringsilicic acid column chromatography were separated into aseries of fractions. After identification of the metabolites inthe fractions by GC/MS and ESI-LC/MS they were isolated

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and purified by semi-preparative HPLC and their struc-tures confirmed by NMR. This has allowed us to formulatea complete pathway of SDG transformation to the mam-malian lignans by the faecal microflora [5,6]. Now, suffi-cient amounts of the mammalin lignans can be isolatedand purified for comprehensive screening of their antican-cer potential.Furthermore in our studies on faecal phenols (1.5) it wasnoted that although many individuals excreted metabolitesof olives and olive oil antioxidant precursors, the lignans(1.2.1) were not detected as part of the phenolic fraction.Therefore studies on the fate of lignans derived from oliveoil, sesame oil and sesame seeds were studied in a simi-lar manner to SDG. The data shows that lignans derivedfrom olive oil (acetoxypinoresinol, pinoresinol), sesame oil(sesamin, sesamolin) and sesame seeds (sesamolinol,lariciresinol, isolariciresinol, matairesinol) are also meta-bolised by the faecal microflora to the mammalian lignansENND and ENNL. This may explain the discrepancy thathas been observed between the intake of SDG (which untilnow was considered to be the only precursor of these me-tabolites) and mammalian lignan formation in the largebowel. Also a number of novel intermediate lignans havebeen detected and isolated and studies are in progrees toelucidate their definitive structures by GC/MS, ESI-LC/MSand NMR.

1.5. Antioxidant content of red winesR.W. Owen, G. Würtele, B. Spiegelhalder, H. Bartsch

The antioxidant profile and content of red wines is well re-searched and a number of different classes have beenidentified. However studies with human volunteers whowere instructed not to consume olive derived products fortwo weeks continued to excrete readily detectable levels oftyrosol and hydroxytyrosol in the faecal matrix. It was con-sidered unlikely that these antioxidants were constituentsof either meat, vegetables or fruit which the volunteerswere allowed to consume. Rather a more likely sourcewas beverages such as tea or wine. Therefore a compre-hensive analysis of the phenolic antioxidant content of tea(green and black) and red wine was conducted. The pro-files in tea were identical to those already published butred wine is found to contain substantial amounts ofhydroxytyrosol and tyrosol along with several other com-pounds which have not been previously described as com-ponents of red wine. The major classes of antioxidantswere first separated by column chromatography into a se-ries of fractions from which their nature was identified byHPLC, GC/MS and ESI-LC/MS. This has enabled a num-ber of various LC/MSD programs to be devised which al-lows the antioxidant profiles of wines to be analysed by di-rect injection (1-20 µL) of the wine (a total of only 200 µL isrequired to obtain a complete profile) into the LC/MSDwithout any prior work-up using single ion monitoring tech-niques. At present over fifty antioxidant substances can bedetected and identified in red wines by this method anddue to the ease of analysis will allow rapid comparisons ofthe antioxidant content of wines Worldwide in our ongoingchemoprevention studies.

1.6. Assessment of the role of reactive oxygenspecies in the aetiology and promotion ofcancer

R.W. Owen, G. Würtele, B. Spiegelhalder, H. Bartsch

HPLC methods have been developed [Owen et al. Gut 38(1996) 591-597] for the reliable determination of phyticacid (PA) and reactve oxygen species (ROS) in humanfaeces. Stool PA content shows a strong correlation withthe excretion of minerals such as iron, calcium and mag-nesium but exhibits no relation with cell proliferation.HPLC [Owen et al. European Journal of Cancer Preven-tion 5 (1996) 233-240] allows the simultaneous monitoringof the hypoxanthine/xanthine oxidase system and ROSgeneration, enables the kinetics of the process to beevaluated in detail and has been successfully applied tothe monitoring of ROS production by the faecal matrix.Recent developments [Owen et al. European Journal ofCancer Prevention 7 (1998) 41-54] utilising a refinedHPLC method has shown clearly that the capacity offaeces to generate ROS is not related to the bacteriapresent. Rather ROS generation is supported by an as yetunidentified soluble factor within the faecal matrix. Studiesare in progress to identify this soluble factor and a refinedassay system has been re-applied to the faecal samplesstudied thus far and to additional patient and populationgroups to finally establish whether or not PA, iron andROS generation alone or in combination within the largeintestine have any bearing on the aetiology of colorectalcancer. This data is currently being evaluated.

1.7. Reactive oxygen species (ROS) and gastriccancer

R.W. Owen, G. Würtele, B. Spiegelhalder, H. Bartsch,H. Bauer* and J. Rudi***Division of Molecular Toxicology, DKFZ; **Department of Gastro-enterology, Krehl Klinik, Heidelberg.

Infection with Helicobacter pylori is assumed to be a riskfactor for the development of gastric carcinomas, but themolecular mechanism for this is still unknown. Thereforethe upregulation of reactive oxygen species (ROS) in gas-tric juice of human gastritis patients and methylation/de-methylation of the p53 gene in the human gastric carcino-ma cell line AGS by H. pylori were studied. Gastric juice of31 patients (with and without gastritis) were analysed fortheir potential to generate ROS as measured by hydroxylradical attack on the aromatic probe salicylic acid. Theproducts (dihydroxy benzoic acids) were analysed by highperformance liquid chromatography. Gastric juice samplesof twenty (64.5%) patients with chronic H. pylori gastritisgenerated elevated concentrations of ROS, compared toonly 8 (21.6%) of gastritis patients without H. pylori infec-tion.The methylation status of wild type p53 gene in the gastrictumor cell line AGS (ATCC:CRL-1739) was also evaluatedwith respect to H. pylori infection (type:60190,vac A geno-type,cag A-positive). In incubation experiments with H. py-lori the 5 methyl dC of CpG regions of p53 was analyzedby the MspI/HpaII restriction enzyme method using 14

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sense/antisense primer pairs at CCGG restriction sites ofp53 intron1 - intron7, exon1 exon5, exon6, exon7, exon8.Methylated cytosines at CpG sites could be detected in in-tron1, exon5, exon7, exon8. After the incubation of cellseither with H. pylori bacteria or the supernatant of culturemedia from H. pylori a higher degree of methylation in theCpG islands of p53 could not be detected. Demethylationwas only shown in intron1. Stomach infections with H. py-lori appear to be associated with the potential for a higherrate of ROS formation by the gastric juice. This may repre-sent one step in the mechanism of cancer formation in thestomach. There was no upregulation of p53 methylation,but the endogenous methylation grade at cytosines is be-ing investigated further.

