R. Balansky 1,2, F. D Agostini 2, A. Izzotti 2, P. Kalpakam 3, V.E. Steele 4, S. De Flora 2
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1 INCON / 10 ICMAA, Guarujà, Brazil, September 26-29, 2010 MECHANISMS OF INHIBITION OF CIGARETTE SMOKE GENOTOXICITY AND CARCINOGENICITY R. Balansky 1,2, F. D Agostini 2, A. Izzotti 2, P. Kalpakam 3, V.E. Steele 4, S. De Flora 2 National Center of Oncology, Sofia, Bulgaria Department of Health Sciences, University of Genoa, Italy National Institute of Nutrition, Hyderabad, India National Cancer Institute, Rockville, MD, USA
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3 ACTIVE SMOKE AND PASSIVE SMOKE MAINSTREAM SMOKE ACTIVE SMOKER SIDESTREAM SMOKE ENVIRONMENTAL TOBACCO SMOKE (ETS) or INDOOR AIR TOBACCO SMOKE (IATS) PASSIVE OR INVOLUNTARY SMOKER
4 CIGARETTE SMOKING AND CANCER Evidence for causal association Lung (90%) Urinary tract (bladder, ureter, and renal pelvis) Oral cavity Nasal cavity and paranasal sinuses Naso-, oro-, and hypopharynx Larynx Oesophagus Stomach Liver Pancreas Uterine cervix Bone marrow (myeloid leukemia) Inconclusive evidence for association Colon-rectum Evidence for lack of association Breast Endometrium Prostate? IARC Vol. 83, 2004 Tobacco Smoke and Involuntary Smoking
5 DEATHS ATTRIBUTABLE TO TOBACCO SMOKE TOTAL TUMORS 30% LUNG TUMORS 85% AERODIGESTIVE TRACT TUMORS 50 70% synergism with alcohol CARDIOVASCULAR DISEASES 30% synergism with other risk factors COPD 75% EU ~ 500,000 deaths / year USA ~ 500,000 deaths / year
6 TOBACCO SMOKE AS A COMPLEX MIXTURE Balansky RM, Blagoeva PM, Mircheva ZI. Investigation of the mutagenic activity of tobacco smoke. Mutat. Res May;188(1):13-9. Balansky RM, Blagoeva PM, Mircheva ZI. The mutagenic and clastogenic activity of tobacco smoke. Mutat. Res Jul;208(3-4): Balansky RM, Blagoeva PM. Tobacco smoke-induced clastogenicity in mouse fetuses and in newborn mice. Mutat. Res May;223(1):1-6. Balansky RM, Blagoeva PM, Mircheva ZI. Modulation of genotoxic activity of tobacco smoke. IARC Sci. Publ. 1991;(105):535-7.
7 PROTECTIVE FACTORS RISK FACTORS STOP EXPOSURE MARKERS Targets for chemoprevention - Addicted current smokers - Passive smokers - Ex-smokers - Other exposures BIOLOGICALLY EFFECTIVE DOSE EARLY BIOLOGICAL DAMAGE DAYS - WEEKS YEARS - DECADES Mutagenicity of escreta Protein adducts DNA adducts 8-oxo-dG Metabolic alterations DNA damage and repair Cytogenetic effects Activation of oncogenes Deletion or mutation of oncosuppressor genes DISEASES Effects on proliferation, differentiation, apoptosis, etc. Multigene expression microrna profiles Proteomic profiles
8 TIME COURSE FORMATION OF DNA ADDUCTS IN RATS EXPOSED TO CIGARETTE SMOKE DNA Adducts/10 8 nucleotides Tracheal epithelium BAL cells Lung Heart Bladder Exposure time (weeks) A. Izzotti et al., Carcinogenesis 20, , 1999
