STUDY 2: CLINICAL PROTOCOL Metabolism of NNK Among Japanese Americans
Conditions: Smoking
Sponsor: Masonic Cancer Center, University of Minnesota

 
 
 
 
 
Protocol Title 
STUDY 2: CLINICAL PROTOCOL 
Metabolism of NNK Among Japanese Americans 
Principal 
Investigator/Faculty 
Advisor 
Name:   
 
Dorothy Hatsukami, Ph.D. 
Department:   
Masonic Cancer Center 
Telephone Number:  612-626-2121 
Email Address:  
hatsu001@umn.edu 
Scientific 
Assessment 
Nationally-based, federal funding organizations 
IND/IDE # (if 
applicable) 
IU0000492 
IND/IDE Holder 
Dorothy Hatsukami 
UMN 
Investigational Drug 
Services #  
5555RO   
Version 
Number/Date: 
Version 1  October 11, 2019 
 

 
REVISION HISTORY 
 
Revision # 
Version Date 
Summary of Changes 
Consent Change? 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Table of Contents 
 
Study Synopsis ........................................................................................................................................ 4 
1.0 
Objectives..................................................................................................................................... 6 
2.0 
Background .................................................................................................................................. 6 
3.0 
Study Endpoints/Events/Outcomes ........................................................................................... 10 
4.0 
Study Intervention(s)/Investigational Agent(s) ......................................................................... 10 
5.0 
Procedures Involved .................................................................................................................. 11 
6.0 
Data and Speciµen Banking ....................................................................................................... 16 
7.0 
Sharing of Results with Participants .......................................................................................... 16 
8.0 
Study Population ........................................................................................................................ 17 
9.0 
Local Number of Participants..................................................................................................... 18 
10.0 Recruitment Methods ................................................................................................................ 18 
11.0 Withdrawal of Participants ........................................................................................................ 18 
12.0 Risks to Participants ................................................................................................................... 19 
13.0 Potential Benefits to Participants .............................................................................................. 20 
14.0 Statistical Considerations ........................................................................................................... 20 
15.0 Provisions to Monitor the Data to Ensure the Safety of Participants ....................................... 21 
16.0 Provisions to Protect the Privacy Interests of Participants ....................................................... 22 
17.0 Consent Process ......................................................................................................................... 23 
18.0 Setting ........................................................................................................................................ 23 
19.0 Multi-Site Research .................................................................................................................... 23 
20.0 References ................................................................................................................................. 24 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Study Synopsis 
 
 
Study Design: 
The risk of lung cancer varies by individual and by ethnic/racial group. In this 
study we will explore how individual differences in the metabolism of a tobacco-
specific lung carcinogen may contribute to the variable risk of lung cancer 
between ethnic/racial groups. 
 
In this 10 day clinical trial, Japanese Americans will smoke a cigarette 
containing deuterium-labeled 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone 
(NNK), a tobacco-specific lung carcinogen. The study cigarette will be smoked 
for 7 days.  
This will allow for NNK metabolic profiling and determining the effect of CYP2A6 
genotype on the level of NNK α–hydroxylation in Japanese Americans smokers 
using [pyridine- D4]-NNK containing cigarettes.    
Primary Aim: 
• 
To characterize the bioactivation of NNK byα-hydroxylation; 
• 
To develop an NNK metabolite profiling method; 
• 
To determine the contribution of the enzyme CYP2A6 to NNK 
bioactivation.  
 
Patient 
Population: 
Japanese American Smokers    
Accrual Goal: 
20 completed participants  
Enrollment 
Period: 
January 1, 2019 through August 30, 2021  
ITP # 
IU0000492 
     
 
 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 5 of 25 
 
ABBREVIATIONS/DEFINITIONS 
AE: 
Adverse Events: is an undesired harmful effect resulting from a medication or other 
intervention / study procedure. 
α-OHNNK Gluc: α-hydroxymethyl NNK glucuronide 
BP:  
Blood Pressure: is the pressure exerted by circulating blood upon the walls of blood vessels 
and is one of the principal vital signs. 
CO:  
Carbon Monoxide: Breath carbon monoxide is the level of carbon monoxide in a person's 
exhalation. 
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 (CYP2A6) 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. 
[PYRIDINE-D4]NNK: NNK labeled with deuterium (heavy hydrogen isotope that is non-radioactive and 
non-hazardous). Adding [pyridine-D4] to the NNK found cigarette tobacco and spiking study 
cigarettes allows evaluation of NNK metabolic activation in humans.  
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 
(cotinine, trans3’`hydroxycotinine and their glucuronide conjugates plus nicotine N-oxide). 
 
