CONTRACTING ORGANIZATION: University of California at Berkeley E.O. Lawrence Berkeley National Laboratory Berkeley, California 94701

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1 * J AD Award Number: DAMD TITLE: Oxidative Damage, CYP1B1 and Breast Cancer PRINCIPAL INVESTIGATOR: Regine Gth-Gldstein, Ph.D. NTRACTING ORGANIZATION: University f Califrnia at Berkeley E.O. Lawrence Berkeley Natinal Labratry Berkeley, Califrnia REPORT DATE: July 2002 TYPE OF REPORT: Final PREPARED FOR: U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland DISTRIBUTION STATEMENT: Apprved fr Public Release; Distributin Unlimited The views, pinins and/r findings cntained in this reprt are thse f the authr(s) and shuld nt be cnstrued as an fficial Department f the Army psitin, plicy r decisin unless s designated by ther dcumentatin. \ * p- If <0

2 REPORT DOCUMENTATION PAGE Frm Apprved OMB N Public reprting burden fr this cllectin f infrmatin is estimated t average 1 hur per respnse, including the time fr reviewing instructins, searching existing data surces, gathering and maintaining the data needed, and cmpleting and reviewing this cllectin f infrmatin. Send cmments regarding this burden estimate r any ther aspect f this cllectin f infrmatin, including suggestins fr reducing this burden t Washingtn Headquarters Services, Directrate fr Infrmatin Operatins and Reprts, 1215 Jeffersn Davis Highway, Suite 1204, Arlingtn, VA and t the Office f Management and Budget, Paperwrk Reductin Prject ( ), Washingtn, DC AGENCY USE ONLY (Leave blank) 2. REPORT DATE 3. REPORT TYPE AND DATES VERED July TITLE AND SUBTITLE Oxidative Damage, CYP1B1 and Breast Cancer Final (1 Jul Jun 02) 5. FUNDING NUMBERS DAMD AUTHOR(S) Regine Gth-Gldstein, Ph.D. 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) University f Califrnia at Berkeley E.O. Lawrence Berkeley Natinal Labratry Berkeley, Califrnia ; R_Gth-Gldstein@lbl.gv 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland SUPPLEMENTARY NOTES Reprt cntains clr 12a. DISTRIBUTION / AVAILABILITY STATEMENT 8. PERFORMING ORGANIZATION REPORT NUMBER 10. SPONSORING / MONITORING AGENCY REPORT NUMBER b. DISTRIBUTION DE Apprved fr Public Release; Distributin Unlimited 13. ABSTRACT (Maximum 200 Wrds) This study addresses the initiating causes and early events f breast cancer. Increased xidative DNA damage has been reprted in breast tissue frm breast cancer patients. This study investigated if the cytchrme P450 enzyme CYP1B1 plays a rle in the frmatin f xidative damage. The CYP1B1 enzyme is invlved in the metablic activatin f envirnmental carcingens, and als metablizes estrgen t a carcingenic metablite. The cncept tested was that high levels f CYP1B1 lead t increased xidative damage, thereby initiating the carcingenic prcess. CYP1B1 expressin and xidative damage were determined in 58 breast tissue specimens frm reductin mammplasties and nn-tumr peripheral tissue frm mastectmies. CYP1B1 expressin was determined by real time RT-PCR relative t the ß-actin gene. 8-hydrxy- 2'dexyguansine (8-xG), a reliable index f verall xidative DNA damage, was determined by 32P-pstlabeling. 8- xg levels varied thirty-fld and CYP1B1 expressin 500-fld amng specimens. In the limited number f specimens analyzed, 8-xG levels were slightly higher in specimens frm cntrls then specimens frm cases. CYP1B1 expressin differed between cases and cntrls nly in wmen ver 50 years. N crrelatin between CYP1B1 expressin and 8-xG levels was bserved. 14. SUBJECT TERMS breast cancer, xidative damage, CYP1B1, RT-PCR 15. NUMBER OF PAGES PRICE DE 17. SECURITY CLASSIFICATION OF REPORT Unclassified 18. SECURITY CLASSIFICATION OF THIS PAGE Unclassified 19. SECURITY CLASSIFICATION OF ABSTRACT Unclassified 20. LIMITATION OF ABSTRACT Unlimited NSN Standard Frm 298 (Rev. 2-89) Prescribed by ANSI Std. Z