1.8. Effect of lignan precursors on risk bio-markers of breast cancer: an interventionstudy in pre- and peri-menopausal womenwith an increased familial breast cancer risk

U. Knust*, K.H. Adzerson*, R.W. Owen,B. Spiegelhalder, H. Bartsch*Frauenklinik der Universität Heidelberg, Abteilung für AllgemeineFrauenheilkunde und Geburtshilfe.

The aim of this study is to evaluate the effect of interven-tion with flaxseed in a two-arm study on serum and urinarylignan levels and urinary oestrogens the levels of whichare related to the risk of breast cancer. It is a randomizeddietary intervention study with 20 high (20g/day) and 20low-flaxseed exposed participants. All participants wereadvised also to increase their daily intake of fruits and veg-etables. The participants are pre- and peri-menopausalvolunteers (25-55 years of age) having a mother or sisterwith breast cancer. The main end-points are serum andplasma enterolignan levels (measured directly by ESI-LC/MS) along with urinary oestrogen profiles (measured byGC-MS) in flaxseed exposed (non-exposed) women. Otherend-points are serum vitamin E and C, folic acid and caro-tenoids. Correlations between the administered (EPIC)food frequency questionnaire (FFQ) utilised, and the end-points will be sought. The study is currently well underwayand should be completed by the end of the year.

1.9. Evaluation of the phenolic antioxidants inwaste products of Thai fruits.

R.W. Owen, R. Haubner, B. Spiegelhalder,H. Bartsch, W.E. Hull*, A. Giacosa**;Y. Sudjoereen*** and S. Chambumrung****Central Spectroscopy, DKFZ; **Istituto Nazionale per la ricercasul cancro, Istituto scientifico per lo studio e la cura del tumori,Genova, Italy; ***Mahidol University, Bangkok, Thailand.

The methods developed in 1.2.1 are being applied toevaluate the profiles and contents of phenolic antioxidantsin waste products (pericarp and stones) of Thai fruits (Lon-gan, Lychee, Rambutan, Jackfruit, Durian, Mangoteen etc.Preliminary data indicate that Longan, Lychee, Rambutanand Mangoteen contain an abundance of different phe-nolic compounds while the stones of Jackfruit and Durianare devoid. While the nature of some are known, the

structures of many are novel and are currently beingcharacterised.

1.10. Characterization of new anti-oxidant fromplant origin and mechanisms of action ascancer chemopreventive agents

M.T.S. Trevisan*; R.W. Owen, G. Würtele,R. Haubner, B. Spiegelhalder, H. Bartsch*Departamento de Quimica Orgânica e Inorgânica, UniversidadeFederal do Ceará, Fortaleza CE, Brazil.

The majority of adult cancers are carcinomas of epithelialorigin with lung, colon, and uterus as the primary sites,which reflects a selective vulnerability of these tissues tocarcinogenic insult as a result of frequent exposure to theexternal environment. Indeed, it is estimated that up to 80to 90% of all cancers are attributable to environmental riskfactors, including chemicals, radiation, and viruses. Thenotion that a majority of human cancers have an environ-mental origin implies an optimistic outlook in terms of can-cer prevention since most cancer-causing substances areintroduced into the environment by human activities andare hence, controllable or removable. The elimination ofenvironmental carcinogens or at least avoiding exposureto them offers the opportunity to prevent most cancers,which is a basis of primary prevention.Chemoprevention is the process of inhibiting, delaying orreversing carcinogenesis in the premalignant phase andaims to halt or reverse the development and progressionof precancerous cells through use of non-cytotoxic nutri-ents and/or pharmacological agents during the lengthytime period between tumor initiation and progression.Recently, considerable attention has been focused onidentifying naturally occurring chemopreventive substan-ces capable of inhibiting, retarding, or reversing multi-stage carcinogenesis. A wide array of phenolic substan-ces, particularly those present in dietary and medicinalplants, have been reported to possess substantial anti-carcinogenic and antimutagenic activities. The majority ofthese naturally occurring phenolics retain antioxidativeproperties which appear to contribute to their chemopre-ventive and chemoprotective activity [Owen et al. Gut 46(2000) 225-232]. Particularly important in oxidant defenseare the radical-scavenging antioxidants. Free radicals arevery reactive chemical species that readily lead to uncon-trolled reactions, resulting in oxidative damage of impor-tant biological macromolecules such as DNA, proteins andlipids [Owen et al. Food Chemical Toxicol 38 (2000) 647-659] .Cancer protective factors are present in several fruits, veg-etables and commonly used spices and herbs. They candivided in several groups based on their chemical struc-ture, e.g. polyphenols, thiols, carotenoids and retinoids,carbohydrates, trace metals, terpenes, tocopherols anddegradation products of glucosinolates.The Mediterranean diet has a chemoprotective effectagainst cancer and also reduces mortality from heart dis-eases [Bartsch et al. Toxicology 153 (2000) 105-114. Amajor component of this diet is olive oil. Other components