9 MODULATION OF DNA ADDUCTS BY DIETARY AGENTS IN THE LUNG OF SMOKE-EXPOSED RATS SHAM ECS ECS + 5,6-BF ECS + D3T ECS + PEITC ECS + NAC ECS + OPZ ECS + NAC + OPZ P < P < P < P < DNA adducts/10 nucleotides P < 0.05 A. Izzotti et al., Cancer Res., 61, , 2001
10 ADDUCTS TO LUNG DNA IN SMOKE EXPOSED RATS mtdna ndna NAC NAC + R. Balansky et al., Cancer Res. 56, , 1996
11 GENOMIC AND TRANSCRIPTIONAL ALTERATIONS IN MOUSE FETUS LIVER SHAM NAC ECS ECS + NAC TRANSPLACENTAL CIGARETTE SMOKE * SHAM NAC ECS ECS + NAC SHAM NAC ECS ECS + NAC * 8-oxo-dG/10 5 nucleotides * 0 0,5 1 1,5 2 2,5 0 2,5 5 7, ,5 DNA adducts/10 8 nucleotides MN PCE ( ) A. Izzotti et al., FASEB J. 17, , 2003
12 INTERPLAY BETWEEN HISTOPATHOLOGICAL ALTERATIONS, CIGARETTE SMOKE AND CANCER CHEMOPREVENTIVE AGENTS IN DEFINING microrna PROFILES IN MOUSE LUNG A. Izzotti et al., Mutat. Res., in press PCA component 2 3 MCS (Carcinoma) 5 MCS (Adenoma) 4 MCS (Normal) 7B MCS + NAC (Adenoma) 7A MCS + NAC (Normal) 1, 2 SHAM (Normal) 6B MCS + PEITC (Carcinoma) 8A, 8B MCS + PEITC (Pneumonia) 6A MCS + PEITC (Normal) PCA component 1
13 FREQUENCY OF MICRONUCLEI ( ) IN SMOKE- EXPOSED MICE R. Balansky et al., Carcinogenesis 20, , Smoke Bone marrow (PCE) Time (weeks) NAC Smoke + NAC Smoke + NAC post-exposure
14 FREQUENCY OF MICRONUCLEI ( ) IN SMOKE- EXPOSED MICE R. Balansky et al., Carcinogenesis 20, , Peripheral blood (NCE) Time (weeks) Smoke NAC Smoke + NAC Smoke + NAC post-exposure
15 FREQUENCY OF MICRONUCLEI ( ) IN SMOKE- EXPOSED MICE R. Balansky et al., Carcinogenesis 20, , Time (weeks) Smoke Respiratory tract (PAM) NAC Smoke + NAC Smoke + NAC post-exposure
16 LUNG TUMOR YIELD IN A/J MICE EXPOSED TO CIGARETTE SMOKE (ECS) ECS + AIR mo. 2 mo. + 7 mo. 5 mo. + 4 mo. 9 mo. + 0 ** P < / 20 6 / 20 8 / 20 ** 15 / 20 ** Incidence (%) Multiplicity (mean ± SE) F. D Agostini et al., Int. J. Oncol. 18, , 2001
17 EFFECTS OF CIGARETTE SMOKE IN MUTANT MICE S. De Flora et al. Molecular alterations and lung tumors in P53 mutant mice exposed to cigarette smoke. Cancer Res. 63, , 2003 A. Izzotti et al. Gene expression in the lung of P53 mutant mice exposed to cigarette smoke. Cancer Res. 64, , 2004 F. D Agostini et al. Early loss of Fhit in the respiratory tract of rodents exposed to environmental cigarette smoke. Cancer Res. 66, , 2006 S. De Flora et al. Molecular and cytogenetical alterations induced by environmental cigarette smoke in mice heterozygous for Fhit. Cancer Res. 67, , 2007
18 INCREASED SUSCEPTIBILITY TO CARCINOGENS AT BIRTH: MECHANISMS Induction of oxidative DNA damage and formation of bulky DNA adducts Overexpression of multiple genes Increased proliferative rate in neonatal organs Alterations of xenobiotic metabolism Lower efficiency of certain DNA repair mechanisms Involvement of stem cells?
