Tobacco Carcinogen and Toxicant Biomarkers

1. Tobacco Carcinogen and Toxicant Biomarkers Stephen S. Hecht, Ph.D. Masonic Cancer Center University of Minnesota

2. Outline of Presentation Tobacco and cancer Background on tobacco carcinogen biomarkers Examples of tobacco carcinogen biomarkers Total NNAL Formaldehyde-DNA adducts

3. Cancers Caused by Smoking:IARC Monograph Series Volume 38, 1986 Lung Oral cavity Pharynx Larynx Esophagus Pancreas Bladder Volume 83, 2004, added: Nasal Cavity Stomach Liver Kidney Ureter Cervix Myeloid leukemia Volume 100E, 2009, added: • Colorectum • Ovary (mucinous)

4. Cancer Deaths Due to Smoking Worldwide: 21% (1,420,000 per year) United States: 33% (185,000 per year) IARC World Cancer Report, 2008

5. Tobacco Use Prevalence, 2008 Adult smokers, U.S.: 46,000,000 (20.6%) Ex-smokers, U.S.: 48,100,000 (21.5%) Smokers, worldwide: 1,300,000,000 Smokeless, worldwide: xxx,000,000 CDC, MMWR, November 13, 2009

6. World Smoking Prevalence: Males O. Shafey, M. Ericksen, H. Ross, J. Mackay (2009) The Tobacco Atlas, 3rd Ed.

7. World Smoking Prevalence: Females O. Shafey, M. Ericksen, H. Ross, J. Mackay (2009) The Tobacco Atlas, 3rd Ed.

8. Overall Goal Elucidate mechanisms of tobacco-induced cancer and apply this knowledge to cancer prevention.

9. Conceptual Framework for Understanding Tobacco Carcinogenesis S.S. Hecht, JNCI, 91:1194-1210 (1999), Nature Rev. Cancer 3:733-744 (2003); Cancer: Principles and Practice of Oncology, 8th Edition, 147-155 (2008)

10. Significantly Mutated Genes in Lung Adenocarcinoma: Based on Sequencing of 623 Genes in 188 Tumors Ding et al, Nature, 455:1069-1075, 2008

11. Other Factors Contributing to Tobacco-Induced Cancer • Receptor mediated effects:nicotine, nitrosamines • Direct activation of EGFR and COX-2 • Down-regulation of FHIT • Hyper-methylation of tumor suppressors • Tumor promotion and co-carcinogenesis • Oxidative damage and inflammation • Cilia-toxicity H. Takahashi et al, Cancer Cell 17: 89 (2010); H. Schuller, Nature Rev. Cancer 9: 195-205 (2009); K.A. West et al, J. Clin. Invest. 111: 81-90 (2003); S.A. Belinsky, Carcinogenesis 26: 1481 (2005); D’Agostini et al, Cancer Res 66: 3936-3941 (2006); Jin et al, Carcinogenesis 29: 1614-1622 (2008); Bhutani et al, Cancer Prev. Res. 1: 39-44 (2008)

12. Conceptual Framework for Understanding Tobacco Carcinogenesis S.S. Hecht, JNCI, 91:1194-1210 (1999), Nature Rev. Cancer 3:733-744 (2003); Cancer: Principles and Practice of Oncology, 8th Edition, 147-155 (2008)

13. IARC Carcinogens in Tobacco Smoke S.S. Hecht, In: DeVita et al, Cancer (2010); IARC Monographs No. 83 (2004); D. Hoffmann and S.S. Hecht, Handbook Exp. Pharmacol. 94:63-102 (1990)

14. Goal • Develop and validate tobacco carcinogen and toxicant biomarkers • Urinary metabolites • DNA and protein adducts • Metabolites in blood, saliva, breath, nails, hair • Use these biomarkers to identify those smokers susceptible to cancer.

15. Outline of Presentation Tobacco and cancer Background on tobacco carcinogen biomarkers Examples of tobacco carcinogen biomarkers Total NNAL Formaldehyde-DNA adducts

16. Definitions Biomarker: A distinctive biological or biologically derived indicator (as a metabolite) of a process, event, or condition (Merriam-Webster’s Collegiate Dictionary) Tobacco carcinogen biomarker: Any quantifiable substance, such as a metabolite, that can be specifically related to human exposure to a given tobacco carcinogen.