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such as spices, of the Mediterranean diet may also be im-portant in the chemoprevention of cancer. Since ancienttimes, spices have been added to different types of food toimprove the flavour and also storage stability. The spicesprevent the oxidation of lipids, in foodstuffs which results inthe development of off-flavour, rendering the product un-acceptable for human consumption.The synthetic antioxidants such as butylated hydroxytolu-ene (BHT) and butylated hydroxyanisole (BHA), that havebeen used widely for many years to retard lipid oxidation infoodstuffs are believed to possess carcinogenic activity.Thus interest in the antioxidative activity of spices has in-creased and has led to an increase in information aboutthe compounds and mechanisms involved. That spices,have importance as chemoproprotective agents has beenreported by Murcovic et al. [Zeitschrift für Lebensmittel-untersuchung und -forschung 207 (1998) 477] applicationof rosemary, thyme sage, garlic and brine reduced thecontent of the heterocyclic aromatic amines, mutageniccompounds, that are formed during heating of meat andfish, in 60% of the amount found in the control. Nalini et al.[Journal of Ethnopharmacology 62 (1998) 15] showedcumin and black pepper may protect the colon from can-cer, by decreasing the activity of beta-glucuronidase andmucinase.The objective of this research project is the isolation ofnatural antioxidants from medicinal plants, fruits andspices. The project has been initiated by a comprehensivescreen of phenolic antioxidants in Cashew nut(Anacardium occidentale L.) and its associated productswhich is one of the most important cash crops of the north-eastern region of Brazil. Approximately, 700,000 ha areplanted with this crop giving employment to more than100,000 people, and providing an annual turn-over of 200million dollars. The processed kernels are the principalcommodity exported to the USA, Europe and Japan[Pessoa et al. In: Cajucultura: Modernas Técnicas deProdução (1975) 23-42]. Juice, jam, alcoholic and softdrinks made from cashew apples (i.e. pear-shaped ediblepeduncle), and toasted kernels are also sources of incomeand food for local populations.Studies are also being extended to various fruit, spicesand medicinal plant extracts from the Amazon.

1.11. The lipid and antioxidant content of argan(Morocco) oil

R.W. Owen, R. Haubner; G. Würtele,B. Spiegelhalder, H. Bartsch, F. Khallouki*,C. Younos*, T. Oster*, Z. Charrouf***Laboratoire D´Ingénierie Moléculaire et de Biochimie Pharma-logique, Université de Metz, Campus Bridoux, Metz, France;**Département de Chimie, Faculté des Sciences, UniversitéMohammed V, Rabat, Morocco.

The aim of this study was to evaluate the fatty acids, toco-pherols, squalene, sterols and phenolic antioxidants inthree types of argan oil (Moroccan food, Moroccan aes-thetic and a French commercial variety) along with a basiccomparison to extravirgin olive and sunflower oil. The fattyacid profiles in the argan oils were very similar, with oleic

acid (43%) and linoleic acid (36%) and their respectivemonacylglycerols predominating. The major vitamer identi-fied was γ-tocopherol with a mean of 483 ± 11 mg/kg, incontrast to α-tocopherol which is the major vitamer in olive(190 ± 1 mg/kg) and sunflower oil (532 ± 6 mg/kg). Thesqualene content of the argan oils was very similar with amean of 313 ± 4 mg/100g which is lower than that of theolive oil (499 mg/100g) but significantly higher than in thesunflower oil (6 mg/100g). In contrast to olive and sun-flower oils in which β-sitosterol is predominant the majorsterols detected in the argan oils were schottenol (mean =147 ± 10 mg/kg) and spinasterol (mean = 122 ± mg/kg).The only phenolic compounds other than the tocopherolvitamers which could be readily detected and quantitatedwere vanillic, syringic and ferulic (probably conjugated toglucose) acid along with tyrosol. In comparison to theextravigin olive oil (793 mg/kg) the concentration of totalphenolic compounds is extremely low (< 5.0 mg/kg). Nev-ertheless argan oil with its high content of the vitamer γ-tocopherol, squalene and oleic acid is likely to providehealth benefit effects in the Moroccan diet [1].

1.12. Effects of moderate versus low impactrehabilitative exercise programs onoxidative DNA damage and repair inpatients with treated colorectal cancer

H. Allgayer*, R.W. Owen, J. Nair, B.Spiegelhalderand H. Bartsch*Oncology Department, Rehabilitation Clinic Ob der Tauber, derLVA Württemberg, Bad Mergentheim.

Increased oxidative DNA products currently are believed tobe involved in early stages of tumor initiation and promo-tion suchas breast and colon cancer. Based on this knowl-edge the so called biomarker concept has been put for-ward assuming that measuring DNA damage products de-rived from interactions with reactive oxygen and/or lipidperoxidation such as 8-oxo-2dg, 8-oxo adenine and/oretheno-DNA base adducts can be used as quantifiableand modifiable risk indicators. Dietary intervention includ-ing low fat, supplementation with vitamins C and E, orshort chain fatty acids, and, the intake of certain plant in-gredients in healthy volunteers, tumor patients and ani-mals has been found to be associated with decreased lev-els of DNA damage products paralleled in most instancesalso by a diminished cancer risk and/or relapse rates.Life style aspects are also increasingly coming into the fo-cus of interest as higher levels of physical exercise arefound to be associated with decreased cancer risk espe-cially breast and colorectal cancer and, in concordancewith the biomarker concept, with decreased levels of DNAdamage products in volunteers and animals.A more detailed knowledge of short and long term effectsof different levels of physical activity on DNA damage andtumor relapse is presently available for cancer patients,but would be of particular importance in further clinicalmanagement of these patients. In addition detailed knowl-edge of exercise effects on DNA repair mechanisms alsoare of clinical relevance, because strengthening such re-pair may further contribute to decreased DNA damage and

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cancer (relapse) risk. As colorectal cancer is the secondmost frequent malignancy in the Western world thisproject will focus on patients with these tumors.The aims are to compare short and long term effects ofmoderate versus low intensity rehabilitative exercise pro-grams in patients with treated (surgery, chemotherapy, ra-diation) colorectal cancer on the levels of DNA damageproducts and repair activity (etheno-DNA adducts in bloodcells, urine and stool samples) and relapse states, using acontrolled and randomized trial. The study has just beeninitiated.

1.13. Unsaturated fatty acids and oestrogenmetabolites: lipid peroxidation andoxidative stress as risk modifiers of breastcancer, a case-control study based on,biomarkers

K.H. Adzerson*, F. Beldmann*, G. Bastert*, J. Nair,R.W. Owen and H. Bartsch*Frauenklinik der Universität Heidelberg, Abteilung für AllgemeineFrauenheilkunde und Geburtshilfe.