 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 6 of 25 
 
1.0 
Objectives 
1.1 Purpose:  
Smoking is the cause of 90% of all lung cancer but only 11-24% of smokers develop the 
disease .1 The risk of lung cancer varies by individual and by ethnic/racial group.2, 3 In 
this study we will explore how individual differences in the metabolism of a tobacco-
specific lung carcinogen may contribute to the variable risk of lung cancer between 
ethnic/racial groups. In this protocol we will specifically test the hypothesis that 
carcinogen bioactivation varies by a smoker's CYP2A6 activity. CYP2A6 activity varies by 
ethnic group and the activity of this enzyme is associated with lung cancer.  Therefore, 
determining the role of the enzyme in the bioactivation of tobacco carcinogens will 
contribute to our understanding of the mechanism by which CY2A6 activity influences 
racial/ethnic differences in lung cancer. 
2.0 
Background 
2.1 Significance of Research Question/Purpose:  
The risk of lung cancer for individual smokers and members of different ethnic/racial 
groups varies significantly. Differences in carcinogen exposure, activation and 
detoxification contribute to this variable risk. In this study, we will characterize the 
metabolic pathways of the tobacco-specific lung carcinogen, NNK, in Japanese American 
(JA) smokers.  JA have a high prevalence CYP2A6 variants that code for low activity and 
non-functional enzyme. 4, 5 CYP2A6 is the primary enzyme that metabolizes nicotine and 
a key enzyme involved in the bio-activation of NNK. 6-8   
There are over 20 identified lung carcinogens in tobacco smoke.1, 9 These include 
polycyclic aromatic hydrocarbons, NNK, volatiles such as butadiene, metals such as 
cadmium, and radioactive 210Po.1  Among these the evidence for a role in human lung 
cancer is strongest for polycyclic aromatic hydrocarbons  and NNK, which reproducibly 
and at modest doses induce lung tumors in laboratory animals.1, 10, 11  NNK, which is the 
focus of this project, is unique in that it is tobacco-specific. NNK is considered 
"carcinogenic to humans" by the International Agency for Research on Cancer and its 
urinary metabolite NNAL, a biomarker of NNK exposure, has been prospectively 
associated with lung cancer.11-13 
The overall hypothesis of the program project grant under which this protocol will be 
carried out is that the differential lung cancer risk across ethnic/racial groups is due to 
differences in exposure and response to tobacco smoke carcinogens. In the prior grant 
period significant differences in carcinogen exposure across ethnic/racial groups were 
documented. 14, 15 Exposures were highest in African Americans (AA) and lowest in 
Japanese Americans (JA) compared to Whites. These data are consistent with the 
relative lung cancer risk in these 3 groups. The lower carcinogen exposure in JA was 
driven by the influence of CYP2A6 activity on nicotine metabolism.5 The focus of this 
study is to determine the influence of CYP2A6 variants on NNK metabolism 
 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 7 of 25 
 