17. Tobacco Carcinogen Biomarkers DNA Adducts Protein Adducts Hemoglobin Albumin Metabolites Breath Saliva Nails and Hair Urine Blood Reviewed in Carcinogenesis 23: 907 and 1979 (2002); Nature Rev. Cancer 3: 733 (2003)

18. Applications of Tobacco Carcinogen Biomarkers Assessing exposure in smokers, smokeless tobacco users, and non-smokers exposed to secondhand smoke Regulation of tobacco products Understanding mechanisms of human carcinogenesis and identifying susceptible individuals Not specifically designed for early detection of cancer, but could have applications in screening

19. A Panel of Tobacco Carcinogen and Toxicant Biomarkers Based on 1.3g creatinine per 24h in smokers and 1.5g creatinine per 24h in non-smokers, or 1.5 l urine per 24h. S.S. Hecht, J-M Yuan, and D. Hatsukami, Chem. Res. Toxicol., 2010

20. Structures of the Urinary Biomarkers

21. A Panel of Biomarkers

22. Biomarker Validation • Analytical • Specificity, sensitivity, accuracy, precision • With respect to tobacco • Decreases upon cessation • Dose-response • With respect to cancer risk

23. Persistence of Biomarkers Study Smokers provide baseline 24h urine samples. Eight days later, they quit smoking and receive nicotine replacement therapy. They provide 24h urine samples on days 3, 7, 14, 21, 28, 42, and 56 after quitting. Urine samples are analyzed for mercapturic acids (by LC-MS/MS) and other biomarkers. S.G. Carmella, M. Chen, S. Han, A. Briggs, J. Jensen, D. K. Hatsukami, and S. S. Hecht Chem. Res. Toxicol., 22: 734-741 (2009)

24. Metabolism of 1,3-Butadiene to Mercapturic Acids a. GSH, GSTs; b. g-glutamyltranspeptidase; c. cysteinylglycine dipeptidase; d. cysteine S-conjugate N-acetyltransferase C.L. Sprague and A.A. Elfarra, Chem. Res. Toxicol., 17: 819-826 (2004)

25. Mean Urinary MHBMA Reduction Upon Smoking Cessation, N=17

26. Metabolism of Acrolein, Crotonaldehyde, Benzene, and Ethylene Oxide to Mercapturic Acids a. GSH, GSTs; b. g-glutamyltranspeptidase; c. cysteinylglycine dipeptidase; d. cysteine S-conjugate N-acetyltransferase

27. Mean Urinary HPMA Reduction Upon Smoking Cessation, N=17

28. Mean Urinary HBMA Reduction Upon Smoking Cessation, N=17

29. Mean Urinary SPMA Reduction Upon Smoking Cessation, N=17

30. Mean Urinary HEMA Reduction Upon Smoking Cessation, N=17

31. Structures of Urinary Biomarkers S.G. Carmella, et al, Chem. Res. Toxicol., 22: 734-741 (2009)

32. Mean Urinary 1-HOP Reduction Upon Smoking Cessation, N=15

33. Mean Urinary Total NNAL Reduction Upon Smoking Cessation, N=17

34. Outline of Presentation Tobacco and cancer Background on tobacco carcinogen biomarkers Examples of tobacco carcinogen biomarkers Total NNAL Formaldehyde-DNA adducts

35. A Panel of Tobacco Carcinogen and Toxicant Biomarkers Based on 1.3g creatinine per 24h in smokers and 1.5g creatinine per 24h in non-smokers, or 1.5 l urine per 24h. S.S. Hecht, J-M Yuan, and D. Hatsukami, Chem. Res. Toxicol., 2010

36. Essential Facts About NNK,A Tobacco-Specific Lung Carcinogen Present in tobacco and tobacco smoke; specific to tobacco products Systemic lung carcinogen in rats, mice, hamsters, and ferrets. Also induces tumors of the pancreas, nasal cavity, and liver in rats Considered to be a cause of lung, oral cavity and pancreatic cancer in people exposed to tobacco products NNK and NNN- Carcinogenic to humans; Group 1 (IARC Volume 89, 2007); reaffirmed (Vol 100E, 2009) S.S. Hecht, Chem. Res. Toxicol. 11:559 (1998); Nature Rev. Cancer 3:733 (2003)

37. Metabolism of NNK by Carbonyl Reduction

38. NNAL Plus NNAL-Glucs (Total NNAL): A Biomarker of NNK Exposure Quantified by GC-TEA or LC-MS/MS High analytical specificity and sensitivity Specific to tobacco product exposure Responsive to dose Measures uptake of a lung carcinogen S.S. Hecht, Carcinogenesis 23 907 (2002); S.G.Carmella et al, CEBP 4: 635 (1995); 12: 1257 (2003); D. Hatsukami et al, Nic. Tob. Res. 8: 169 (2006)

39. GC-TEA Chromatogram of NNAL in a Smoker's Urine

40. Applications of the Total NNAL Biomarker Cessation of smoking or smokeless tobacco Reduction of smoking Carcinogen uptake from new and old tobacco products: Omni, light and ultra-light cigarettes Snus and other smokeless products Ultra low nicotine cigarettes Evaluation of carcinogen dose in various groups Reducers vs. light smokers Smokers of differing numbers of cigarettes Ethnic groups, gender, and teen-age smokers Smokeless vs. smokers Duration of smokeless use Carcinogen uptake from secondhand cigarette smoke Relationship to lung cancer D. Hatsukami, J. Jensen, A. Joseph, S. E. Murphy, S.G. Carmella, S.S. Hecht, and co-workers. Cancer Res., JNCI, CEBP, Nic. Tob. Res., 1999-2008