Epidemiologic and experimental data show correlationsbetween an elevated risk for breast cancer and the dietaryintake of long-chain fatty acids (LCFA). While consumptionof fat per se is not strongly associated, evaluation of intakeof individual fatty acids show that diets high in polyunsatu-rated long-chain fatty acids (PUFA) of the n-6 series is del-eterious while high intake of PUFA of the n-3 series andmonounsaturated long-chain fatty acids (MUFA) may beprotective. It is imperative that the associations betweenfat intake in terms of the relative proportions of the specificn-3, n-6 and, n-9 LCFA is clearly elucidated so that futurechemopreventive strategies can be devised for high riskgroups.To this end a collaborative study between the Departmentof Gynecology and Obstetrics, University of HeidelbergWomens Clinic and the Division of Toxicology and CancerRisk factors has been designed involving 240 cases andcontrols. The intake of the various LCFA classes will beevaluated by dietary questionairre and related to a varietyof end-points such as fatty acid composition and estrogenprofiles in breast adipose tissue, exocyclic DNA adducts inbreast epithelial tissue and white blood cells and serumantioxidant status (carotenoids, ascorbic acid, tocopherolsand polyphenols). This project is funded by World CancerResearch Fund, London, UK.

1.14. Phenolic antioxidant profile of CaromaxH. Huang, R.W. Owen, R. Haubner, G. Würtele,B. Spiegelhalder, H. Bartsch, B. Haber*, D. Cremer*,G.-W. von Rymon Lipinski**Nutrinova, Nutrition Specialties & Food Ingredients GmbH,Industriepark Höchst, Frankfurt.

The Division of Toxicology and Cancer Risk Factors has aspecial interest in cancer chemopreventive agents and apilot study aimed at evaluating the role of Caromax (a jointproject in collaboration with Nutrinova, Nutrition Special-ties & Food Ingredients, Frankfurt) as an important lead

functional food in this domain has been already been con-ducted successfully. Over 20 potential antioxidant com-pounds have been identified (definitive) and quantitatedbut a range of other compounds which are also present insolvent extracts of Caromax have yet to be identified.From the standpoint of preliminary investigations it is obvi-ous that Caromax may have a very important role to playin future cancer chemopreventive strategies. To justify thisclaim the following is being conducted1. Activity guided fractionation of Caromax extracts to es-

tablish basic cancer chemopreventive activity by aHPLC hypoxanthine/xanthine oxidase based method.

2. Isolation of the compounds from the active fractions es-pecially for tests on the individual substances.

3. Subject both positive fractions and individual com-pounds to a battery of further in-vitro testing systemsrelevant to cancer prevention (e.g. inhibition of Phase-1and induction of Phase-2 enzymes, Frap and Trap as-says, etc) to fully evaluate the mechanisms of potentialchemoprotective effects.

4. Isolation, identification and testing (as above) of the asyet unknown substances in Caromax.

Publications (* = external co-author)[1] Khallouki, F.*; Younos, C.*; Oster, T.*; Charrouf Z.*;Spiegelhalder, B.; Bartsch, H.; Owen R.W. Consumption of arganoil (Morocco) with its unique profile of fatty acids, tocopherols,squalene, sterols and phenolic compounds shpuld confer valu-able cancer chemopreventive effects. European Journal of Can-cer Prevention 12 (2003) 67-75.[2] Owen, R.W.; Haubner, R.; Mier, W.*; Giacosa, A.*; Hull, W.E.;Spiegelhalder, B; Bartsch, H. The isolation, structural elucidationand antioxidant potential of the major phenolic compounds inbrined olive drupes. Food Chem Toxicol (2003), in press.[3] Bartsch, H; Nair J.; Owen, R.W. Exocylic DNA adducts as oxi-dative stress markers in colon carcinogenesis: Role of lipidperoxidation, dietary fats and antioxidants. J. Biol. Chem 383(2002) 915-921.[4] Little, J.L.*; Owen, R.W.; Fernandez, F.*; Hawtin, P.G.*; Hill,M.J.*; Logan, R.F.A.*; Thompson, M.H.*; Hardcastle, J.D.*: As-ymptomatic colorectal neoplasia and fecal characteristics: a casecontrol study of subjects participating in the Nottingham fecal oc-cult blood screeing trial. Dis. Colon Rectum 45 (2002) 1233-1241.[5] Owen, R.W.; Haubner, R.; Hull, W.E.; Thompson, L.U.*;Spiegelhalder, B; Bartsch, H. Complete pathway of mammalianlignan formation from secoisolariciresinol diglucoside. WholeGrain and Human Health, VTT Symposium 213 (2001) 81-84.[6] Owen, R.W.; Würtele, G.; Haubner, R.; Hull, W.E.; Giacosa,A.*; Spiegelhalder, B; Bartsch, H. Formation of the mammalianlignans enterodiol and enterolactone from (+)-pinoresinol, a majorlignan present in olive oil. Whole Grain and Human Health, VTTSymposium 213 (2001) 85-88.[7] Owen, R.W.; Giacosa, A.*; Hull, W. E.; Haubner, R.; Würtele,G.; Spiegelhalder, B; Bartsch, H. Olive oil consumption andhealth: the possible role of antioxidants. Lancet Oncology 1(2000) 107-112.[8] Bonithon-Kopp, C.*; Kronborg, O.*; Giacosa, A.*; Räth, U.*;Faivre, J.*; [Experts: Milan, C.*; Fenger, C.*; Piard, F.*; BelghitiC.*; Owen, R. W.; Pignatelli, M.*.] Calcium and fibre supplemen-tation in the prevention of colorectal adenoma recurrence: a pla-cebo-controlled intervention trial from the European Cancer Pre-vention Organisation (ECP). Lancet 356 (2000) 1300-1306.