2.2 Preliminary Data:  
In this project we will characterize the metabolic pathways of the tobacco-specific lung 
carcinogen, NNK, in JA smoking D4-pyridyl NNK-containing cigarettes.  The rationale for focusing 
our study on NNK metabolism in JA is: 1) NNK is a tobacco-specific lung carcinogen 2) CYP2A6 is 
a catalyst of NNK bioactivation (by α-hydroxylation) 3) CYP2A6 activity and genotype is 
associated with lung cancer, and 4) CYP2A6 copy number variants and other loss of function 
alleles are present at relatively high frequency in JA compared to other ethnic groups.5 
D4-pyridyl NNK-containing cigarettes.  Preliminary data from a prior study that was identical in 
design to the one proposed here, demonstrated a significant level of interindividual variation in 
NNK α-hydroxylation across smokers. The prior study that used Marlboro Virginia Blend 
cigarettes (as well as a previously published study with Quest cigarettes16), added [pyridine-
D4]NNK to the study cigarettes so that the sum of NNK and [pyridine-D4]NNK was not higher 
than the level of NNK in conventional cigarettes. In this study American spirit cigarettes will be 
used.  The beauty of using [pyridine-D4]NNK is that the products of NNK α-hydroxylation (such 
as hydroxy acid the primary urinary metabolite, Fig. 2) may be distinguished from common 
metabolites formed from nicotine. This is critical as nicotine is present in cigarettes at levels 
that far exceed those of NNK.  
In the prior study of African Americans and Whites, the ratio of D4-hydroxy acid to total D4-
NNAL was used as a measure of the total NNK and NNAL α-hydroxylation (fig 2). This ratio varied 
about 6 fold among the 114 subjects analyzed, suggesting that significant inter-individual 
variation occurs. However, since hydroxy acid is a metabolite of both NNAL and NNK this ratio is 
not ideal to establish a relationship of CYP2A6 activity with NNK bioactivation. In this project, α-
hydroxymethyl NNK Gluc will be used a direct measure of the flux of NNK metabolism through 
the α-hydroxylation pathway. In addition, by carrying out the study in JA smokers with no 
CYP2A6 activity we will be able to determine the importance of CYPA6 to NNK bioactivation. 
Preliminary evidence of an association of P450 2A6 genotype with NNK α-hydroxylation. To test 
the hypothesis that CYP2A6 activity will influence the extent of NNK metabolism by α-
hydroxylation we compared total NNAL levels, corrected for smoking dose (TNE), in smokers 
homozygous for CYP2A6 loss of function alleles to homozygous "normal" smokers.  The 
geometric mean ratio of total NNAL/TNE excreted by L/L individuals (n=54) was lower, 0.0358 
(95% CI, 0.0.33-0.038) than the ratio for N/N individuals, 0.0427 (0.036-0.051). This difference 
was borderline significant (p=0.09). Our interpretation of these data is that smokers with no 
CYP2A6 activity metabolize NNK less by α-hydroxylation and more by reduction to NNAL.   
Given the limitation of these data, (not directly measuring α-hydroxylation, further metabolism 
of NNAL, and the use of TNE as a proxy for NNK dose) it was encouraging that the effect was in 
the direction predicted. In this protocol, we will minimize these limitations by quantifying a 
more direct measure of NNK α-hydroxylation, the α-hydroxymethyl NNK Gluc as well as 
quantifying the total NNK metabolite profile, and therefore the total dose of NNK more 
accurately by using [pyridine-D4] NNK. 
 
