Quantitation of Oral Cell DNA Adducts in Smokers
Conditions: Lung Cancer
Sponsor: University of Minnesota

CPRC #: 2017NTLS019 
Ethnic/Racial Differences in Lung Cancer in Smokers 
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Study 1 Observation Study  
Mechanisms of Ethnic/Racial Differences in Lung Cancer 
Due to Cigarette Smoking 
part of the Clinical and 
Biomarkers Core 
 
CLINICAL PROTOCOL 
 
 
CPRC # 2017NLS019 
 
 
Principal Investigators: 
Dorothy Hatsukami, Ph.D. 
Loic Le Marchand, MD, PhD 
UMN Department of Psychiatry 
University of Hawaii Cancer Center 
 
Irina Stepanov, Ph.D.* 
Charles Rosser, MD 
Division of Environmental Health Sciences 
UH Cancer Center 
*non-clinical - will not consent participants 
 
 
Biostatistician: 
Daniel Stram, Ph.D. 
University of Southern California 
 
 
Version 4  
Date: July 2, 2020 

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Revision History 
 
 
 
 
 
 
Revision 
# 
Version 
Date 
Summary Of Changes 
Consent 
Changes 
1 
1-20-2017 
original to CPRC-IRB - rejected 
2 
4-17-2017 
Revised for Study 1 only 
 
3 
8-10-2017 
Removed reference to investigational product 
No 
4 
7-2-2020 
COVID-19 modifications to reduce contact and enforce social 
distancing 
x 
Discontinue carbon monoxide 
x 
Some of the questionnaires done at home  
x 
Addition of COVID-19 screening questions 
x 
Temperature taken before visit 
x 
Use of PPE during visits 
Compensation changed to  $75 
Yes 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Principal Investigator Contact Information: 
Dorothy Hatsukami, Ph.D. 
717 Delaware St SE #260 
hatsu001@umn.edu 
612-626-2121 

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Table of Contents 
Table of Contents 
Abbreviations ...................................................................................................................................... 4 
Study Synopsis ................................................................................................................................... 5 
1 
Study Objectives ...................................................................................................................... 6 
1.1 
Primary Objectives for Projects Requiring Recruited Subject Samples* .......................... 6 
2 
Background and Significance .................................................................................................. 6 
3 
Rationale .................................................................................................................................. 8 
4 
Study Design Overview .......................................................................................................... 10 
5 
Participant Selection .............................................................................................................. 10 
5.1 
Inclusion Criteria ............................................................................................................. 10 
5.2 
Exclusion Criterion .......................................................................................................... 11 
6 
Participant Registration .......................................................................................................... 11 
7 
Study Procedures for Recruitment: ........................................................................................ 11 
7.1 
Observation Study .......................................................................................................... 11 
14 
Adverse Event Reporting ....................................................................................................... 16 
15 
Study Data Collection and Monitoring .................................................................................... 16 
15.1 
Data Management .......................................................................................................... 16 
15.2 
Case Report Forms ........................................................................................................ 16 
15.3 
Data and Safety Monitoring Plan (DSMP) ...................................................................... 17 
15.4 
Record Retention ............................................................................................................ 17 
16 
Statistical Considerations ....................................................................................................... 17 
17 
Data and Biospecimen Sharing Plan ..................................................................................... 20 
18 
Conduct of the Study ............................................................................................................. 21 
18.1 
Good Clinical Practice .................................................................................................... 21 
18.2 
Ethical Considerations .................................................................................................... 21 
18.3 
Informed Consent ........................................................................................................... 21 
References ....................................................................................................................................... 22 
Appendix A: Eligibility Checklist: Observation Study ................................................................. 27 
Eligibility Checklist – page 1 of 2 ...................................................................................................... 27 
 

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Abbreviations 
 
AE: 
Adverse Events: is an undesired harmful effect resulting from a medication or other 
intervention / study procedure. 
BP: 
Blood Pressure: is the pressure exerted by circulating blood upon the walls of blood vessels 
and is one of the principal vital signs. 
CYP2A6: 
This gene encodes a member of the cytochrome P450 superfamily of enzymes. The 
cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in 
drug metabolism. The enzyme is known to metabolize nicotine and nitrosamines. Individuals 
with certain allelic variants are said to have a poor metabolizer phenotype, meaning they do 
not efficiently metabolize nicotine. 
FTND: 
Fagerstrom Test for Nicotine Dependence: is a 6-item standard instrument for assessing the 
intensity of physical addiction to nicotine and includes an evaluation of cigarette 
consumption, the compulsion to use, and dependence. 
HR: 
Heart Rate: a measure of the number of heart beats per minute (bpm) 
MEC 
Multiethnic Cohort 
NIAAA: 
National Institute on Alcohol Abuse and Alcoholism: part of the U.S. National Institutes of 
Health that supports and conducts biomedical and behavioral research on the causes, 
consequences, treatment, and prevention of alcoholism and alcohol-related problems. 
NMR: 
Nicotine Metabolite Ratio: is a urinary measure of the ratio of nicotine metabolites, which 
indicates speed of nicotine metabolism. 
NNAL: 
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol: Human exposure to NNK can be measured 
by analyzing the sum of NNAL and its glucuronides (total NNAL) in urine. 
NNK 
 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone:  One of the tobacco specific nitrosamines, 
a potent lung carcinogen in tobacco products, which are formed from nicotine during the 
curing and processing of tobacco. 
NNN: 
N'-nitrosonornicotine: One of the tobacco specific nitrosamines formed from nicotine 
during the curing and processing of tobacco and a known carcinogen. 
PAH: 
Polycyclic Aromatic Hydrocarbons: PAH, a product of combustion, are mong the most 
important carcinogens in tobacco smoke. 
SAE: 
Serious Adverse Events: generally, any event which causes death, permanent damage, birth 
defects, or requires hospitalization is considered an SAE. 
TLFB: 
Timeline Follow-Back: is a method that can be used as a clinical and research tool to obtain 
a variety of quantitative estimates of marijuana, cigarette, and other drug use by asking 
clients to retrospectively estimate their usage prior to the interview date. 
TNE: 
Total nicotine equivalents: a urinary measure of nicotine and its metabolite concentrations. 

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Study Synopsis 
 
The Clinical and Biomarkers Core of the Program Project entitled “Mechanisms of Ethnic/Racial 
Differences in Lung Cancer Due to Cigarette Smoking” will recruit smokers in Hawaii to collect 
biological samples and will carry out analyses of tobacco constituent biomarkers for four 
projects. The biological samples collected will be used to evaluate and compare biomarkers of 
tobacco exposure across Japanese Americans, Whites, and Native Hawaiians on multiple 
urinary or blood components to assess tobacco related exposures. 
 
The samples will either be procured from the existing biorepository of the Multiethnic Cohort 
MEC Study for life-long non-smokers or obtained from smokers who are recruited from the MEC 
or general population by this Core. This protocol includes the scope of work for the Clinical 
and Biomarkers Core related to recruitment of human subjects (Observation Study) for 
the 3 projects that will use and/or store the biorepository samples. Biorepository samples 
will be available to the PPG research team or other qualified investigators examining issues 
related to cancer and/or tobacco exposure biomarkers. 
 