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[9] Haines, A.*; Hill, M.J.*; Thompson, M.H.*; Owen, R.W., Will-iams, R.E.O.*; Meade, T.W.*; Wilkes, H.*: A prospective study offaecal bile acids and colorectal cancer. European Journal of Can-cer Prevention 9 (2000) 317-323.[10] Rudi, J.*; Bruchhausen B.*; Kuck D.*; Stremmel, W.*; vonHerbay, A.*; Bauer, H.*; Berger, M.*; Owen, R.W. Reactive oxy-gen species analysis in human gastritis patients and p53 methyla-tion analysis in gastric tumor cell line ags infected by Helicobacterpylori. Adv Exp Med Biol 500 (2001) 199-202.[11] Owen, R.W.; Giacosa, A.*; Hull, W.E.; Haubner, R.;Spiegelhalder, B.; Bartsch, H. The antioxidant/anticancer potentialof phenolic compounds isolated from olive oil. European Journalof Cancer 36 (2000) 1235-1247.[12] Owen, R.W., Mier, W.*; Hull, W.E.; Giacosa, A.*;Spiegelhalder, B.; Bartsch, H. Identification of lignans as majorcomponents in the phenolic fraction of olive oil. Clinical Chemistry46 (2000) 976-988.[13] Owen, R.W., Mier, W.*., Giacosa, A.*; Hull, W.E.;Spiegelhalder, B.; Bartsch, H. Phenolic compounds and squalenein oilive oils: the concentration and antioxidant potential of totalphenols, simple phenols, secoiridoids, lignans and squalene.Food Chem Toxicol 38 (2000) 647-659.[14] Owen, R.W., Spiegelhalder, B.; Bartsch H. Generation of re-active oxygen species by the faecal matrix. Gut 46 (2000) 225-232.[15] Bartsch, H.; Nair, J.; Owen, R.W. Dietary polyunsaturatedfatty acids and cancers of the breast and colorectum: emergingevidence for their role as risk modifiers. Carcinogenesis 20 (1999)2209-2218.[16] Fadden, K.*; Hill, M.J.*; Latymer, E.*; Low, G.*; Owen, R.W.Steroid metabolism along the gastrointestinal tract of the pig. Eu-ropean Jounral of Cancer Prevention 8 (1999) 35-40.[17] Trevisan, M.T.S.; Scheffer, J.J.C.*; Verpoorte, R.*: Peroxi-dase Activity in Hop Plants after Infestation by Red Spider Mites(2003) in press.[18] Bandeira, P.N.*; Pessoa, O.D.L.*; Trevisan, M.T.S.; Lemos,T.L.*: Metabólitos Secundários de Protium heptaphyllum March.Química Nova (2003) in press.[19] Trevisan, M.T.S.; Macedo, M.W.*; Meent, M.*; Rhee,I.K.*;Verpoorte, R.*: Seleção de Plantas com AtividadeAnticolinesterase para Tratamento da Doença de Alzheimer.Química Nova (2003) in press.[20] Owen R.W., Spiegelhalder, B., Bartsch, H. Possible molecu-lar targets for exogenous factors. In: Exogenous Factors in Co-lonic Carcinogenesis - Falk Symposium 128. Scheppack, W. andScheulen, M. (eds.) Kluwer Academic publishers (Lancaster, UK)(2003) pp 69-72.[21] Owen R.W. Biomarkers in colorectal cancer. In: Biomarkersin cancer chemoprevention. Miller, A.B. et al. (eds.). IARC Scien-tific Publications No 154 (Lyon, France) (2001) 101-112.[22] Owen R.W. The role of nutritional factors: colon cancer. In:Carcinogenic and anticarcinogenic factors in food. Eisenbrand, G.et al. (eds.), DFG, Wiley-VCH (2000) 43-75.

B) Genetic susceptibility markers (C0207)Group Leader: Dr. Bertold Spiegelhalder

A. Risch, H. Bartsch

Genetic variability in metabolic enzymes, such as thosemetabolising carcinogens from cigarette smoke may in-crease susceptibility to a number of human environment-related cancers. N-acetyltransferases, glutathione-S-trans-ferases and cytochrome-P450 enzymes are just a few ex-amples of tobacco-carcinogen-relevant metabolic en-zymes displaying such genetic polymorphisms, which are

known to affect the enzyme activity (Bartsch et al, CancerEpidemiology, Biomarkers and Prevention 9 (2000) 3-28).Worldwide, lung cancer has a very high incidence for men,and its incidence is increasing among females. Lung can-cer has a bad prognosis, as therapeutic measures haveonly limited success. While tobacco smoking is stronglyassociated with lung cancer risk, only 20% of all heavysmokers develop lung cancer. Many genetic and molecu-lar biological studies point towards a polygenic heritablepredisposition for lung tumors. Xenobiotic metabolism ofenvironmental carcinogens and DNA-repair processes aretwo important ways in which individual susceptibility to en-vironmental carcinogenesis can be affected. Genetic poly-morphisms in enzymes involved in detoxification of pro-carcinogens, such as cytochrome-P450-monooxygenasesCYP1A1, CYP2A6 and CYP2E1, glutathione-S-transfer-ases GSTM1, GSTT1 and GSTP1, N-acetyltransferasesNAT1 or NAT2, and myeloperoxidase (MPO) or in DNA-repair enzymes may affect lung cancer susceptibility. Thecombination of homozygous mutated CYP1A1 and theGSTM1*0/*0 genotypes, for instance, has been shown tolead to a stronger increase of anti-benzo(a)pyrene diol-ep-oxide DNA adduct levels than CYP1A1 and GSTM1 wild-type, in individuals with similar exposure levels (Rojas etal, Pharmacogenetics 8 (1998), 109-118). At-risk geno-types can therefore be used as susceptibility markers, withthe aim of identifying high risk individuals. There are indi-cations that interindividual differences as a result of ge-netic polymorphisms may be of particular importance tocancer risk at low dose exposures. Among a group ofsmokers with low cigarette consumption (low nicotine-cotinine levels in blood) those with the slow NAT2 geno-type had higher adduct levels than fast acetylators, whilethis difference was much less marked in heavier smokers(Vineis et al, Nature 396 (1994), 154-156). Similar resultswere observed in smokers in Japan in connection withCYP1A1 and GSTM1 polymorphisms and lung cancer.The better characterisation of the relevance of gene-envi-ronment interactions in the context of carcinogenesis is ofgreat importance for preventive measures such as the set-ting of exposure threshold values, public health cam-paigns, and chemopreventive approaches.The major objective of our work is the identification of ge-netic susceptibility markers for different human cancersand tumor subtypes. To this end a number of studies in-vestigating tumors in different organs, with known or sus-pected environmental aetiology are being conducted.