3 FOREWORD Opinins, interpretatins, cnclusins and recmmendatins are thse f the authr and are nt necessarily endrsed by the U.S. Army. Where cpyrighted material is quted, permissin has been btained t use* such material. Where material 'frm dcuments designated fr limited distributin is quted, permissin has been btained t use the material. Citatins f cmmercial rganizatins and trade names in this reprt d nt cnstitute an fficial Department f Army endrsement r apprval f the prducts r services f these rganizatins. N/A In cnducting research using animals, the investigatr(s) adhered t the "Guide fr the Care and Use f Labratry Animals," prepared by the Cmmittee n Care and use f Labratry Animals f the Institute f Labratry Resurces, natinal Research Cuncil (NIH Publicatin N , Revised 1985). X Fr the prtectin f human subjects, the investigatr(s) (RST adhered t plicies f applicable Federal Law 45 CFR 46. vtf N/A In cnducting research utilizing recmbinant DNA technlgy, the investigatr (s) adhered t current guidelines prmulgated by the Natinal institutes f Health, N/A In the cnduct f research utilizing recmbinant DNA, the investigatr (s) adhered t the NIH Guidelines fr Research Invlving Recmbinant DNA Mlecules. N/A In the cnduct f research invlving hazardus rganisms, the investigatr (s) adhered t the CDC-NIH Guide fr Bisafety in Micrbilgical and Bimedical Labratries.

4 Table f Cntents Cver 1 SF298 2 Frewrd 3 Table f Cntents 4 Intrductin 5 Bdy 6 Key Research Accmplishments 9 Reprtable Outcmes 10 Cnclusins 10 References 11 List f Persnnel receiving Pay frm Research Effrt 13 Bibligraphy f PI 14 Appendices 16

5 Oxidative damage, CYP1B1 and Breast Cancer Intrductin Oxidative damage t DNA has been shwn t ccur in viv indicating that DNA can be damaged as a cnsequence f nrmal metablism (1). Because a large number f DNA base mdificatins are caused by xidative stress, it is difficult t measure the whle spectrum f mdified lesins. One f the mre abundant lesins, 8-hydrxy- 2'dexyguansine (8-xG), which is knwn t cause a G t T transversin, is used as a reliable index f verall xidative DNA damage. DNA bases mdified by xidative stress can lead t mutatins, chrmsmal abnrmalities and altered gene expressin. Cntinuus xidative stress, which leads t the frmatin f free radicals frm mlecular xygen, has been implicated in varius stages f chemical carcingenesis (1). The etilgy f the majrity f human breast cancers is nt understd. Evidence supprting a link between xidative damage and breast cancer includes the finding that xidative DNA damage is increased in breast tissue frm cancer patients cmpared t cntrls (2) and that cells with BRCA1 deficiencies lack transcriptin-cupled repair f xidative damage (3). Suggested breast cancer risk factrs such as expsure t envirnmental carcingens, dietary fat intake and estrgen metablism culd all act thrugh an xidative stress mechanism. The xidative metablism f envirnmental cntaminants, like plycyclic armatic hydrcarbns (PAHs) by the cytchrme P450 enzymes CYP1A1 and CYP1B1, creates reactive xygen species that result in xidative damage t DNA. The amunt f xidative damage far exceeds that f direct PAH adduct frmatin (4). Endgenus surces f xidative damage include the xidative metablism f sterid hrmnes, such as estrgen. Estradil is cnverted t catechl estrgens thrugh hydrxylatin by varius cytchrme P450 iszymes, leading predminantly t frmatin f 2- hydrxyestrgen (5). Only the iszyme CYP1B1 catalyzes the predminant frmatin f 4-hydrxyestrgen (4-OH-E2). It has been suggested that estrgen exerts sme f its effects in target tissues like breast thrugh lcally frmed estrgen metablites. The estrgen metablite 4-OH-E2 might fulfill such a rle (6). 4-OH-E2 has als been implicated in estrgen-assciated cancers in varius rdent mdels and is knwn t elicit an xidative stress respnse induced by free radicals generated by metablic redx cycling reactins (6). We have fund previusly that CYP1B1 is expressed in breast and that expressin levels were significantly higher in nn-tumr breast tissue frm breast cancer patients than frm healthy individuals, suggesting a pssible rle f CYP1B1 in the etilgy f breast cancer. The cncept investigated here is that high levels f CYP1B1 lead t increased xidative damage, thereby initiating the carcingenic prcess.