Study Design: 
The Clinical and Biomarkers Core will include two components: the Clinical 
Recruitment and Data Collection component and the Biomarker Analysis 
component. The purpose of the Clinical Recruitment. Data Collection component 
is to provide human biological samples and measurements from people of 
various ethnic and racial backgrounds for Projects 2, 3 and 4 (Project 1 is 
conducted at the University of Southern California and University of Hawaii on 
samples from the MEC Biorepository, Southern Community Cohort Study 
(SCCS), Singapore Cohort Study, Shanghai Chinese Health Study and 
Atherosclerosis Risk in Communities (ARIC). This Core provides a centralized 
approach to subject recruitment, biological sample collection, and analysis of 
validated biomarkers applying well-established protocols and using standardized 
methodologies. The subject population is recruited from the Multiethnic Cohort 
Study (MEC) and the general population. The samples collected under this 
protocol will be used to evaluate and compare biomarkers of tobacco exposure 
across Japanese Americans, Whites, and Native Hawaiians and to establish a 
biorepository to develop or assess future biomarkers. In addition, for Project 2, 
we will conduct a short clinical study with a subsample of Japanese Americans 
using deuterium-labeled tobacco-specific lung carcinogen 4- 
(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) cigarettes for NNK 
metabolic profiling 
 
This protocol is for Study 1 – Observation Study. 
Primary Aim: 
The Clinical and Biomarkers Core will facilitate the research by collecting 
biological samples and biomarker data from 300 smokers that will be used by 
individual projects to achieve their Specific Aims. 
Patient 
Population: 
Cigarette smokers who are part of the targeted ethnic/racial groups including 
Japanese Americans, Non-Hispanic Whites and Native Hawai’ians. Subjects will 
be recruited from male and female participants of the Multiethnic Cohort (MEC) 
Study or the general population (recruited through local advertisements). 
Accrual Goal: 
Years 1 -3: 100 subjects in the Observation Study. 
Enrollment 
Period: 
September 1, 2017 through  December 31, 2020 

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1 Study Objectives 
1.1 
Primary Objectives for Projects Requiring Recruited Subject 
Samples* 
x 
Project 2: Identify and recruit a subset of Japanese smokers (N=20 selection 
based on slow and fast metabolizers based on CYP2A6 activity) to determine the 
effect of CYP2A6 genotype on NNK metabolic activation and will be conducted 
under a separate Study 2: Clinical Study protocol. 
x 
Project 3: Obtain biomarker samples and observational data on 300 Native Hawaiian, 
White, and Japanese American smokers (100 in each group) to investigate ethnic 
differences in butadiene-DNA adducts and determine the relationship of DNA adducts 
to lung cancer and the influence of carcinogen metabolizing genes on DNA adduct 
formation, repair, and toxicity/mutagenicity. 
x 
Project 4: Obtain oral mucosa cell samples on 300 Native Hawaiian, White, and 
Japanese American smokers to quantify DNA adducts and investigate urinary 
biomarkers of acrolein, crotonaldehyde, inflammation, and oxidative damage 
among smokers and non-smokers. 
(*Project 1 will use existing samples from the existing biorepository and are not part 
of this Core). 
 
2 Background and Significance 
The major goal of this project is to provide insights into the underlying mechanisms of ethnic/ 
racial differences in lung cancer due to cigarette smoking. In a previous study, we carried out 
extensive carcinogen metabolite phenotyping studies, genome wide association studies 
(GWAS), and mechanistic investigations to test the hypothesis that these differences in lung 
cancer susceptibility are due to ethnic and racial variations in exposure and response to 
cigarette smoke carcinogens. 
Multiethnic Cohort Study (MEC) 
The MEC is a prospective cohort study of 215,251 men and women between the ages of 45 to 
75 at baseline, primarily belonging to five racial/ethnic groups: African American, Japanese 
American, Latinos, Non-Hispanic, and whites. This cohort was established to investigate the 
association of lifestyle and genetics with chronic diseases in a multiethnic population. 61 
Between 1993 and 1996, potential participants were identified in Hawai’i and California 
(primarily Los Angeles County) through drivers’ license files, voter registration lists, and Health 
Care Financing Administration files. Each participant completed a mailed, self-administered 
questionnaire regarding demographic, dietary, lifestyle, and other exposure factors at baseline 
and follow-up questionnaires updating smoking and other exposures every five years. 
Tobacco constituent biomarkers to be assessed: 
Nicotine is the major known addictive constituent of cigarette smoke and its uptake determines 
smoking behaviors in individual smokers.1 Since each dose of nicotine inhaled with cigarette 
smoke is accompanied by numerous toxic and carcinogenic compounds,2 nicotine uptake is 
critical in determining carcinogen dose in smokers. Total nicotine equivalents (TNE) is the sum 
of urinary nicotine, nicotine-N-oxide, cotinine, and 3'-hydroxycotinine and their glucuronides, 
which together account for 73 – 96% of the nicotine dose.1 This biomarker is an outstanding 
monitor of nicotine uptake and level of smoking.1 Nicotine metabolite ratios can also provide 
other important information, such as differences in glucuronidation and in cytochrome P450 
2A6 activity. The ratio of total 3'-hydroxycotinine to cotinine will be used for this purpose in this 
program.3,4 

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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen in every 
species tested, inducing lung tumors independent of the route of administration.5,6 Based on 
animal data, mechanistic studies, and epidemiological evidence, the International Agency for 
Research on Cancer (IARC) classified NNK and the related carcinogen NNN as human 
carcinogens.7 Human exposure to NNK can be measured by analyzing the sum of NNAL and 
its glucuronides (total NNAL) in urine.8-10 Total NNAL correlates with cigarettes per day, 
nicotine equivalents in urine, and cotinine,6,11 and is an effective uptake marker for NNK.12 
Consistent with the known carcinogenicity of NNK, three nested case-control studies in 
smokers have demonstrated that NNAL is related to lung cancer.13-15 Thus, NNAL is both an 
exposure and a risk biomarker. 
 
Polycyclic aromatic hydrocarbons (PAH) are among the most important carcinogens in tobacco 
smoke.16 Phenanthrene tetraol (PheT) is a metabolite of the non-carcinogenic PAH 
phenanthrene, which is the simplest polycyclic aromatic hydrocarbon (PAH) with a “bay region”, 
a structural feature associated with carcinogenicity.17-19 PheT forms through the diol epoxide 
metabolic pathway of phenanthrene, mimicking the carcinogenic pathway of the prototypic 
carcinogenic PAH benzo[a]pyrene (Figure 2).20-26 The end product of this pathway for 
phenanthrene is PheT in urine, which can serve as a biomarker of PAH exposure and 
metabolic activation.23 PheT levels correlate with those of 1-hydroxypyrene, an established 
biomarker of PAH exposure,22 and are 2-3 times higher in smokers than in non-smokers.24 
Urinary PheT is related to lung cancer in a nested case-control study in male smokers.15 
Another phenanthrene metabolite excreted in urine is 3-hydroxy phenanthrene (3-PheOH); this 
biomarker represents PAH exposure and detoxification.27 The ratio PheT:3-PheOH can 
therefore be a phenotypic indicator of an individual’s ability to metabolically activate. 
 