 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 8 of 25 
 
2.3 Existing Literature:  
Our previous studies support an association of CYP2A6 activity with lung cancer. This 
association is, at least in part, driven by the influence of CYP2A6 activity on nicotine 
metabolism and smoking intensity (ref). CYP2A6 also catalyzes the metabolism NNK. 7, 8 
Therefore, individuals deficient in CYP2A6 activity may not only smoke less but may also be 
protected from NNK carcinogenesis 
Lung cancer and CYP2A6 
Meta-analyses of the association between CYP2A6 genetic polymorphisms and risk of lung 
cancer reported a statistically significant approximately 50% lower crude odds ratio of lung 
cancer for poor metabolizers (e.g., carriers of two loss of-function allele or one loss-of-function 
allele and one decreased-function allele) in Asians smokers.17, 18  In a recent analysis on the 
small number of lung cancer cases that have occurred in the multi ethnic cohort (MEC) for 
whom we have analyzed nicotine metabolism, CYP2A6 activity was associated with an 
increased risk of lung cancer.19  Also, in, a genome wide association study by the 
Transdisciplinary Research in Cancer of the Lung (TRICL) consortium a SNP in CYP2A6, which 
was associated with lower CYP2A6 activity, was significantly associated with the decreased risk 
of lung cancer.20  Importantly, the populations included in the TRICL consortium are 
predominantly White. Together these studies support an association of CYP2A6 activity with 
lung cancer. This association is, at least in part, driven by the influence of CYP2A6 activity on 
nicotine metabolism and smoking intensity. In this project we will begin to test the hypothesis 
that the relationship of CYP2A6 to lung cancer may also be mediated by its key role in the 
bioactivation of the tobacco specific lung carcinogen NNK. 
Nicotine metabolism 
In most smokers the predominant pathway of nicotine metabolism is CYP2A6-catalyzed C-
oxidation15 (Fig. 1). The product of this reaction is oxidized to cotinine, which is then 
metabolized to trans 3'-hydroxycotinine (3-HCOT). Nicotine, cotinine and 3-HCOT are all 
glucuronidated (Fig. 2). The sum of these metabolites plus nicotine and nicotine N-oxide (Fig. 2) 
is referred to as total nicotine equivalents (TNE) and account for >85% of the nicotine dose. TNE 
are an objective measure of smoking.16  CYP2A6 is the primary catalyst of the oxidation of 
cotinine to 3-HCOT, and the ratio of 3-HCOT to cotinine (in plasma or urine) has been used as a 
measure of CYP2A6 activity.15,17  This ratio will be used in the current project to quantify 
CYP2A6 activity, which will allow us to identify individuals with little or no activity and those 
with relatively high or normal activity. 
In our prior studies of the MEC, nicotine metabolism was comprehensively characterized in five 
ethnic groups.18 Nicotine C-oxidation was significantly lower in JA than in Whites or AA. JA 
excreted the highest concentration of nicotine and the lowest concentration of TNE, adjusted 
for cigarettes per day (CPD). These data are consistent with the relatively low risk of lung cancer 
in JA compared to Whites. When TNE are adjusted for CYP2A6 activity (total 3-HCOT/cotinine) 
there is no longer a statistically significant difference between the TNE excreted by JA and 
Whites.19  We analyzed CYP2A6 genotype in these smokers and demonstrated in JA the direct 
relationship between CYP2A6 genotype and both the 3-hydroxycotinine to cotinine ratio 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 9 of 25 
 
(CYP2A6 activity) and TNE. These data 
support the hypothesis that in JA 
smoking intensity (and therefore 
carcinogen dose) is significantly 
influenced by CYP2A6-catalyzed nicotine 
metabolism.  
 
 
NNK metabolism 
There are three pathways of NNK 
metabolism; reduction to NNAL, α-
hydroxylation and N-oxidation21 (Fig. 
2). NNAL, also a lung carcinogen7 is further metabolized by α-hydroxylation, N-oxidation and 
two pathways of glucuronidation.22 NNAL glucuronidation is a detoxification pathway for 
NNK.23 Two NNAL glucuronides are formed: NNAL-O-Gluc or NNAL-N-Gluc (Fig. 3). 
Bioactivation of NNK and NNAL occurs by α-hydroxylation of either the methyl or methylene 
carbon next to the nitroso group. The α-hydroxy products of these pathways decompose to 
highly reactive DNA-binding diazohydroxides. The DNA adducts generated, if not repaired may 
initiate the carcinogenic process. The products of NNK α-hydroxylation excreted in the urine,- 
hydroxy acid and keto acid - are minor metabolites of nicotine. Therefore, these major urinary 
NNK metabolites cannot be used as measures of NNK bioactivation (the α-hydroxylation 
pathway). NNK metabolites  that still contain the nitroso group could be used as biomarkers of 
NNK metabolism.  The α-OH-NNK Gluc is the only characterized metabolite of NNK α-
hydroxylation, and its level in smokers should be dependent on CYP2A6 activity, and could 
serve as a measure of NNK bioactivation. The relationship of α-OH-NNK Gluc to total α-
hydroxylation will be determined in the two groups (normal and low/no CYP2A6 activity) of JA 
smokers of [pyridine-D4]-NNK cigarettes in this protocol. 
Cytochrome P450 enzymes (CYP) catalyze the α-hydroxylation of both NNK and NNAL.21 These 
enzymes include CYP2A6, CYP2B6 and CYP1A2.24,25 In human liver microsomes, up to 70% of 
NNK α-hydroxylation is inhibited by CYP2A6 antibodies.24 CYP2B6 is similar in catalytic efficiency 
to CYP2A6, but is typically less abundant in the liver.24,26  These data support an important 
(albeit not exclusive) role for CYP2A6 in the catalysis of NNK α-hydroxylation. In this project we 
will take advantage of the high prevalence of CYP2A6 copy number variants (CNV) and loss of 
function alleles in JA smokers to quantify the role of this enzyme in the metabolism of NNK. Our 
preliminary data (described above) provides some evidence for an association of CYP2A6 
genotype with NNK α-hydroxylation.  
We have recently developed a targeted metabolomics approach for urine from rats 
administered [pyridine-D4]-NNK (ref). This method will be applied to the analysis of the JA 
smokers of [pyridine-D4]-NNK containing cigarettes to determine the relative amounts of all 
NNK metabolites (fig.2).  This will be the first characterization of an NNK metabolic profile in 
smokers.  
 