6 Bdy f Final Reprt A cllectin f histlgically nrmal breast tissue specimens frm mastectmy patients and frm reductin mammplasties has been analyzed bth fr expressin levels f the CYP1B1 gene and fr markers f xidative damage. Specimens: A tissue bank established by Peralta Cancer Center in and maintained by Aern Bitechnlgy Inc. was btained. Only histlgically nrmal breast tissue specimens (as determined by pathlgy reprt) were used fr these studies. The specimens had been dissected and islated frm adipse and cnnective tissue, s that nly epithelial material was stred frzen as rganids (8). The cntrl grup cnsisted f 18 reductin mammplasty specimens (Table 1). The case grup cnsisted f 38 peripheral nn-tumr tissue specimens btained frm mastectmy patients and 1 tissue specimen cntralateral t the tumr. The average age f the cntrl grup is 18 years yunger than the case grup and is a ptential cnfunder in the statistical analysis f the specimen. Stratifying the grups by age slves this prblem, but reduces the sample size cnsiderably. Table 1: Descriptin f specimen. Grups Specimen Analysed (n) Average Age (y) Cntrls Cntrls >50y Cases Cases >50y Ttal Measuring CYP1B1 expressin levels: Ttal RNA was islated frm 56 breast epithelial specimens using TRI Reagent as recmmended by the manufacturer. Tw ßg f ttal RNA was reverse transcribed at 37 C fr 1 hur using (lig dt) 16 and Mlney murineleukemia virus reverse transcriptase. The cdna was stred as several small aliquts at - 80 C. A PCR-based assay fr calibratr-nrmalized relative quantitatin was develped using a rapid micr-capillary cycler with real-time prduct detectin by flurescence (LightCycler, Rche Mlecular Bichemicals). The cdna was amplified with ß-actin primers (9) and with CYP1B1 primers (10,11). Bth primer sets are designed t span an intrn, thus excluding amplificatin f any cntaminating genmic DNA and generated prducts f 158 bp fr CYP1B1, and 273 bp fr ß-actin. cdna frm primary cultures f a human mammary epithelial cell line (HMEC184) was included in each PCR reactin as a calibratr. CYPIB1 levels are expressed as a rati t a reference gene: ß-actin. This crrects fr sample variatins in the amunt and quality f the islated RNA. The rati f CYPlBl/ß-actin fr the sample is divided by the CYPlBl/ß-actin rati fr the calibratr

7 in rder t crrect fr run-t-run variatins and mst imprtantly, differing amplificatin efficiencies between the tw primer sets. The reliability f the assay t measure CYP1B1 expressin was determined by re-analyzing 10% f the cdna specimens. The CYP1B1 levels had a variance f 12%. Cmparisn f CYP1B1 expressin in breast cancer patients and cntrls: In breast tissue specimens frm cases and cntrls, there was a 500-fld variatin in the CYP1B1 expressin level (range 0.05 t 25.87). The arithmetic means f the CYPlBl/ß-actin rati f the cases and cntrls (see Table 2) were cmpared using a tw-tailed student's t-test. N significant difference in CYP1B1 levels between the tw grups was fund (p=0.7770). In a previus study, we did discver a statistically significant difference (p = ) in CYP1B1 values between case and cntrl grups but the sample size was much larger (32 cntrls and 24 cases) (14). This finding did nt repeat in this smaller set f samples. When the tw grups are stratified by age (< 50 and > 50 years), which rughly crrespnd t menpausal status, the p-value f the grup ver 50 years is , a significant result if the size f the cntrl grup ver 50 years was larger. Als, the range f CYP1B1 levels was nticeably mre restricted in cntrls ver 50, pssibly indicating that endgenus estrgen levels might influence CYP1B1 expressin. Table 2: Statistical summary f CYP1B1 gene expressin levels in nn-tumr breast tissue f breast cancer patients and healthy cntrls. CYPlBl/actin rati Grups Mean (SD) Median Range Cntrls 2.28 (4.05) Cntrls >50y 0.98 (0.55) Cases 2.65 (5.30) Cases >50y 3.02 (5.79) Ttal 2.53 (4.90) A scatter plt f CYP1B1 expressin levels f all specimen and age shws n crrelatin (Figure 1, appendix). The Pearsn's prduct mment crrelatin cefficient was determined t be r = Determinatin fxidative damage: DNA was islated frm the 56 breast specimens by the newly develped sdium idide methd that has been shwn t minimize, if nt eliminate, the xidative damage t DNA that ccurs during islatin (12). The 8-xG cntent (a reliable index f verall xidative DNA damage) was determined by 32P-pstlabeling in cllabratin with Dr. Dnghui Li, UT, MD Andersn Cancer Center (13). Cmparisn fxidative damage in breast cancer patients and cntrls: The relative 8- xg levels varied abut 30-fld amng the 56 specimens tested (Table 3). A cmparisn f the mean xidative damage level between cases and cntrls (using a tw-tailed