Volatile compounds such as acrolein and crotonaldehyde may also contribute to lung cancer 
induction in smokers.28 Acrolein is a highly toxic agent in cigarette smoke, causing cilia- 
toxicity among other effects in the lung.29 One study has shown that acrolein causes a pattern 
of DNA damage in the p53 tumor supressor gene similar to that observed in lung tumors of 
smokers.30 We have demonstrated the presence of substantial quantities of acrolein-DNA 
adducts in human lung DNA.31 Crotonaldehyde, a structural homologue of acrolein, also 
forms DNA adducts in the human lung, and these are known to be mutagenic, although 
crotonaldehyde has not been shown to be carcinogenic to the lung.32 Acrolein and 
crotonaldehyde are detoxified by reactions with glutathione, and the resulting conjugates are 
excreted in the urine as 3-hydroxypropylmercapturic acid (3-HPMA) and 3-hydroxy-1- 
methylpropylmercapturic acid (HMPMA), respectively. These mercapturic acids can be 
readily measured by established and analytically validated methods, and are related to 
cigarette smoking.33-36 Previous research shows levels of these mercapturic acids were 
unusually elevated in Native Hawaiians, an aspect that will be further investigated in Project 
4. 
Cadmium (Cd) is a human lung carcinogen, with cigarette smoking being one of the major 
sources of exposure.37 Urinary Cd increases with age and smoking, as major determinants of 
its levels.10 A statistically significant association between urinary Cd levels and lung cancer 
mortality in men from the NHANES III cohort has been reported.38 While there is some 
overlap in urinary levels of Cd between smokers and non-smokers, the levels of this 
biomarker have been demonstrated to increase with smoking.10,39,40 Exposure to Cd has been 
shown to cause hypermethylation of tumor suppressor genes41 and was inversely associated 
with LINE1 methylation, which is a common epigenetic event in carcinogenesis.42 
Oxidative stress, inflammation, and related biomarkers 
Chronic inflammation and oxidative stress are important interlinked contributing factors in the 
pathogenesis of cigarette smoke-associated diseases, including lung cancer.43-48 Oxidative 

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damage is likely involved in tumor promotion and other deleterious effects of smoking 
(reviewed in 49,50). Cigarette smoke contains high levels of prooxidants, such as reactive 
oxygen and nitrogen species, as well as catechols and related compounds which can induce 
oxidative damage and lead to inflammatory state.51,52 Inflammation involves infiltration of 
lymphocytes, macrophages, and neutrophils into tissues under stress, and induces lipid 
peroxidation, these processes resulting in the generation of a spectrum of reactive oxygen 
and nitrogen species capable of causing extensive damage to DNA and proteins, and 
resulting in toxic and mutagenic events.53,54 In smokers, inflammation and oxidative stress will 
be assessed by measuring urinary metabolites and DNA adducts. 
Inflammation leads to the induction of the immediate-early response gene prostaglandin G/H 
synthase-2 (COX-2).55 The product of COX-2, prostaglandin E2, is metabolized to PGEM, a 
urinary metabolite and promising biomarker of inflammation which has been associated with 
cancer development in a variety of studies.55 Levels of PGEM are reported to be significantly 
higher in current smokers than in former smokers than in never-smokers, which is thought to 
reflect inflammation in the lung due to induction of COX-2.56,57 Oxidative stress-induced 
peroxidation of arachidonic acid results in the production of F2-iosprostanes, among which 
urinary 8-iso-3*)Į is an accepted biomarker of oxidative damage.58,59 Many studies 
consistently report that levels of 8-iso-PGFĮare elevated in smokers (reviewed in 39,60-62). In 
DNA, guanine is the major target for the direct oxidation by the inflammation-induced 
radicals. The oxidation produces several DNA adducts including 8-oxo-7,8-dihydro-ƍ- 
deoxyguanosine (8-oxo-dG), which can lead to chromosomal aberrations and the induction of 
mutations, and is widely used as biomarker for oxidative stress.63 
Clinical and Biomarkers Core’s Role 
The Clinical and Biomarkers Core will provide services, biomarker samples and tools for all 
projects in this program project grant to test new hypotheses put forth and will provide new 
insights into the ethnic/racial differences in lung cancer risk due to smoking. 
 
3 Rationale 
Smoking-related lung cancer is the leading cause of cancer death in the United States and 
worldwide. For a given lifetime exposure to cigarette smoking, the risk of lung cancer is 
greatest in Native Hawaiians and lowest in Japanese Americans compared to Whites. The 
results of this program project will provide unique and critical data relevant to the 
mechanism(s) underlying the high risk of Native Hawaiian smokers for lung cancer; thus, 
potentially generating new insights for lung cancer prevention strategies. Using a biomarker 
approach, previous research found a direct link of tobacco carcinogens NNK and PAH to 
lung cancer, and NNN to esophageal cancer in humans. These novel findings are strongly 
consistent with the results from experimental animal models and implicate the carcinogenic 
role of these specific tobacco constituents in the development of lung and esophageal 
cancers.  This project will build on these results and further provide rationale to regulate 
and control these constituents in tobacco products to reduce the harmful effects of tobacco 
smoking. 
The purpose of Study 1: Observation Study under the Clinical and Biomarkers Core of the 
“Mechanisms of Ethnic/Racial Differences in Lung Cancer Due to Cigarette Smoking” 
program project is to provide human biological samples and measurements from people of 
various ethnic and racial backgrounds for Projects 2, 3 and 4. These samples will be used to 
evaluate and compare biomarkers of tobacco exposure across Japanese Americans, 
Whites, and Native Hawaiians and to add to the Multiethnic Cohort MEC biorepository to 
develop or assess future biomarkers. 

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Clinical and Biomarker Core Project Aims 
Project 1 in this program will investigate the relationship of biomarkers of tobacco constituent 
exposure and early-biological effects with the risk of lung cancer. MEC and other pre-existing 
samples will be used for this analysis. 
Project 2 will focus on characterization of NNK bioactivation across different ethnic groups and 
the effect of P450 2A6 activity on this process. This requires recruitment of smokers from three 
ethnic/racial groups (Native Hawaiians, Whites, and Japanese Americans) for the analysis of 
ELRPDUNHUVRI11.Į-hydroxylation (bioactivation) pathway, as well as recruitment of a 
subgroup of Japanese American smokers for participation in a clinical study in which 
deuterium-labeled NNK will be used to specifically trace NNK-derived metabolites. The Clinical 
and Biomarkers Core will carry out subject recruitment and the clinical study. 
Project 3 will investigate the role of 1,3-butadiene metabolic activation and deactivation in lung 
cancer risk among various ethnic/racial groups. This project will require urine samples from 
smokers and nonsmokers from the three ethnic/racial groups recruited by the Clinical and 
Biomarker Core for the analysis of 1,3-butadiene DNA adducts. Data on nicotine intake 
(urinary TNE) in these subjects as well as in 400 lung cancer cases and 400 controls from 
Project 1 will be also required for this project. 
Project 4 will investigate whether DNA adduct levels in oral cells, themselves or together with 
urinary biomarker levels, correlate with lung cancer risk across various ethnic/racial groups. 
This project will require biological samples from smokers and nonsmokers from these 
ethnic/racial groups, as well as data on urinary tobacco constituent biomarkers and PGEM and 
8-iso-PGFĮ, in these subjects. 
 
This protocol will recruit smokers, and will generate the biomarker data required. The 
Coordinating site at the University of Minnesota will not recruit subjects, but provide 
oversight for the study team recruiting subjects at the University of Hawai’i. 