 
Figure 1. Major pathways of nicotine metabolism. 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 10 of 25 
 
The goals of this study of a small group of JA smoking [pyridine-D4]-NNK cigarettes is 1) to 
determine the influence of CYP2A6 activity on NNK a-hydroxylation pathways and 2) to 
determine if the α-OH-NNK Gluc reflects differences in a-hydroxylation measured in these two 
groups.  This will allow us to determine the contribution of CYP2A6 to NNK bioactivation, and 
ideally to identify α-OH-NNK Gluc(s) as potential measures of NNK bioactivation.  We will 
validate the NNK metabolic profiling approach in participants with little or no CYP2A6 activity, 
who we predict will have decreased metabolism through the α-hydroxylation pathway and 
increased metabolism by NNAL and the NNK-N-oxide pathways. 
 
3.0 
Study Endpoints/Events/Outcomes 
3.1 Primary Endpoint/Event/Outcome:  
To determine the effect of CYP2A6 genotype on the level of NNK α–hydroxylation in JA 
smokers administered [pyridine-D4]-D4 NNK. 
 
To characterize the NNK metabolic profile in JA smokers receiving [pyridine-D4]-NNK  
3.2 Secondary Endpoint(s)/Event(s)/Outcome(s):  
 NA 
4.0 
Study Intervention(s)/Investigational Agent(s) 
4.1 Description:  
The goal of this trial is to determine the effect of CYP2A6 genotype/phenotype on NNK 
α-hydroxylation in Japanese American (JA) smokers administered [pyridine-D4]NNK for 7 
days. Japanese American smokers (N=20 completers) will be recruited from the 100+ 
Japanese Americans participating in Study 1: Observation Study part of “Mechanisms of 
Fig. 2  NNK metabolism 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 11 of 25 
 
Ethnic/Racial Differences in Lung Cancer Due to Cigarette Smoking” Clinical and 
Biomarkers Core. 
4.2 Drug/Device Handling: 
The study will use a commercial tobacco product that has [pyridine-D4]NNK added to 
the tobacco thereby making it an investigational tobacco product.  Natural American 
Spirit-Tan cigarettes (king size, hard pack) are commercially manufactured by the Santa 
Fe Natural Tobacco Company currently marketed by RJ Reynolds and are available for 
purchase in retail stores and online.  The American Spirits-Tan were chosen because 
they have approximately 11% of the level of NNK found in other manufactured 
cigarettes. Natural American Spirit-Tan cigarettes will be modified by injecting [pyridine-
D4]NNK into the tobacco rod of these cigarettes so that the sum of the naturally present 
NNK and the added [pyridine-D4]NNK is similar to or below the NNK levels found in 
typical commercial U.S. cigarettes.  
 