8 student's t-test) shws a significant difference between the tw grups (p = ) with xidative levels f DNA being higher in the cntrl grup than the cases. A scatter plt f xidatin levels and age f all specimen (Figure 2, appendix) des indicate a slight crrelatin that is inversely related t age (Pearsn's prduct mment crrelatin cefficient r = ). When the tw grups are stratified by age, the difference in xidative damage fund in the lder grups is n lnger significant (p-value = ). Table 3: Statistical summary f xidative damage in nn-tumr breast tissue f breast cancer patients and healthy cntrls. 8-xG/dG (xl(f) Grups Mean (SD) Median Range Cntrls (43.7) Cntrls >50y (47.0) Cases 113.5(46.4) Cases >50y 108.3(45,4) Ttal 121.2(46.5) Cmparisn f xidative damage and CYP1B1 expressin levels between breast cancer patients and cntrls: The distributin f xidative damage levels ver the range f CYP1B1 levels appears t be nrmal (Figure 3, appendix). In specimen with high CYP1B1 expressin (rati > 5), the xidative damage appears als t be nrmally distributed and nt dependent n high expressin f the CYP1B1 gene. Overall, these results n CYP1B1 expressin and xidative damage d nt supprt ur hypthesis that higher levels f CYP1B1 leads t increased xidative damage. Future gals We plan t determine the genetic plymrphism f the CYP1B1 gene in the specimens t determine if a certain CYP1B1 gentype crrelates with xidative damage levels. We will als determine the PAH DNA-adduct levels t evaluate the relatinship f PAH expsure t xidative damage levels.

9 Key Research Accmplishments A sensitive and reliable RT-PCR assay was develped t determine expressin f CYPlBlby real-time PCR. A 500-fld variatin in expressin f CYP1B1 in 56 nn-tumr breast tissue samples was determined. Mre breast cancer cases ver 50 years ld than cntrls in that age grup shwed high CYP1B1 expressin. Oxidative damage was measured in the same 56 specimens and was fund t vary by a factr f 30 with mean 8-xG levels slightly higher in cntrls than cases. N crrelatin between CYP1B1 expressin and xidative damage culd be bserved.

10 Reprtable Outcmes Abstracts An abstract n this study was submitted fr presentatin at the Era f Hpe, Department f Defense Breast Cancer Research Prgram Meeting in September Cnclusins Because f the ptential imprtant rle f CYP1B1 in the activatin f envirnmental and endgenus cmpunds t carcingenic intermediates, it was hypthesized that high CYP1B1 expressin culd result in high levels f xidative damage. We fund a very large inter-individual variatin in CYP1B1 expressin and a much smaller variatin in levels f xidative damage. N crrelatin between CYP1B1 expressin and xidative damage was bserved. 10