1. Genetic polymorphisms as modifiers of lungcancer risk

A. Risch, H. Dally, H. Wikman, K. Gassner, S. Thiel,B.Jäger, P. Schmezer, N. Rajaee-Behbahani,R. Godschalk, J. Nair, B. Spiegelhalder, H. BartschIn cooperation with Prof. Dr. med. Peter Drings, Prof. Dr. med.Hendrik Dienemann, Prof. Dr. Dr. med. Klaus Kayser, Prof. Dr.med. V. Schulz, PD Dr. med. Jürgen Fischer, Thoraxklinik, Heidel-berg-Rohrbach und Dr. Lutz Edler, DKFZ - Biostatistik.This work is/was partly supported by funding from the DeutscheKrebshilfe (H.D., B.J.), the Verein zur Förderung der Krebsfor-schung in Deutschland e.V. (A.R.), the EU-Marie-Curie Fellowship

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Programme (H.W.) and the Gottlieb Daimler und Karl BenzStiftung (H.W.).

In a hospital based case-control study the frequency of dif-ferent genetic polymorphisms notably in xenobiotic meta-bolising enzymes and of differences in DNA-repair capac-ity is being investigated. Occupational and smoking historyof lung cancer patients and cancer free hospital controls isrecorded, and blood and tissue samples are collected.Samples are genotyped for different known genetic poly-morphisms including NAT1, NAT2, GSTM1, GSTT1,GSTM3, GSTP1, CYP1B1, CYP1A1, hOGG1 and MPO,as well as newly identified genetic polymorphisms. Over1500 samples have been collected, and over 700 havebeen genotyped for N-acetyltransferases and Glutathione-S-transferases, where it could be shown that it is importantto distinguish between the different histological types oflung tumors, to evaluate the risk associated with differentgenotypes. [3,5]. Analysis of lung tissue from smokersshowed an association of aromatic DNA-adduct levels withthe combined NAT and GST genotypes [4] and a correla-tion between aromatic and O4-ethylthymidine adducts [10].The MPO genotype was identified as modifier of risk ofsmall cell lung cancer [9]. Mutagen sensitivity and DNA re-pair capacity were also measured in lymphocytes of thesame patients, and identified as risk modifiers for non-small cell lung cancer [1,2]. The overall future aim is toevaluate the relevance of different enzyme polymorphismsin a) the risk of developing different types of lung cancer b)the chemotherapy-sensitivity and c) the prognosis of lungcancer patients. Patient recruitment has just been startedwithin a EU-funded collaborative study, where it is alsoplanned to investigate genetic susceptibility factors in agroup of high-risk patients with second primary lung tu-mors from across Europe.

2. Development of higher-throughput genotypingmethods, and identification of new alleles

A. Risch, H. Dally, B. Jäger, B. Spiegelhalder,H. BartschFunded in part, by the Verein zur Förderung der Krebsforschungin Deutschland e.V. (A.R.)

In order to identify the role that genetic polymorphismsmay play in individual risk assessment, large case-controlstudies need to be conducted. PCR-RFLP methods up un-til recently were state of the art for the screening of largesample numbers for known genetic polymorphisms, how-ever, as the number of polymorphisms to be investigatedincreases, even larger studies, and higher throughputgenotyping are required. Methods employing fluores-cence-based capillary PCR followed by melting curveanalysis for the detection of mutations (Roche, Light-Cycler) have been developed for many genes, includingNAT1, NAT2 [3] and MPO [9]. Further such methods, aswell as those for the identification of new previously un-known alleles are under development.

3. Exposure to cigarette smoke and geneticpolymorphisms as risk markers for breastcancer in the general population

A. Risch, B. Spiegelhalder, H. BartschIn cooperation with PD Dr. Jenny Chang-Claude, Dr. Silke Kropp,Maren Rohrbacher, DKFZ Clinical EpidemiologyThis study was in part funded by the Deutsche Krebshilfe.

Genetic polymorphisms in metabolising genes may alsobe relevant for breast cancer in identifying high risk sub-jects. NAT1 and NAT2 metabolise aromatic heterocyclicamines, which are found in cigarette smoke at relativelyhigh concentrations. GSTT1 is responsible for the detoxifi-cation of ethylene oxide formed from ethene in cigarettesmoke as well as for glutathione-dependent activation ofcertain halomethanes also present in cigarette smoke. Asignificantly increased risk of breast cancer in women whoactively smoke and are slow (NAT2) acetylators had previ-ously been shown. There are also indications that enzymepolymorphisms may have a particular impact on cancerrisk at low-level carcinogen exposure. Given that one intwelve women in the Federal Republic of Germany will suf-fer from breast cancer in her lifetime and the prevalence ofsmoking in the population is high, the association of smok-ing with breast cancer is of both etiologic and public healthimportance.The objectives of this study are to determine if geneticpolymorphisms in metabolising genes such as N-acetyl-transferases NAT1 and NAT2 alter the ability of women todetoxify or activate tobacco-related carcinogens andthereby increase their susceptibility to breast cancer.Questionnaire data on active cigarette smoking and bloodsamples collected and stored for subsequent moleculargenetic analysis were already available from a population-based case-control study of breast cancer diagnosed bythe age of 50. Data on exposure to environmental tobaccosmoke have now been collected for an overall assessmentof active and passive smoke exposure of the study sub-jects. Genotyping has been carried out for a number ofgenes, including NAT2 using PCR-RFLP and fluores-cence-based melting curve analysis on breast cancer pa-tients and age-matched controls from the general popula-tion. Results from the statistical analysis of NAT2 genotypedata suggest that the NAT2 status has differential effect onthe association of active and passive smoking with breastcancer, demonstrating the need to consider possible dif-ferent mechanisms associated with exposure to main- andsidestream tobacco smoke [8].

4. Relevance of genetic polymorphisms to risk oflaryngeal carcinoma

A. Risch, V. Raedts, P. Schmezer,N. Rajaee-Behbahani, H. BartschIn Collaboration with Dr. H. Ramroth, DKFZ-Clinical Epidemiol-ogy, Prof. Dr. H. Becher, Institut für Tropen-Hygiene, Univ. Heidel-berg, OA Dr. med. habil. A. Dietz, Kopfklinik Heidelberg.Partly funded by: Bundesministerium für Bildung, Wissenschaft,Forschung und Technologie; Verein zur Förderung derKrebsforschung in Deutschland e.V. (A.R.)