Preparation of study cigarettes 
The addition of [pyridine-D4]NNK to study cigarettes will be carried out by following our 
previously established methods 16  American Spirit-Tan cigarettes will be purchased by 
University of Minnesota (king size, hard pack) which contain 0.21 - 0.37 μg NNK/g wet 
weight tobacco. The cigarettes will be modified by adding 0.300 μg [pyridine-D4]NNK to 
each cigarette so that the amount of total (deuterated plus unlabeled) NNK in these 
cigarettes is below 0.700 μg/g tobacco which is the average NNK level in popular 
commercial cigarette brands. [Pyridine- D4]NNK has been synthesized in our laboratory.  
The addition of [pyridine-D4]NNK to the cigarettes will be carried out with a specially 
designed microsyringe applicator system. The prepared cigarettes will be conditioned at 
25°C and 60% relative humidity for 2 days, placed back in their original packs, 20 
cigarettes per pack, and stored at 4°C until being dispensed to study participants. 
 
The cigarettes will be processed and stored at the Masonic Cancer Center. Once the 
cigarettes are spiked with the deuterated NNK they will be transferred to the University 
of Hawai’i study site.  The study cigarettes will be kept in a locked refrigerator and only 
study staff will have access. Detailed records of study cigarettes received, dispensed and 
returned will be kept. Any study product remaining after the completion of the trial will 
be destroyed per the University of Hawaii’s institutional Chemical Waste procedures. 
 
5.0 
Procedures Involved 
5.1 Study Design: 
In this clinical trial, the selection and recruitment will be determined based on the 
urinary ratios of CYP2A6-genreated nicotine metabolites total 3-HCOT to cotinine. Little 
or no-activity is defined as a ratio of <0.6 (expected 12% for JA from previous MEC 
sample and "relatively high" is defined as a ratio of >3.0. (28% had a ratio >3 and 15% 
<0.6 expected for JA smokers in the previous MEC sample and had a median ratio of 
1.8). 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 12 of 25 
 
Japanese American smokers will be recruited for a study where they will smoke 
cigarettes that contain [pyridine-D4] NNK.  One group will be smokers with very low or 
no CYP2A6 activity and the other will have relatively high activity.  Participants are 
selected from Study 1: Observational Study which provided human biological samples 
and measurements from Native Hawaiian, Japanese Americans and Whites.   
In this study, participants will smoke the [D4-pyridine]-NNK study cigarettes for 7 days 
and 24 hour urine samples and blood samples will be collected to assess NNK and 
nicotine metabolism (see Figure 1 for study schema). 
Figure 1 
Study Day: Day 1 
Day 2 
Day 3  Day 4 
Day 5 
Day 6 
Day 7 
Day 8 
Day 9  Day 12 
Smoke Study 
Cigarettes 
 
 
 
 
 
 
 
 
 
 
Collect 24h 
urine 
 
 
 
 
 
 
 
 
 
 
Blood 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Follow-
up Call 
 
5.2 Study Procedures:  
Clinical Study visits will be scheduled at the University of Hawai’i Cancer Center clinic, 
however, if participants are unable to attend the clinic visit, home visits will be 
completed.     
 
Visit 1 (Baseline) 
At the first Clinical Study visit, the following procedures will be administered:   
 
1. Participants will be provided detailed information about the study, and asked to sign 
the Study 2 Consent Form.  Medical History, Concomitant Medications and Tobacco 
Use Exposure Update will be reviewed for any changes in health since last contact 
following consent procedures. 
2. Timeline Follow-Back will be administered to assess the last two weeks of cigarettes 
and other tobacco/nicotine products, other combusted products or other drugs. 
3. Provide the following supplies and instructions for the collection of the 24 hour 
baseline urine sample to obtain baseline sample while smoking usual brand 
cigarettes:  