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4 Study Design Overview 
This cross-sectional, observational study will primarily recruit smokers from the MEC study. 
The biological samples from these smokers are being collected since they do not currently 
exist in the biorepository (e.g. buccal cells). Potential smokers from the three racial groups 
who meet specific inclusion criteria will be invited to participate in the study. Should 
recruitment from the MEC sample be insufficient, smokers who meet the criteria will be 
recruited from the general population. This study will involve one to two home visits where 
tobacco use and medical history and biological samples will be collected including blood, 
buccal cells and urine.  
 
5 Participant Selection 
Approximately 300 smokers will be recruited from the Multiethnic Cohort (MEC) study or the 
general population in Hawai’i. 100 participants from each of the three designated 
ethnic/racial groups with equal distribution by gender. All participants will be recruited from 
Hawai’i to reduce variation due to geographical location and maximize our ability to recruit 
the targeted ethnic/racial groups. Based on the data collected from the MEC, participants 
who are at least 21 years old and who currently smoke greater than 5 cigarettes per day 
(current smokers) will be selected and contacted in the MEC standard method by sending a 
letter inviting them to participate in this study. The letter states that a researcher will be in 
contact with them to provide further information and obtain verbal consent to submit to the 
telephone screening questionnaire, if interested. In the MEC database, the approximate 
number of Oahu participants that might be available for each of the three categories of 
participants is listed in Table 2. From past experience with Oahu MEC participants, including 
smokers64 we expect a participation rate of ~50%. Several hundreds of additional MEC 
current smokers are also available from the neighboring islands. 
 
Table 2. Number of potential Oahu MEC participants available for each category. 
Race/ethnicity 
Smokers* 
Japanese Americans 
810 
Non-Hispanic Whites 
397 
Native Hawaiians 
385 
* As determined from the most recent follow-up questionnaire 
 
Recruitment beyond the MEC sample will be expanded to the general population of smokers in 
Hawai’i. Recruitment will conducted via local print, radio, TV, or internet advertisements and 
posted flyers. 
5.1 
Inclusion Criteria 
a) One of the three targeted ethnic groups: 
o Japanese American - two parents of Japanese descent 
o Non-Hispanic Whites - two parents of non-Hispanic white descent 
o Native Hawaiians will include individuals with at least one parent of 
Hawaiian descent; 
b) Smoke 5 cigarettes per day over the past three months; 
c) >21 years of age; 

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d) Consumes 14 or fewer drinks of alcohol per week; 
e) Generally stable and good health (determined by review of medical history); 
f) Able to provide written voluntary consent before performance of any study  
related procedure. 
5.2 
Exclusion Criterion 
a) Current use of other nicotine containing products for > 4 times per month (and no 
use of any nicotine-containing products except cigarettes for 2 weeks prior to their 
study visits); 
b) Acute or uncontrolled medical or psychiatric conditions; 
c) Currently taking any medications that affect relevant metabolic enzymes or anti- 
inflammatory medications such as ibuprofen (this will be reviewed by study 
investigators on a case-by-case basis); 
d) Active infection (e.g., influenza, cold, respiratory infection, sinus infection) at the 
time of the visit; 
e) Pregnant or nursing or planning on becoming pregnant during the study; 
f) Unable to read and understand English. 
 
6 Participant Registration 
Registration will occur after the subject consent form is signed and eligibility is confirmed, 
but before any study samples or data are collected.  To be eligible for registration to this 
study, the participant must meet each criteria listed on the eligibility checklist based on the 
documented eligibility assessment. 
Registration with the Masonic Cancer Center Clinical Trials Office 
 
Upon completion of obtaining written consent, screening evaluation, and eligibility 
confirmation, the study coordinator or designee will enroll the subject and enter 
registration information into the study registration system. This data will be provided to 
the Masonic Cancer Center. 
 
7 Study Procedures for Recruitment: 
7.1 
Observation Study 
Participants recruited from the MEC pool who meet eligibility will be sent a letter 
informing them of the study. After receipt of the letter, staff will contact the subject 
and obtain verbal consent for telephone screening to further assess eligibility. If the 
subject is interested and eligible they will be scheduled for an in-home visit or a visit 
in the clinic, whichever is preferred. Subjects recruited from the general population 
will contact the clinic in response to advertisements to be screened over the 
telephone for eligibility. 
7.1.1 Observation Study-Visit 1 Procedures: (see Table 3 Procedures by Visit) 
Visits 1 and 2 may be combined if biological samples can be collected at Visit 1.  
During the first visit, potential participants will be more fully informed about the 
study and written consent will be obtained. 
Prior to any visit, COVID-19 symptoms or potential exposure to COVID-19 will be 
assessed.  Prior to entering the clinic, participants will have their temperature 
taken and asked to wear a mask. Staff will be using appropriate PPE during the 
visits. 
 

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Once informed consent is obtained, more extensive and updated information will be 
obtained. The following measures will be reviewed by the researcher to assist in 
confirming eligibility: 
 
 
 
Questionnaires completed by participant at home or clinic or by interview: 
1. Tobacco Use History, Tobacco Dependence and Exposure Interview. 
2. Brief medical history. 
3. Medication use (current and last 30 days). 
4. NIAA Alcohol Use Questionnaire (12 month and 1 month) 
5. Timeline Follow-back assessing tobacco, nicotine, other combustibles or 
alcohol use for last 14 days. 
Physiological Measures: 
1. Blood pressure. 
2. Heart rate. 
3. Body weight (in kilograms). 
4. Height. 
5. Calculate body mass index and possible body fat composition. 
 
Scheduling Visit 2 
At the end of Visit 1, the second visit will be scheduled approximately 1 week after 
Visit 1 (+7 to 14 days). The interviewer will provide the subject with: 
1. A urine container for first morning void sample. 
2. A specimen collection commode (female participants only). 
3. A cooler and two packs of blue-ice. 
4. Instructions to freeze the blue-ice in advance of the collection, collect the first 
morning urine void upon rising the morning of the second visit, and store the 
sample in the cooler on the blue-ice. 
5. Instructions to avoid grilling foods two days before the biomarker sample 
collection visit (to avoid exposure to PAH in cooked meats). 
6. Smokers will be reminded not to use smoked products (e.g., cigars, marijuana, 
etc.) except cigarettes before the next visit. 
7. Avoid any other tobacco/nicotine product, including electronic cigarettes prior to 
the visit. 
7.1.2 Observation Study-Visit 2 Procedures (Visit +7 to 14 days)  
Visit 1 and 2 are typically combined. 
At the second visit, the following exposure questionnaires completed by participant 
at home or in the clinic or by interview will be administered: 
1. Environmental Tobacco Smoke Questionnaire. 
2. Environmental and Occupational Exposure History (including volcanic fumes). 
3. Grilling, smoking or barbecuing meat or fish in the past 48 hours. 
4. Timeline Follow-Back assessing tobacco, nicotine, other combustibles or alcohol 
and drug use since Visit 1. 
5. Medication and nutritional supplements (including name, dose per pill, number of 
pills and indication for use) will be obtained by visual inspection of the products. 
Collection of biological samples. 
1. First morning void urine samples will be collected for analysis of biomarkers. 
2. Blood sample (non-fasting):  Three tubes of blood (2- 10mL lavender EDTA 
tubes and 1 10 mL red top tube) will be drawn by trained staff. If the participant is 

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seen in their home, the phlebotomist visit may occur on a separate day due to 
scheduling concerns. 
3. Oral cell samples collected. 
7.1.3 Compensation: 
Participants will be compensated $75 per visit. 
 