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As part of a population-based case-control study to inves-tigate the aetiology of laryngeal carcinoma, blood samplesand details on occupational exposure information as wellas on exposure resulting from personal habits are avail-able from laryngeal carcinoma cases and controls. Com-parison of 245 cases and 251 matched population controlsconfirmed alcohol- and tobacco consumption as significantrisk factors, while statistical analysis of genotyping analy-sis for alcohol dehydrogenases ADH1B and ADH1C andGlutathione-S-transferases GSTM1 and GSTT1 showedthat the genetic variants investigated did not significantlymodify this risk, emphasising the importance of the envi-ronmental exposure as risk factors for laryngeal tumors.All studies should contribute to a better understanding ofthe aetiology of different cancers, and may provide cluesto the mechanisms of activation of pro-carcinogens in hu-mans. In order to tackle questions for which much largersubject numbers are required, anonymised genotype dataare additionally pooled for analysis in international collabo-rative studies [6,7]. The better characterisation of the rel-evance of gene-environment interactions in the context ofcarcinogenesis is of great importance for preventive mea-sures such as the setting of exposure threshold valuesand public health campaigns. A medium-term aim is toevaluate their predictive value in connection with preven-tive measures such as chemoprevention, and clinical ap-plications such as chemotherapy, and, if applicable, tovalidate them for clinical use.

Publications (* = external co-author)[1] Schmezer, P.; Rajaee-Behbahani, N.; Risch, A.; Thiel, S.*;Rittgen, W.; Drings, P.*; Dienemann, H.*; Kayser, K.W.*; Schulz,V.*; Bartsch, H. Rapid screening assay for mutagen sensitivityand DNA repair capacity in human peripheral blood lymphocytes.Mutagenesis 16 (2001) 25-30.[2] Rajaee-Behbahani, N.; Schmezer, P.; Risch, A.; Rittgen, W.;Kayser, K.W.*; *Dienemann, H.*; Schulz, V.*; Drings, P.*; Thiel,S.*; Bartsch, H. Altered DNA repair capacity and bleomycin sensi-tivity as risk markers for non-small cell lung cancer. InternationalJournal of Cancer 95 (2001) 86-91.[3] Wikman, H.; Thiel, S.*; Jäger, B.; Schmezer, P.; Spiegelhalder,B.; Edler, L.; Dienemann, H.*; Kayser, K.*; Schulz, V.*; Drings,P.*; Bartsch, H.; Risch, A. Relevance of N-acetyltransferase 1 and2 (NAT1, NAT2) genetic polymorphisms in non-small cell lungcancer susceptibility. Pharmacogenetics 11 (2001) 157-168.[4] Godschalk, R.W.L.; Dallinga, J.W.*; Wikman, H.; Risch, A.;Kleinjans, J.C.S.*; Bartsch, H.; Van Schooten, F.-J.*: Modulationof DNA and protein adducts in smokers by genetic polymor-phisms in GSTM1, GSTT1, NAT1 and NAT2. Pharmacogenetics11 (2001) 389-398.[5] Risch, A.; Wikman, H.; Thiel, S.*; Schmezer, P.; Edler, L.;Drings, P.*; Dienemann, H.*; Kayser, K.*; Schulz, V.*;Spiegelhalder, B.; Bartsch, H. Glutathione-S-transferase M1, M3,T1 and P1 polymorphisms and susceptibility to non-small-celllung cancer subtypes and hamartomas. Pharmacogenetics 11(2001) 757-764.

[6] Garte, S.*; Gaspari, L.*; Alexandrie, A.-K.*; Ambrosone, C.*;Autrup, H.*; Autrup, J.L.*; Baranova, H.*; Bathum, L.*;Benhamou, S.*; Boffetta, P.*; Bouchardy, C.*; Breskvar, K.*;Brockmöller, J.*; Cascorbi, I.*; Clapper, M.L.*; Coutelle, C.*; Daly,A.*; Dell�Omo, M.*; Dolzan, V.*; Dresler, C.M.*; Fryer, A.*;Haugen, A.*; Hein, D.W.*; Hildesheim, A.*; Hirvonen, A.*; Hsieh,L.-L.*; Ingelman-Sundberg, M.*; Kalina, I.*; Kang, D.*; Kihara, M.*;Kiyohara, C.*; Kremers, P.*; Lazarus, P.*; Le Marchand, L.*;Lechner, M.C.*; van Lieshout, E.M.M.*; London, S.*; Manni, J.J.*;Maugard, C.M.*; Morita S.*; Nazar-Stewart V.*; Noda K.*; Oda Y.*;Parl F.F.*; Pastorelli R.*; Persson I.*; Peters, W.H.M.*; Rannug,A.*; Rebbeck, T.*; Risch, A.; Roelandt, L.*; Romkes, M.*; Ryberg,D.*; Schoket, B.*; Seidegard, J.*; Shields, P.*; Sim, E.*; Sinnet,D.*; Strange, R.C.*; Stucker, I.*; Sugimura, H.*; To-Figueras, J.*;Vineis, P.*; Yu, M.C.*; Taioli, E.*: (2001) Metabolic Gene Polymor-phism Frequencies in Control Populations. Cancer Epidemiology,Biomarkers and Prevention 11 (2001), 1239-1248.[7] Vineis, P.*; Marinelli, D.*; Autrup, H.*; Brockmöller, J.*;Cascorbi, I.*; Daly, A.K.*; Golka, H.*; Okkels, K.*; Risch, A.;Rothman, N.*; Sim, E.*; Taioli, E.*: Smoking, Occupation, N-acetyltransferase-2 and Bladder Cancer: a pooled Analysis ofGenotype based studies. Cancer Epidemiology, Biomarkers andPrevention 10 (2001) 1249-1252.[8] Chang-Claude, J.; Kropp, S.; Jäger, B.; Bartsch, H.; Risch A.Differential effect of NAT2 on the association between active andpassive smoke exposure and breast cancer risk. Cancer Epidemi-ology, Biomarkers and Prevention 11 (2002) 698-704.[9] Dally, H.; Gassner, K.; Jäger, B.; Schmezer, P.; Spiegelhalder,B.; Edler, L.; Drings, P.*; Dienemann, H.*; Schulz, V.*; Kayser,K.*; Bartsch, H.; Risch, A. Myeloperoxidase (MPO) genotype andlung cancer histologic types: The MPO -463 A allele is associatedwith reduced risk for small cell lung cancer in smokers. Interna-tional Journal of Cancer 102 (2002) 530-535.[10] Godschalk, R.; Nair, J.; van Schooten, F.-J.*; Risch, A.;Drings, P.*; Kayser, K.*; Dienemann, H.*; Bartsch H. Comparisonof multiple DNA adduct types in tumor adjacent lung tissue: ef-fect of cigarette smoking. Carcinogenesis 23 (2002) 2081-2086.