11 References 1. Ames, B.N., and Gld, L.S. (1991) Endgenus mutagens and the causes f aging and cancer. Mutat. Res. 250: Malins. D. et.al. (1993) The etilgy f breast cancer. Characteristic alteratins in hydrxyl radical-induced DNA base lesins during ncgenesis with ptential fr evaluating incidence risk. Cancer. 71: Gwen, L. et.al. (1998) BRCA1 required fr transcriptin-cupled repair f xidative DNA damage. Science. 281: Leadn, S. et.al. (1988) Prductin f xidative damage during metablic activatin f benz[a]pyrene in human mammary epithelial cells crrelates with cell killing. Prc. Natl. Ac. Sciences. 85: Hayes, C.L., Spink, D.C., Spink, B.C.,Ca, J.Q., Walker, N.J. Sutler, T.R. (1996) 17 ß-estrdil hydrxylatin catalyzed by human cytchrme P4501B1. Prc.Natl. Aca. Sciences USA 93: Zhu, B. T. and A. H. Cnney. (1998) Functinal rle f estrgen metablism in target cells: review and perspectives. Carcingenesis. 19: Gnzalez, F.J. and H. V. Gelbin. (1994) Rle f human cytchrmep450 in the metablic activatin f chemical carcingens and txins. Drug Metab. Rev.26: Stampfer, M. (1985) Islatin and grwth f human mammary epithelial cells. J. Tissue Culture Methds. 9: Hrikshi, T., Danenberg, K., Stadibauer, T., Vlkenandt, M., Luke, C. S., Aigner, K., Gustavssn, B. Leichman, L., Fresing, R., Ray, M., Gibsn, N. W., Spears, C. P., Danenberg, P. (1992) Quantitatin f thymidylate synthase, dihydrflate reductase, and DT-diaphrase gene expressin in human tumrs using the plymerase chain reactin. Cancer Res., 52: Larsen, M. C. Angus, W. G., Brake, P. B., Eltm, S. E., Sukw, K. A., Jefcate, C. R. (1998) Characterizatin f CYP1B1 and CYP1A1 expressin in human mammary epithelial cells: rle f the aryl hydrcarbn receptr in plycyclic armatic hydrcarbn metablism. Cancer Res., 58: Keshava, C, Whipkey, D. L., Westn, A. (2002) Transcriptinal signature f primary nrmal human mammary epithelial cells in respnse t benz[a]pyrene expsure. Prceedings f the American Assc. fr Cancer Research. 43: Hamiltn, M.L., Gu,Z.M., Fuller, C. D., Van Remmen, H., Ward W., Austad, S. N., Tryer, D.A., Thmpsn, I. and Richardsn, A. (2001) A reliable assessment f 8-x-2- dexyguansine levels in nuclear and mitchndrial DNA using the sdium idide 11

12 methd t islate DNA. Nucleic Ac. Res. 29: Zhang, W., Chang, P., Sahin, A., Singletary, E. S., Bndy, M., Hazra, T., Mitra, S., DiGivanni, J., and Li, D. (2001) Oxidative stress and repair f xidative DNA damage in human breast tissues. Prceedings f the American Assc. fr Cancer Research. 41: Gth-Gldstein, R., Erdmann, CA., Russell, M. R. Cytchrme P4501B1 expressin in nrmal breast tissue. Submitted tpac. 12

13 List f persnnel receiving pay frm the research effrt Regine Gth-Gldstein, Principal Investigatr Marin Russell, Senir Research Assciate 13

14 Bibligraphy f PI Publicatins since 1999:.Hlman, H.Y., Zhang, M., Gth-Gldstein, R., et al. (1999) Detecting expsure t envirnmental rganic txins in individual cells: twards develpment f a micrfabricated device. In: Micr-and Nanfabricated Structures and Devices fr Bimedical Envirnmental Applicatins II, SPIE Vl. 3606, Gth-Gldstein, R., Hlman, H.Y., Russell, M. (1999) In vitr mdel fr intestinal uptake f benz(a)pyrene. LBNL reprt #43752 Erdmann, CA., Petreas, M.X., Caleffi, M., Barbsa, F.S., Gth-Gldstein, R. (1999) Cmparisn f Organchlrine Chemical Bdy Burdens f Female Breast Cancer Cases with Cancer Free Wmen in Ri Grande d Sul, Brazil - Pilt Study. LBNL reprt # Hlman, H.Y., Ma, Y., Gth-Gldstein, R. (2000) Applicatin f in vitr ral biavailability test prtcl t 10 field sils. In: Final Prject Reprt: PERF Research Area N.l, 'Envirnmentally Acceptable Treatment Endpints fr Hydrcarbn-Cntaminated Sils', Appendix 12A. Gth-Gldstein, R., Russell, M. and Hlman, H.Y. (2000) Examinatin f transprt and metablism f benz(a)pyrene thrugh Cac-2 cells. In: Final Prject Reprt: PERF Research Area N.l, 'Envirnmentally Acceptable Treatment Endpints fr Hydrcarbn-Cntaminated Sils', Appendix 12B. Hlman, H.Y., Gth-Gldstein, R., Blakely, E., Bjrnstad, K., Martin, M.C. and McKinney, W.R.(2000) Individual Human Cell Respnses t Lw Dses f Chemicals Studied by Synchrtrn Infrared Spectrmicrscpy. In: Bimedical Spectrscpy: Vibratinal Spectrscpy and Other Nvel Techniques, SPIE Vl. 3918, Hlman, H.Y., Gth-Gldstein, R., Martin, M., Russell, M.L., McKinney W.R. (2000) Lw-dse respnses t 2,3,7,8,-tetrachlrdibenz-p-dixin in single living cells measured by synchrtrn infrared spectrmicrscpy. Env. Science & Tech. 34: Gth-Gldstein, R., C. Erdmann, M. Russell (2000) CyplBl expressin in nrmal human breast tissue specimen. Prc. Am. Ass. Cancer Res.41: #807. Gth-Gldstein, R., Stampfer, M.R., Erdmann, CA., Russell, ML. (2000) Interindividual variatin in CYP1A1 expressin in breast tissue and the rle f genetic plymrphism. Carcingenesis 21: Gth-Gldstein, R., Erdmann, C, Russell, M. (2001) CYP1B1 expressin as a risk factr fr breast cancer. Abstr., 32nd Annual Meeting, Envirnmental Mutagen Sciety, San Dieg, CA. Envirnmental Mutagenesis36, Suppl