8 
Subjective and Exposure Forms for Observation Study  
8.1 
Questionnaires: 
a. Demographics questionnaire will assess age, gender, race, ethnicity, income, 
education, current occupation and usual occupation. 
b. Tobacco Use Exposure and History Interview assesses variables such as 
number of cigarettes smoked per day, age of initiation of smoking, duration of 
quit attempts, duration of smoking, pack years and other tobacco product use. 
 
c. The Fagerstrom Test for Nicotine Dependence is the most widely used and 
psychometrically tested scale for dependence.65 
d. Medical History and Current Health Status list current diagnoses, symptoms and 
past health problems. 
e. Concomitant Medications includes any medications taken within 30 days prior to 
the first visit and throughout study participation in Project 2. 
f. 
NIAAA Alcohol Use Questionnaire examines rate of alcohol use for the past 
month.67-68 
g. Environmental Tobacco Smoke Questionnaire consists of 6 questions related to 
smoke exposure at home, work and socially.69 
h. Environmental and Occupational Health History assesses environmental and 
occupational toxicant exposures, which might affect our biomarker measures.70 
i. 
48 hour Food Recall will be administered by trained study staff to assess 
ingestion of any foods that have might affect the biomarkers that are assessed 
(e.g., grilled foods or smoked meats, cruciferous vegetables). 
j. 
Adverse Effects Checklist lists several nicotine exposure and toxicity and 
respiratory symptoms related to smoking. 
k. Health Changes Questionnaire. 
l. 
Timeline Follow-Back. 
m. Daily Diary card provides daily number of cigarettes, other tobacco/nicotine 
products and number of alcoholic drinks per day. 
 
9 Laboratory Procedures for processing of biological samples: 
All collection containers will be labeled with study number, subject number, collection date 
and type of biological sample (urine, blood, buccal cells). 
9.1 
Blood Collection and Processing 
Blood collected into purple top tubes will be processed within 12 hours of phlebotomy. The 
tubes will be centrifuged at 1,200 × g for 10 minutes in a refrigerated centrifuge to separate 
plasma, buffy coat, and red blood cells. The separated components will be transferred into 
cryogenic vials, labeled (with information described above) and stored in a -80°C freezer. 
The urine samples will be aliquoted into cryotubes and frozen at -70°C. Tubes with 
mouthwash and buccal cell samples will be centrifuged at 5000 x g for 15 minutes to pellet 
the cells and the supernatant will be discarded. The cell pellets will be washed by adding 1 
mL of 1X TE buffer (pH 7.4), swirling the tube gently, repeating the centrifugation, and 
discarding the buffer. The washed buccal cell pellet will be re-suspended in 200 µL cold 1X 
TE buffer, transferred into a pre-labeled cryovial, and stored at -80°C until isolation of DNA 

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for adduct analyses in Project 4. 
9.2 
Oral Cell Collection and Processing 
Participants will be asked to brush their teeth at the beginning of the visit by using a 
provided commercial individually packed pre-pasted toothbrush (ReadyBrushTM) and 
asked to not eat, drink, smoke or chew gum for 30 minutes prior to obtaining oral cells. Two 
types of oral cell collection methods will be used on each subject: an oral rinse with 
mouthwash and buccal cell scrapings. Sample collection with mouthwash will be carried out 
first; participants will be provided with 10 mL of mouthwash or saline with approximately in 
a sterile 50-mL polypropylene tube and asked to swish the mouthwash throughout the 
mouth for 45 seconds. The swishing will be performed in the following manner: 15 seconds 
on the left side, 15 seconds on the right side, and 15 seconds throughout the mouth, 
including the mouth floor and the area between the lips and gums. This technique will 
ensure the most complete collection of exfoliated cells from different parts of the oral cavity, 
and minimize possible variations in the efficiency of sample collection by different subjects. 
The mouthwash sample will be expectorated back into the 50-mL polypropylene tube. 
Buccal cells will be collected by scraping the oral mucosa inside the mouth with a 
cytobrush. Two cytobrushes will be used on each subject to collect two separate samples 
from the left and the right inner cheek. After scraping the mucosal surface, each cytobrush 
will be immediately placed into a separate pre-labeled sterile polypropylene tube containing 
5 mL of Scope mouthwash, swirled for several seconds, and tapped against the walls of the 
tube to ensure the cells are transferred from the brush to the Scope. Samples will be stored 
in a -20 freezer. 
9.3 
Urine Collection and Processing 
Subjects are instructed to collect their first morning void on the day of the visit and keep it in 
the provided cooler and on freezer packs. Staff will collect the sample and verify with the 
subject when the sample was collected.  Urine will be aliquoted into 4.5 mL cryo vials 
labeled with study specific scanner labels upon returning to the laboratory and stored in a - 
20C freezer. Vials will be stored by assay type and shipped upon request. 
9.4 
Biomarker Management Website 
Throughout the study, biological samples (blood, buccal cells, urine) will be collected, 
labeled and registered in the University of Hawai’i’s MEC Tracking Website.  Samples will 
be sent in split batches to the University of Southern California (USC) Biomarker Core for 
processing and aliquots sent to Masonic Cancer Center, University of Minnesota, to be 
stored in the Biorepository with de-identified information. Some samples will remain with 
the PI at the University of Hawaii.  The UHCC MEC, USC Biomarker Core and Masonic 
Cancer Center Tobacco Biorepositories have established policies and procedures 
consistent with NCI guidelines. Samples will be stored until fully used, no longer usable, or 
destroyed upon subject request. A discussion of the storage, future use, and sharing of 
samples is contained in the informed consent for this study. A Data and Biospecimen 
Sharing Plan is also provided in section 17. 
 
10 Clinical Data Collection 
Sources of materials: 
The primary source of clinical data will be the protocol specific case report forms and a web 
database. Subjective questionnaires will be completed by the subject directly into the 
internet based database that is password protected and meets HIPAA standards. All data 
from the subjects will be on a secure web-site or sent to the University of Minnesota, and all 
information will be de-identified. All biosamples sent to the University of Minnesota will also 
be de-identified and location and disposition will be kept on the Biomarker Management 
Website housed on the Masonic Cancer Center server. 
 

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For the Observational Study, subjects will be asked to provide information on their tobacco 
use history, medical history, medication use and other measures that might affect 
biomarkers such as alcohol or other drug use, food, secondhand smoke exposure and 
occupational history. Biological samples including urine and oral cells will be collected. 
 
 
All records will be confidential. All shared data will be de-identified. University of Hawaii 
Cancer Center personnel will ensure all the study records are accurate and that all 
consents have been signed, thereby having access to names of individuals. University of 
Minnesota staff will monitor progress through a web-based tracking database and regular 
research phone calls. University of Minnesota staff may also visit Hawaii to review protocol 
adherence and forms. However, no record of this identifying information will leave the 
premises. Identifying information will be kept locked in a secure area. 
 
Human subject consent forms will indicate corresponding subject ID and will not be stored 
with the data. Paper and pencil subjective forms, and other Case Report Forms (CRF) will 
be identified with subject number and initials, and visit number and date will be entered 
onto appropriate forms. Data entered into the study database and will be de-identified, with 
only subject number as a link. Biomarker samples will be labeled with a scanner barcode 
indicating subject number, visit number, specimen type (e.g., urine, serum, saliva) and 
intended assay or biobank samples. 
 