C) Genotype Dependence of CarcinogenDNA Adduct Levels in humans (C0207)Group Leader: Dr. Bertold Spiegelhalder

1. CYP1A1 and GSTM1 genotypes affectbenzo[a]pyrene DNA adducts in smokers� lung:comparison with aromatic/hydrophobic adductformation[1]

K. Alexandrov*, I. Cascorbi**, M. Rojas,G. Bouvier***, E. Kriek***** Résidence du Grimpre, Villebon s/ Yvette, France; **ErnstMoritz Arndt University of Greifswald, Medical Faculty, Greifswald;***Product Team Responsible, GALDERMA R&D, SophiaAntipolis, France; ****GWAmstelveen, The Netherlands

Benzo[a]pyrene diol-epoxide (BPDE)-DNA adducts are in-volved in the induction of p53 mutations and probably inthe causation of human lung cancer associated with ciga-rette smoking. The ratio between CYP1A1 and GSTM1 en-zyme activities is a critical determinant of the target doseof carcinogenic BPDE and other DNA-reactive PAH me-tabolites. In the review, we summarized the published andour own data on modulation of (+)-anti-BPDE-DNA adductlevels in smokers� lungs by CYP1A1*2 genotypes alone orin combination with GSTM1 polymorphism and comparethese results with those reported for aromatic/hydrophobic(bulky) DNA adducts. The data published so far show only

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a trend for a non-significant increase in bulky DNA adductlevels in subjects with GSTM1*0 or the CYP1A1*2-GSTM1*0 genotype combination. In contrast, a clear de-pendence of (+)-anti-BPDE-DNA adduct levels was foundas a function of the CYP1A1 and GSTM1 genotypes: Inlung parenchyma, this adduct was more pronounced inpersons with the GSTM1*0 genotype, and CYP1A1*2-GSTM1*0 carriers had higher (+)-anti-BPDE-DNA adductlevels than those with CYP1A1*1/*1-GSTM1*0. The ho-mozygous CYP1A1*2/*2 carriers in the GSTM1*0 grouphad the highest (+)-anti-BPDE-DNA adduct levels. Ouranalysis leads to the conclusion that the risk-modifying ef-fects of metabolic genotypes and of gene interactionsmight be more easily identifiable if specific markers ofstructurally defined adducts were used, such as the(+)-anti-BPDE-DNA adduct. These results are also consis-tent with the hypothesis that BP (PAH) induce G:C to T:Atransversion mutations in the hotspot codons of the p53tumor suppressor gene and are thus involved in malignanttransformation of the lung tissue of smokers.

2. Myeloperoxidase -463A variant stronglyreduces benzo[a]pyrene diol-epoxide DNAadducts in skin of coal tar treated patients [2]

M. Rojas, R. Godschalk, K. Alexandrov** Résidence du Grimpre, Villebon s/ Yvette, France

In cooperation with : I. Cascorbi, Ernst Moritz Arndt University ofGreifswald Medical Faculty; Judith Ostertag, University HospitalMaastricht: Department of Dermatology, The Netherlands

The skin of atopic dermatitis patients provides an excellentmodel to study the role of inflammation in benzo[a]pyrene(BaP) activation, since these individuals are often topicallytreated with ointments containing high concentrations ofBaP. In this study, we determined by HPLC-fluorescencedetection (Alexandrov et al., Cancer Res., 52 (1992), 6248-6253) the BaP-diolepoxide (BPDE)-DNA adduct levels inhuman skin after topical treatment with coal tar and theirmodulation by the -463G→A myeloperoxidase (MPO) poly-morphism, which reduces MPO mRNA expression. BPDE-DNA adduct levels were 2.2 and 14.2 adducts per 108

nucleotides for MPO-463AA/AG and -463GG, respectively.The predominant BaP tetrol observed was tetrol I-1, whichis derived after hydrolysis of the anti-BPDE-DNA adduct.The tetrol I-1/ II-2 ratio corresponding to the anti/ syn ratiowas 6.7. The 32P-postlabelling assay was also performedand thin layer chromatograms showed a major spot withthe chromatographic location corresponding toBPDE-DNA. The mean values of the BPDE-DNA adductspots were 3.8±2.4 per 108 nucleotides for MPO-463AA/AG (n=3) and 18.4±11.0 per 108 nucleotides for MPO-463GG (n=7), respectively (P=0.03). One individual withthe homozygous mutant genotype (-463AA) even had a13-fold lower adduct level (1.4 per 108) as compared toMPO-463GG subjects. In conclusion, these data showedfor the first time (i) the in vivo formation of BPDE-DNA ad-ducts in human skin treated with coal tar and (ii) that theMPO-463AA/AG genotype strongly reduced BPDE-DNAadduct levels in human skin.

Publications (* = external co-author)[1] Alexandrov, K.*; Cascorbi, I.*; Rojas, M.; Bouvier, G.*; Kriek,E.*; Bartsch, H. CYP1A1 and GSTM1 genotypes affectbenzo[a]pyrene DNA adducts in smokers� lung: comparison witharomatic/hydrophobic adduct formation. Carcinogenesis 23(2002) 1969-1977.[2] Rojas, M.; Godschalk, R.; Alexandrov. K.*; Cascorbi, I.*; Kriek,E.*; Ostertag, J.*; Van Schooten, F.-J.*; Bartsch, H.Myeloperoxidase -463 A variant reduces benzo[a]pyrene diol-ep-oxide DNA adducts in skin of coal tar treated patients. Carcino-genesis 22 (2001) 1015-1018.

division toxicology and cancer risk factors (c0200 / c010) · exploration of markers in large-scale...

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