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 13 of 25 
 
a. Two 24h urine collection 1500 mL containers (no preservative) to collect 
the urine for Sample 1 (the second container is provided in case the 
participant collects over 1500 mL of urine). 
b. For women:  Specimen collection commode that can be placed over the 
toilet that will allow easy capture of urine.  
c. Instructions to begin the 24 hour urine collection with the second void the 
day before the next visit and continue to collect ALL urine through the first 
void of the visit day.  
d. Instructions for urine container storage:  Keep container in a cool place, 
such as near the toilet.    
e. At Home Urine Collection Questionnaire assessing start and end time of 
the collection and verifies any missed collections. 
4. Obtain vital signs (heart rate, blood pressure).  
5. Obtain exhaled alveolar carbon monoxide (CO) levels; obtain time of last cigarette 
and number of cigarettes smoked the day of the visit.  
6. Provide Daily Tobacco Use Diary to record each day’s cigarette, other tobacco or 
nicotine use, and number of alcoholic drinks daily until the end of the study.  
a. Inform participants to refrain from using any other nicotine-containing or 
combustible product, however ask them to record these products should 
any be used.  
7. Schedule Visit 2 for day of starting study cigarettes. (Provide Visit Calendar 
Handout). 
Procedures to be performed at Visits 2 - 6: 
The following procedures will be performed: 
1. Administer Health Changes Questionnaire to assess any change in health since last 
visit. 
2. Administer Timeline Follow-Back. 
3. Administer Adverse Event checklist to assess for any untoward symptoms. 
4. Obtain vital signs (heart rate, blood pressure).  
5. Obtain carbon monoxide (CO) levels; obtain time of last cigarette and number of 
cigarettes smoked the day of the visit.  
6. Provide two 24 hour urine sample collection containers for starting the next 24h 
sample (Visits 3, 4 and 5).  
• Instruct the participant to start the 24h sample starting with the second morning 
void and ending with the first morning void on the day of the visit.  This sample 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 14 of 25 
 
will be brought into the clinic the day it is completed. The second container will 
be used to start the next 24h collection with the second void.  
7. Collect 24h urine sample and review collection times and volume and verify if any 
collection points were missed (Visits 2, 4, 5, 6). 
8. Collect and review Daily Tobacco Use Diary.  
Additional Procedures to be performed at Visit 2, 3, 4, and 5: 
1. Dispense deuterated NNK study cigarettes to last until the next visit. Number of 
cigarettes to be dispensed is based on the maximum number of cigarettes smoked 
per day as reported in the participant’s baseline Daily Tobacco Use Diary.   
2. Complete the Study Product Dispensing Log 
3. Instruct participant to: 
• Smoke only the study cigarettes and to use no other nicotine-containing 
products or combustibles over the 7-day intervention period. 
• Smoke approximately the same number of cigarettes per day as when 
smoking their usual brand of cigarettes and to try to maintain this rate of 
smoking for the duration of the study.  
• Collect all study cigarette spent filters (butts). Return of all used cigarette 
butts in dated ziplock collection baggies at next clinic visit. Return all 
unsmoked cigarettes and empty cigarette packs. This allows for verification 
of amount of study cigarettes smoked.  
Additional Procedures for Visit 4 and 5   
1. Blood draw (2 lavender and 1 red top) 
Additional Procedures for Visit 6   
1. Provide tobacco cessation materials to the participant and advise to set a quit date.  
2. Arrange for study compensation. 
Phone follow-up – Visit 7  
The participant will be called two to four days after going off of the study cigarettes to 
assess for any health changes or adverse events.  
1. Administer Health Changes Questionnaire to assess any change in health since last 
visit. 
2. Administer Adverse Event checklist. 
 
 
 
 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 15 of 25 
 
 
 
Table of Study Procedures 
 
 
CLINICAL STUDY 
(N=20 Japanese Americans) 
OBS1  BL2 
Days on Study Cigarettes 
STUDY DAY 
 
 
0  
1 
2 
3 
4 
5 
6 
7 
8 
11 
Clinic Visits 
 
 
1 
2 
 
 
3 
 
4 
5 
6 
Phone 
Follow
-up 
Telephone screening 
X 
 
 
 
 
 
 
 
 
 
 
 
Consent form 
 
X 
X 
 
 
 
 
 
 
 
 
 
Questionnaires3 
 
X 
 
 
 
 
 
 
 
 
 
 
Tobacco Exposure and 
History Interview 
 
X 
X4 
 
 
 
 
 
 
 
 
 
Environmental Exposures 
 
X 
  
 
 
 
 
 
 
 
 
 
48 food recall 
 
X 
 
 
 
 
 
 
 
 
 