15 Gldstein, L., Gth-Gldstein, R., Russell, M., Singh, R., Weyand, E. H. (2001) 7ff- Benz[c]flurene a candidate carcingenic PAH with majr health implicatins. Abstr. 32nd Annual Meeting, Envirnmental Mutagen Sciety, San Dieg, CA. Envirnmental Mutagenesis 36, Suppl. 36. Gth-Gldstein, R., Russell, M., Singh R., and Weyand, E.H., (2001) 1H- Benz[c]flurene:DNA adduct frmatin in MCF7 and HepG2 Cells. The Txiclggists, 60:1817. Li, D., Wang, M., Firzi, P.F., Chang, P., Zhang, W., Baer-Dubwska, W., Mrthy, B., Vulimiri, S.V., Gth-Gldstein, R., Weyand, E. H., and DiGivanni, J. (2002) Characterizatin f a majr armatic DNA adduct detected in human breast tissues, Env. Ml. Mutagenesis 39: Hlman, H.Y., Gth-Gldstein, R., Aastn, D., Ma, Y., and Kengsntra, J. (2002) Evaluatin f gastrintestinal slubilizatin f petrleum hydrcarbn residues in sil using an in vitr physilgically-based mdel. Env. Science & Tech. 36: Gth-Gldstein, R., Russell, M. L., Parim B., and Weyand, E.H. (2002) 1H- Benz[c]flurene DNA adduct frmatin in different human cells in culture. Prc. Am. Ass. Cancer Res. 43, #1719. Russell, M. L., Gth-Gldstein, R., Apte, M. and Fisk, W. (2002) Size distributin f airbrne rhinvirus. Prceedings f the 9th Internatinal Cnference n Indr Air Quality and Climate, Indr Air 102, Wldegirgis, S., Ahmed, R. C, Zhen, Y., Erdmann, C. A., Russell, M. L., Gth- Gldstein, R. (2002) Genetic Plymrphism in Three Glutathine 5-transferase Genes and Breast Cancer Risk. LBNL reprt # Gth-Gldstein, R., Erdmann, CA., Russell, M. R. Cytchrme P4501B1 expressin in nrmal breast tissue. Submitted tpac. 15

16 Appendix Cver Sheet List f cntent: Figure 1. CYP1B1 Gene Expressin in Nn-tumr Breast Tissue and Age f Dnrs. Figure 2. Oxidative Damage Measured in Nn-tumr Breast Tissue and Age f Dnrs. Figure 3. CYP1B1 Expressin Levels as a functin f Oxidative Damage in Nn-tumr Breast Tissue f Cases and Cntrls. 16

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18 <-> CD c Ü Ü O E 3 *t C z (0 c ki c Q 3 *- (0 (0 0> 5 O) f < 0) O) (0 i. E (0 (0 0) 3 Q V (A > 1-4-> (0 (0 (0 X 0) O k. CD CM 0) >- 3 CD U_ a0l.x(p/ooxo-8)

19 m CM V > E Q > + " 4-> >< C O c D C r CD 3 Ll- H 3 0) > 1- _l * > c 1- CQ 1-1- Q. X E UJ 3 +* m c Q. z >- c <D 1-3 O) Cases Cntrls i 4 V i ii «I CM LO in t O O X I 00 in CM CM m (u!pe/.ai.dao)!ieh m