11 Risks of Study Participation 
Potential risks: The potential risks for subjects are minimal. For both studies, most 
physiological and subjective measures will be noninvasive and should present no 
psychological or medical risk to the subject. Some questionnaires may be of a sensitive 
nature assessing subjects’ alcohol use. Subjects will be told that they may refuse to 
answer, however, refusal may effect continued participation in the study. 
 
In the observational study, subjects will be using their own products so no additional 
product risk is imposed. The additional risk in this study is loss of privacy due to a breach in 
confidentiality. 
 
Protection against Risks 
Potential subjects will be told the nature of the research over the phone and during 
screening. Subjects will be required to demonstrate an understanding of the study purpose 
and procedures prior to signing the consent form. This consent form must be signed before 
the research is started. They will be told they may discontinue participation at any time and 
will not be discriminated against if they choose to do so and that discontinuing this study 
will not affect their further participation as a MEC participant. 
 
Protection against breach of confidentiality: 
Subjects will be told their participation in the project will be strictly confidential, that any 
identifying information will only be available to the site investigators, coordinating center 
study manager, an auditor for the Food and Drug Administration, study sponsor or 
University of Hawai’i’s institutional oversight. No identifying information concerning the data 
and results will be made known. Subjects will have written assurance that while de-
identified individual subject data may be available to other researchers for research 
purposes, only a summary of the results will ever be published or otherwise publicly 
released. They will also be informed that all raw data will be coded with numbers and any 
identifiers will be kept in locked file cabinets and only appropriate study personnel will have 
access. 
 
All data will be de-identified and transferred through a secure, password-protected website 
that is only available to the investigators. All identifying information will be in a locked in a 

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secure place and accessible only to relevant study staff. 
 
Risk with phlebotomy: 
A trained laboratory technician will obtain blood samples to minimize the risk associated 
with a blood draw. Blood samples will be obtained by a trained phlebotomist with single- 
use, sterile blood collection supplies. 
 
Throughout their participation in the study, adverse symptoms will be recorded at each 
clinic visit and monitored by the PIs and study medical personnel. Subjects who experience 
any significant adverse events will be sent or accompanied to the emergency clinic or 
referred to their physician, depending on the nature and severity of the adverse event. 
 
12 Effect on Patient Care 
This study has no effect on patient care. 
 
13 Duration of Study Participation 
Participation will consist of one to two visits in the Observation Study over a one day to 
three week period (if two visits are conducted, they will be approximately one week apart 
as the preferred time frame, however we will accommodate the participant schedule as 
needed). 
14 Adverse Event Reporting 
This study carries minimal risk; however it will comply with the University of Minnesota and 
University of Hawai’i IRB reporting requirements. Events requiring prompt reporting include 
any adverse event that requires a change to the protocol or consent form, any unauthorized 
disclosure of confidential information, any unresolved subject complaint or any protocol 
deviation resulting in harm or the unanticipated death of an enrolled subject. All study 
related adverse events of a non-serious nature will be reported to each institution’s IRB at 
the time of IRB renewal application submission. 
 
Reportable adverse events or unanticipated problems will be reported to the IRB by phone 
within 24 hours of when the investigator becomes aware of the event and written report 
within 10 days of receipt of information regarding the event at UH and submitted within 5 
business days at UMN. The Data and Safety Monitoring Board will review all serious or 
unexpected adverse events and provide recommendations. 
 
Any event requiring prompt reporting to the IRB will also be reported to the Masonic Cancer 
Center’s SAE Coordinator, University of Hawai’i Cancer Center and the funding agency. 
 
15 Study Data Collection and Monitoring 
15.1 Data Management 
This study will use a tracking database to follow the progress of each subject’s 
participation. Patient demographics and study specific data will be entered into a database 
maintained by the UH Cancer Center. 
15.2 Case Report Forms 
Participant data will be collected using protocol specific electronic case report forms and 
pen and paper questionnaires. The case report forms will be developed and purchased by 
the coordinating site and distributed to the University of Hawaii site. The case report form 
books will be created to ensure capture of all data points. Each visit will have a checklist of 
all the measures that need to be taken and the order by which these measures are 
administered. 

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15.3 Data and Safety Monitoring Plan (DSMP) 
The study coordinator and the supervising physician (Charles Rosser, MD) at the site of 
data collection will be responsible for the daily oversight of subject safety. Entrance criteria 
will be reviewed following screening. Subjects will be under supervision while in the study 
and seen on an ongoing basis by our research staff who will assess adverse events and 
vital signs and make appropriate referrals to the study physician. 
 
Dr. Hatsukami and Joni Jensen will conduct a study initiation meeting and provide internal 
monitoring of subject safety. If any safety issues are identified, the investigator and staff at 
the University of Hawaii will be contacted immediately. 
 
A Data and Safety Monitoring Board will not be implemented for this observational study. 
 
15.4 Record Retention 
The investigator will retain study records including source data, copies of case report form, 
consent forms, HIPAA authorizations, and all study correspondence in a secured facility for 
at least 6 years after the study file is closed with the IRB and FDA. 
 
16 Statistical Considerations 
Listed below are the projects using this protocol’s collected samples. Statistical 
considerations related to Project 1 are found in the protocol related to that study. 
 
3URMHFW11.Į–hydroxyl glucuronides, metabolic profiling and activation 
Aim 1: is to determine the relationship between CYP2A6 activity and NNK 
-hydroxylation 
among two groups of Japanese smokers one with very low measured CYP2A6 activity and 
the other with normal levels (about 10 per group). Our comparison will be between the 
means (or if the distribution is highly skewed the medians or geometric means) of the two 
groups (null versus average CYP2A6). The power of the test depends on the size of the 
group difference compared to the within-group variability, i.e. to the standard deviation of 
NNK- hydroxylation..Assuming that a normal distribution holds we will have 80 percent of 
WKHGLVWULEXWLRQRI11.Į–hydroxylation (33 percent of variation explained). In these 
analyses we will adjust for potential confounders including age, sex, race, total nicotine 
equivalents, BMI, etc. 
 
Aim 2: will test for ethnic differences in Į–hydroxyNNK glucuronides among Japanese- 
Americans, Native Hawaiians, and white smokers recruited in Core B. In addition the 
glucuronides will be related to total urinary NNL and NNK DNA adducts analyzed in Project 
4 (Aims 1 and 2). We will use all 300 smokers recruited by the Clinical Studies Core (100 in 
HDFKRIWKUHHHWKQLFJURXSV)RUWKHVHVPRNHUVĮ–hydroxyNNK glucuronides will be 
measured in this project and related to NNAL, TNE, and NNK DNA adducts measured by 
Project 4. For each of the glucuronides we will examine the data for outliers and failed QC 
values, evaluate batch differences, heteroscedasticity and the appropriateness of log or 
other transformation to achieve normality of the outcome measures. Linear regression 
methods will be used to characterize the association between (possibly transformed) 
glucuronides and the variables (NNAL, TNE, NNK adducts) of interest. We will adjust for 
age, sex, BMI and ethnicity, and examine independent effects of NNAL or NNK adducts 
with and without adjusting for TNE. With 300 smokers having data available we have 80 
percent power to detect a variance explained of approximately 3 percent. 
 
Aim 3: This is a laboratory aim (which will develop a NNK metabolic profiling method) and 
does not specifically test hypotheses needing statistical analysis. 
 