19 INDUCTION OF LUNG TUMORS BY CIGARETTE SMOKE IN MICE EXPOSED EARLY IN LIFE No. of lung tumors/mouse Males Incidence = 14/22 (63.6%) Multiplicity = 6.1 ± Females Incidence = 12/16 (75.0%) Multiplicity = 13.6 ± Spontaneous deaths Including one malignant tumor Time (months) R. Balansky et al., Carcinogenesis 28, , 2007
20 HISTOPATHOLOGICAL ALTERATIONS IN THE LUNG OF MCS EXPOSED MICE Hyperplasia of alveolar epithelium Papillary hyperplasia of bronchial epithelium Adenoma Carcinoma in situ Small cell carcinoma Tumor containing adenocarcinomatous and small cell areas Adenosquamous carcinoma Low differentiated carcinoma
21 INCIDENCE OF LUNG TUMORS IN MICE EXPOSED TO CIGARETTE SMOKE Mouse strain Exposure period End of exp. Adenomas Cigarette Smoke Adenomas* Malignant t. Swiss H 0-4 mo. 7 mo. 0/36 15/38 (39.5%) 7/38 (18.4%) Swiss H 0-4 mo. 7 mo. 1/52 (1.9%) 16/55 (29.1%) 6/55 (10.9%) Swiss H 0-2 mo. 8 mo. 0/36 9/32 (28.1%) 3/32 (9.4%) Unpublished Swiss H 3-7 mo. 11 mo. 0/30 2/60 (3.3%) 0/60 Unpublished Ref. Balansky et al., 2007 Balansky et al., 2009 Swiss H 0-4 mo. 7 mo. 0/34 24/69 (34.8%) 9/69 (13.0%) Unpublished Swiss H 0-4 mo. 8 mo. 3/64 (4.7%) 16/111(14.4%) 2/111 (10.8%) Unpublished Swiss ICR 0-4 mo. 7 mo. 2/57 (3.8%) 25/82 (30.5%) 0/82 Unpublished DBA/2 0-4 mo. 8 mo. 0/18 3/16 (18.8%) 0/16 Unpublished C57BL 0-4 mo. 8 mo. 0/19 1/22 (4.5%) 0/22 Unpublished C57BL 3-7 mo. 11 mo. 0/30 0/27 0/27 Unpublished
22 CARCINOGENICITY OF MAINSTREAM CIGARETTE SMOKE IN STRAIN H MICE Controls (64 mice) MCS transpl. (61 mice) MCS neonatal (111 mice) MCS transpl. + neonatal (105 mice) Ethanol transpl. + MCS neonatal (72 mice) P < MALIGNANT P < P < Incidence of lung tumors (%) P < 0.001
23 CHEMOPREVENTION OF CIGARETTE SMOKE INDUCED TUMORS Experimental design Pregnancy NAC or AsA Birth Weanling NAC PEITC Budesonide NAC PEITC Budesonide PEITC Budesonide CS Prenatal Current smokers Ex smokers Time (days)
24 CHEMOPREVENTION OF CIGARETTE SMOKE INDUCED TUMORS R. Balansky et al., Int. J. Cancer 126, , 2010 MCS MCS + NAC (current smokers) MCS + Budesonide (current smokers) MCS + PEITC (current smokers) MCS + Budesonide (ex smokers) MCS + PEITC (ex smokers) M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F P < 0.1 P < 0.05 P < 0.01 P < Incidence (%) Multiplicity (mean ± SE)
25 PRENATAL N ACETYLCYSTEINE (NAC) PREVENTS CIGARETTE SMOKE INDUCED LUNG TUMORS IN NEONATAL MICE MCS NAC MCS MCS NAC MCS MCS NAC MCS MCS NAC MCS R. Balansky et al., Carcinogenesis 30, , 2009 Total lung tumors Microadenomas Adenomas Bronchoalveolar carcinomas Total lung tumors Microadenomas Adenomas Bronchoalveolar carcinomas Incidence (%) Multiplicity (mean ± SE)
26 PRENATAL N ACETYLCYSTEINE (NAC) PREVENTS CIGARETTE SMOKE INDUCED HISTHOPATOLOGICAL ALTERATIONS IN NEONATAL MICE MCS NAC MCS MCS NAC MCS MCS NAC MCS R. Balansky et al., Carcinogenesis 30, , 2009 Alveolar epithelium hyperplasia Emphysema Urinary bladder epithelium hyperplasia Incidence (%)
27 PRENATAL ASCORBIC ACID (AsA) PREVENTS CIGARETTE SMOKE INDUCED LUNG TUMORS IN NEONATAL MICE MCS AsA MCS MCS AsA MCS MCS Asa MCS Malignant tumors Microadenomas Adenomas Microadenomas Adenomas Malignant tumors Incidence (%) Multiplicity (mean ± SE)
28 CHEMOPREVENTION OF CIGARETTE SMOKE INDUCED LUNG ADENOMAS BY NATURAL PRODUCTS MCS MCS + Aronia (current smokers) MCS + Strawberry (current smokers) M F M+F M F M+F M F M+F M F M+F Incidence (%) Multiplicity (mean ± SE)
29 CHEMOPREVENTION OF SMOKE-RELATED ALTERATIONS OF micrornas AND LUNG TUMORS PIOGLITAZONE BEXAROTENE
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