41. Tobacco Harm Reduction: Continuum of Risk Conventional cigarettes Modified tobacco cigarettes Cigarette reduction Cigarette-like delivery devices; Extra-low nicotine cigarettes Smokeless tobacco products Nicotine delivery devices Smoking Cessation Most toxic Least toxic D. Hatsukami et al., Nicotine Tob. Res.9:S537-S553 2007

42. Applications of the Total NNAL Biomarker Cessation of smoking or smokeless tobacco Reduction of smoking Carcinogen uptake from new and old tobacco products: Omni, light and ultra-light cigarettes Snus and other smokeless products Ultra low nicotine cigarettes Evaluation of carcinogen dose in various groups Reducers vs. light smokers Smokers of differing numbers of cigarettes Ethnic groups, gender, and teen-age smokers Smokeless vs. smokers Duration of smokeless use Carcinogen uptake from secondhand cigarette smoke Relationship to lung cancer D. Hatsukami, J. Jensen, A. Joseph, S. E. Murphy, S.G. Carmella, S.S. Hecht, and co-workers. Cancer Res., JNCI, CEBP, Nic. Tob. Res., 1999-2008

43. Non-Smokers’ Exposure to NNK Throughout Life by Measurement of Urinary Total NNAL a based on 2 pmol/ml total NNAL in smokers S.S. Hecht, Carcinogenesis 23:907 (2002); S.S. Hecht et al, CEBP 15:988 (2006)

44. Total NNAL measurements in secondhand smoke-exposed non-smokers have impact • It can only come from secondhand smoke. • It represents uptake of a lung carcinogen. • It is found in the urine of non-smokers. • It is the only lung carcinogen biomarker consistently elevated in exposed non-smokers. • These studies should spur clean air legislation in the remaining countries, states and localities.

45. Median Serum Cotinine Levels in Non-Smokers, by Age Group: 1988-2002 CDC NHANES Study; MMWR 55: 1130 (2006)

46. Regulation of Indoor Smoking and Tobacco Control • Regulation of indoor smoking • Reduces cues for smoking • Reduces amount smoked • Can change social norms • Regulation of indoor smoking, along with counter-advertising and taxation, are the most effective methods in tobacco control.

47. Applications of the Total NNAL Biomarker Cessation of smoking or smokeless tobacco Reduction of smoking Carcinogen uptake from new and old tobacco products: Omni, light and ultra-light cigarettes Snus and other smokeless products Ultra low nicotine cigarettes Evaluation of carcinogen dose in various groups Reducers vs. light smokers Smokers of differing numbers of cigarettes Ethnic groups, gender, and teen-age smokers Smokeless vs. smokers Duration of smokeless use Carcinogen uptake from secondhand cigarette smoke Relationship to lung cancer D. Hatsukami, J. Jensen, A. Joseph, S. E. Murphy, S.G. Carmella, S.S. Hecht, and co-workers. Cancer Res., JNCI, CEBP, Nic. Tob. Res., 1999-2008

48. Relationship of Urinary NNAL to Lung Cancer in Two Prospective Cohorts of Cigarette Smokers Collaboration with Professors Mimi Yu and Jian-Min Yuan Two prospective cohorts of Chinese cigarette smokers: Shanghai and Singapore Nested case control study of 246 cases of lung cancer and 245 matched controls Total NNAL and cotinine quantified in stored urine samples collected prior to lung cancer diagnosis

49. Joint Effect of Urinary Total NNAL and Cotinine on Lung Cancer Risk 1 No. of cases/no. of controls 2 Odds ratios (OR) were adjusted for age, year of interview, year of sample collection, gender and dialect group, study location (Shanghai versus Singapore), number of cigarettes smoked per day, and number of years of smoking; CI, confidence interval. J. Yuan, M. Yu, S.E. Murphy, S. Carmella, S.S. Hecht et al., Cancer Res.,69: 2990 (2009)

50. Conclusions of the Shanghai and Singapore Study • Total NNAL significantly associated with risk of lung cancer in a dose-dependent manner, after adjustment for smoking history and urinary cotinine. • Cotinine was independently associated with lung cancer, consistent with previous data. • Smokers in the highest tertiles of urinary total NNAL and cotinine exhibited an 8.5 fold increased risk for lung cancer, relative to those with comparable smoking history, but in the lowest tertiles. J. Yuan et al, Cancer Res. 69: 2990 (2009)