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Project 3: Ethnic/Racial Differences in metabolism and DNA Adduct Formation by 
1,3-Butadiene 
Aim 1: This Aim concentrates upon a subset of the participants recruited in the Clinical 
Core (the 100 smokers from each of three ethnic groups) with the objective being to 
characterize differences in metabolites of 1,3butadiene as well as DNA adduct formation by 
race/ethnicity. Levels of metabolites and of DNA adducts of 1,3-Butadiene will be compared 
between Japanese-Americans, Native Hawaiians, and non-Hispanic Whites. Adjustments 
for age, sex, TNE, CPD, and BMI will be performed using the “LS means” option in Proc 
GLM in SAS. Prior to model-fitting we will examine QC metrics and estimate laboratory 
variation in the outcomes based on duplicate samples from the same individual and time 
period. We will identify batch effects and individual outliers. If required, polynomial or log 
transformations will be used to normalize the distribution of metabolite and DNA adduct 
levels prior to analysis. With 100 smokers from each of the three groups we will have 80 
percent power to detect a difference between any two ethnic groups in these outcomes 
which amounts to approximately 6 percent of the total variance of the outcome. Here we 
correct (using the Bonferroni method) for making 3 such comparisons by requiring p<.017 
in order for any single comparison to be declared significant. 
 
Aim 2: This aim investigates the association between urinary butadiene DNA adducts and 
lung cancer risk among White and African-American participants (smokers) in project 1.  
One hundred cases and one hundred controls for each ethnic group will be assessed. 
Controls will be frequency matched to the cases by age and sex. The primary issue is 
whether DNA adducts convey independent information beyond that which is contained in 
smoking dose, either self-reported (CPD) or measured (TNE). We will utilize unconditional 
logistic regression to evaluate the association between risk and DNA adducts after 
including CPD and TNE into the models. With 200 cases and 200 controls total (pooling 
both ethnicities) in this analysis we will be able to detect a 2.25 fold lung cancer risk 
difference between the highest and lowest quintiles of the distribution of DNA adducts 
using a test for log-linear trend. Effects specific to a single ethnic group must be larger (3-
fold between top and bottom quintiles) than this to be detectable. 
 
Aim 3 of this project is to relate inter-individual differences in metabolic inactivation, DNA 
adduct formation/repair and toxicity/mutagenicity of butadiene-derived epoxides measured 
in 40 Hapmap cell lines first to GSTT1 and EPHX1 activity level (low versus high for each) 
with the cell lines chosen as follows: 
 
Cell lines number 
GSTT1 activity 
EPHX1 activity 
10 
High 
High 
10 
High 
Low 
10 
Low 
High 
10 
Low 
Low 
 
In addition we will investigate the relationship between these phenotypes and genetic 
polymorphisms in specific candidate genes for carcinogen metabolism and repair. There 
are several important issues to be dealt with in this project by the Statistical Core. These 
include (1) implementing procedures for summarizing mutagenicity and toxicity for each of 
the 40 cell lines, and (2) selection of an appropriate power or log transformation that will 
normalize the distribution of the estimated values. 
 
For the genetic analyses we chose the HapMap cell lines because of the extensive 
genotyping and other phenotyping (expression array studies etc.) already performed for 
these samples. We will only use founders (no offspring will be selected). Here we attempt 
to elate the cytotoxic, genotoxic and DNA repair endpoints to the genotypes available for 
these same cell lines (www.hapmap.org). For this problem, we simply fit 

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R1: Yi = a + b1Gi + ei 
where Gi represents the main effects of a particular genetic marker (genotyped for 
individual i) Yi can represent any one of the cytotoxic, genotoxic, or DNA adduct/repair 
phenotypes. 
 
Power: 
For the comparisons of metabolites, adducts, etc. by GSTT1 and EPHX1 activity level and 
assuming no interactions between the two activities we will be able to detect with 20 
individuals per group approximately a 1.2 standard deviation difference between the means 
of each phenotype. In our power calculations for the genetic analyses of Aim 3 we 
distinguish between analyses of candidate markers within a small number of candidate 
genes compared to the candidate markers and analyses of all 4 million markers genotyped 
in the HapMap. Our search for genetic markers related to the phenotypes of interest will be 
done in two stages. The first stage will focus only on SNPs in or near specific candidate 
genes, i.e. those genes previously identified as being highly likely to be related to each 
phenotype. It is expected that approximately 100 candidate SNPs will be identified for each 
phenotype triggered by each activated carcinogen. For the second stage all (approximately 
4 million) SNPs will be considered in the performance of a genome-wide association scan 
for each phenotype. The statistical methods for these two stages are identical (based on 
model R1). The only difference will be the type I error rate at which candidate SNPs will be 
considered to be globally significant versus the remainder of the SNPs. For the analysis of 
candidate SNPs we will use a p-value for significance of 0.0005 for the 100 candidate 
SNPs and 2.5 x 10-8 for genome wide significance. The second p-value is based on 
assuming, as described by Pe’er et al [1], that the testing burden for testing all common 
SNPs in the HapMap in a single experiment is equivalent to performing approximately 1 
million tests. For the candidate genes we will have approximately 80 percent power to 
detect HapMap SNPs which explain approximately 38 percent of the variance of a given 
phenotype after correcting for all associations tested. For the remainder of the SNPs only 
extremely strong associations (63 percent of variance explained) will be detectable at the 
genome wide level of significance. 
 
Project 4: Oral cell DNA adducts and urinary biomarkers to investigate ethnic/racial 
differences in lung cancer susceptibility. 
 
Aim 1: Evaluation of ethnic differences in DNA adducts in oral cells quantified in 100 
smokers from Core B in each of three ethnic groups. This aim will quantify DNA adducts of 
acrolein, crotonaldehyde, NNN, NNK, and formaldehyde and identify ethnic differences in 
levels of these adducts. After QC, outlier identification, analysis of duplicate samples for 
laboratory variation and batch effects we will fit linear regression models to the (possibly log 
or power-transformed) adduct levels to allow for differences by age, sex, BMI, TNE and 
CPD while examining ethnic group differences. The SAS procedure GLM will be used to 
provide adjusted means (LS means) and to test for the significance of the observed ethnic 
differences in those means. 
 
As in Project 3, Aim 1, for a given outcome (DNA adduct) we will have 80 percent power to 
detect a difference between any two ethnic groups which amounts to approximately 6 
percent of the total variance of a given outcome. We can also examine the joint effects of 
smoking and ethnicity in the total of 300 Core B participants and will have 80 percent power 
to detect ethnicity x smoking interactions that explain at least an additional 1 percent of 
variation for any outcome. 
 
Aim 2: There are two sub aims to Aim 2. The first is to compare urinary metabolites in both 
smokers and nonsmokers in the three ethnic groups. The main hypothesis is that acrolein 
and crotonaldehyde metabolites, as well as metabolites from oxidative damage and 

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inflammation are higher in Native Hawaiian non-smokers than in the other 3 ethnic groups, 
and that this is the reason that acrolein and crotonaldehyde metabolites were higher in 
Native Hawaiian smokers than Japanese American smokers in our published study. We will 
also compare the levels of the urinary metabolites in the smokers from the three ethnic 
groups. We will also compare smokers to non-smokers from each ethnic group (for 
metabolites of acrolein, and crotonaldehyde and biomarkers of oxidative damage and 
inflammation). Sub aim 2 is to relate DNA adducts measured for the 300 participants in 
Core B to urinary metabolites of TNE, total NNAL, mercapturic acids of acrolein, and 
crotonaldehyde measured in the same individuals. After QC, examinations of batch effects 
and laboratory variation, outlier detection, transformation to normality, we will estimate the 
ethnic differences and provide linear regression estimates of the relationships between the 
DNA adducts and metabolites. For sub aim 1 we will compare the urinary metabolite levels 
in each group of 100 with the other groups. We will use a p-value for any pairwise 
comparison of 0.003 as protection against type I error. For sub aim 2 since we anticipate 
that many different analyses will be performed (each DNA adduct against each relevant 
metabolite) we will use a Bonferroni-corrected p-value of 0.05/50 = 0.001 to declare 
significance in these analyses. Detectable effects will correspond to approximately 3 
percent of variance of the DNA adducts explained. 
 
Aim 3: Identification and quantification of known and previously unknown DNA adducts. 
This aim has two sub-aims. The first is to identify known and unknown DNA adducts related 
to smoking status among 50 current smokers and never smokers of single ethnic group 
(Whites). The second sub aim is to determine ethnic differences in DNA adduct levels 
found to be elevated in smokers in the first sub-aim. DNA oral cell adducts will be 
measured in smokers from Core B. Because of cost and time considerations 20 smokers 
with high levels of the (acrolein and crotonaldehyde) adducts measured in Project 4 Aim 1 
will be selected for analysis of sub aim 2. 
 
Statistical Analysis and Power. For sub aim 1 we will test for differences in DNA adduct 
levels between smokers and never smokers using parametric or (if appropriate) non- 
parametric single degree of freedom tests (t-test, Wilcoxon, etc.) Because it is anticipated 
that several hundred adducts (known + unknown) will be measured we will use a Bonferroni 
criteria of .05/200 = 0.00025 in order to declare that the observed differences are 
statistically significant. With 50 participants per group we will be able to detect (with 80 
percent power) a smoking difference explaining 18 percent of variation of the adduct level, 
equivalent to about 1 standard deviation of the within group (smokers, never-smokers) 
variation of a given DNA adduct. 
 
For sub aim 2 we analyze 20 participants per group and test for ethnic differences in adduct 
levels for the adducts shown to be elevated in smokers. Our main comparison is between 
the high risk group (native Hawaiians) and the other groups (Japanese and Whites). 
Assuming that 50 of the adducts are elevated in smokers we will use a p value of 
0.05/50=.001 in our hypothesis testing as protection against multiple comparisons. For a 
given adduct we will be able to detect a difference of about 1.2 standard deviations 
amounting to 25 percent of variability explained by ethnicity (Native Hawaiian vs Japanese 
American + White). 
 
17 Data and Biospecimen Sharing Plan 
The institutions involved in this project are committed to the sharing of research data and 
samples, consistent with the regulations and policies of the NIH. The investigators involved 
in this project intend to share findings from its research through publications and 
presentations. Institutions and individuals wishing to access any resources or data must 
contact the Principal Investigator (Hatsukami). Data will be available in two formats. One 

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will be a summary of the data, with graphs and tables, posted as pdf files. Also, data will be 
available as raw individual-level data for analysis. However, this will not be available until 
papers are accepted for publication. Data generated by this grant will be made to outside 
investigators, according to NIH Guidance. Persons requesting data must do so in writing, 
identifying the affiliation and how the data will be used. When data are shared, there will be 
no limits placed on how the data will be used, and co-authorship is not required as a 
condition for receiving data. Users will agree, however, that the recipient must not transfer 
the data to other users and that the data are only to be used for research purposes. The PI 
will require requestors of data to sign a data and biospecimen sharing agreement that will 
ensure (1) Use of the data and/or samples only for research purposes unless otherwise 
agreed and not to identify any individual or personal information (2) Data security using 
appropriate technology/firewalls, (3) Destruction or return of data after data analysis and 
(4) Proper citation in publications or other written materials. A record of transfer of data and 
a copy of the dataset that was distributed will be kept by University of Minnesota. 
 
18 Conduct of the Study 
18.1 Good Clinical Practice 
The study will be conducted in accordance with the appropriate regulatory requirement(s). 
Essential clinical documents will be maintained to demonstrate the validity of the study and 
the integrity of the data collected. Master files will be established at the beginning of the 
study, maintained for the duration of the study and retained according to the appropriate 
regulations. 
18.2 Ethical Considerations 
The study will be conducted in accordance with ethical principles founded in the 
Declaration of Helsinki. The UMN and UH IRBs will review all appropriate study 
documentation in order to safeguard the rights, safety and well-being of the patients. The 
protocol, consent, written information given to the patients, safety updates, annual progress 
reports, and any revisions to these documents will be provided to the IRBs for review by the 
investigators. 
18.3 Informed Consent 
All potential study participants will be given a copy of the IRB-approved consent to review. 
The investigator or designee will explain all aspects of the study in lay language and 
answer all questions regarding the study. If the participant decides to participate in the 
study, he/she will be asked to sign and date the consent document. Patients who refuse to 
participate or who withdraw from the study will be treated without prejudice. 
 
 

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Lea, R. A., Dickson, S., and Benowitz, N. L. (2006) Within-subject variation of the salivary 
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Carmella, S. G., Akerkar, S., and Hecht, S. S. (1993) Metabolites of the tobacco-specific 
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Appendix A: Eligibility Checklist: 
Observation Study 
 
Participant initials 
Patient ID 
 
 
Eligibility Checklist – page 1 of 2 
 
INCLUSION CRITERIA 
 
 
A “NO” response to any of the following disqualifies the participant from study entry. 
 
Yes 
No 
1. 
One of the three targeted ethnic groups: 
a. Japanese American - two parents of Japanese descent. 
b. Non-Hispanic Whites -- two parents of non-Hispanic white descent. 
c. Native Hawaiians will include individuals with at least one parent of 
Hawaiian descent; 
 
 
2. 
Smokes 5 cigarettes per day in the past three months; 
 
 
3. 
\HDUVRIDJH 
 
 
4. 
Consumes 14 or fewer drinks of alcohol per week; 
 
 
5. 
Generally good health (determined by review of medical history) 
 
 
6. 
Able to provide written voluntary consent before performance of any study 
related procedure. 
 
 

Date from cover page 
Page 39 of 39 
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Eligibility Checklist – page 2 of 2 
 
EXCLUSION CRITERION 
 
 
A “YES” response to the following disqualifies the participant from study entry. 
Yes 
No 
7. 
Current use of other nicotine containing products for > 4 times per month (and no 
use of any nicotine-containing products except cigarettes for 2 weeks prior to their 
study visits); 
 
 
8. 
Acute or uncontrolled medical or psychiatric conditions; 
 
 
9. 
Currently taking any medications that affect relevant metabolic enzymes or anti- 
inflammatory medications such as ibuprofen (this will be reviewed by study 
investigators on a case-by-case basis); 
 
 
10. Active infection (e.g., influenza, cold, respiratory infection, sinus infection) at the 
time of the visit; 
 
 
11. Pregnant or nursing or planning on becoming pregnant during the study;. 
 
 
12. Unable to read and understand English. 
 
 
 
 
 
Date consent form signed by participant and research staff:  
 
 
Having obtained consent and reviewed each of the inclusion/exclusion criteria, I verify that 
this participant is: 
 
Eligible 
Ineligible 
Date registered  
 
 
 
 
 
Signature of person verifying eligibility 
Date