 
First void urine  
 
X 
 
 
 
 
 
 
 
 
 
 
Oral cell collection 
 
X 
 
 
 
 
 
 
 
 
 
 
Blood draw 
 
X 
 
 
 
 
 
 
 
X 
X 
 
Body Mass Index (ht & wt)  
X 
 
 
 
 
 
 
 
 
 
 
Medical History 
 
 
X4 
 
 
 
 
 
 
 
 
 
Concomitant Medications 
 
 
X4 
 
 
 
 
 
 
 
 
 
Carbon Monoxide 
 
X 
X 
X 
 
 
X 
 
X 
X 
X 
 
Vital signs  
 
X 
X 
X 
 
 
X 
 
X 
X 
X 
 
Adverse effects 
 
 
 
X 
 
 
X 
 
X 
X 
X 
X 
Health changes 
&Timeline Follow-Back  
 
 
X 
X 
 
 
X 
 
X 
X 
X 
X 
Dispense study 
cigarettes 
 
 
 
X 
 
 
X 
 
X 
X 
 
 
Smoke study cigarettes5 
 
 
 
X4 
X 
X 
X 
X 
X 
X 
X 
 
Collect study cigarette 
butts 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Return study cigarette 
butts 
 
 
 
 
 
 
X 
 
X 
X 
X 
 
Start 24h urine collection 
 
 
X 
 
 
 
 
X 
X 
X 
 
 
Return 24h urine 
 
 
 
X 
 
 
 
 
X 
X 
X 
 
Daily smoking diary 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
1OBS= Observational Sample Collection Study Data collected during Study 1 will be used for Study 2; 
2BL=Baseline; 3Demographics, Fagerstrom Test for Nicotine Dependence, NIAAA Alcohol Use 
Questionnaire; 4Abbreviated questionnaires assessing changes from Study 1; 5Participants will attend visit 
before noon and will begin smoking study cigarettes immediately after the clinic visit and before the last 
clinic visit for a full 7 days of smoking study cigarettes.  
 
5.3 Individually Identifiable Health Information: 
 
Identifiable health information will be collected at the University of Hawai’i. 
Identity of participants will not be shared with other investigators. 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 16 of 25 
 
  
6.0 
Data and Specimen Banking 
6.1 Storage and Access:  
Biomarker specimens will be collected and stored at the study site in Hawai’i until 
shipment to the Masonic Cancer Center’s Drs. Stephen Hecht and Sharon Murphy 
laboratories for analysis or long term storage. Shipments may be requested at 
approximately quarterly intervals. Samples that are not used for the primary 
analysis of study biomarkers will be banked for future use. The banked samples 
will be stored until requested by a qualified investigator and used up or destroyed 
if it is determined they are no longer needed. The samples, including DNA or RNA, 
may be stored up to a maximum of 20 years from the study’s end. A participant 
has the right to withdraw consent at any time by informing the Principal 
Investigator by following the instructions provided in the consent documents. If 
this occurs, any remaining identifiable research sample(s) will be destroyed. 
6.2 Data:  
Biomarker samples (blood components and urine) that are banked after the 
completion of the primary analyses will be stored at the Masonic Cancer Center 
Hecht and Murphy laboratories located at the Cancer and Cardiovascular Research 
Building for future use. 
Local data will be stored at the study site at the University of Hawai’i Cancer 
Center. This data will be provided to the other Program Project Investigators as 
needed to conduct their study. Shared data will be de-identified and includes 
demographics, tobacco/nicotine exposure history and current use, health and 
medications, participative questionnaire data, and biomarkers analyses.   
6.3 Release/Sharing:  
We intend to share findings from this research through publications and 
presentations. Institutions and individuals wishing to access any resources or data 
must contact the Principal Investigator. Data will be available in two formats. One 
will be a summary of the data, with graphs and tables, posted as pdf files and as 
raw individual-level data for analysis. Data generated by this grant will be made to 
outside investigators, according to NIH Guidance. 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. A record of transfer of data and a copy of the dataset that was 
distributed will be kept by University of Minnesota. 
7.0 
Sharing of Results with Participants 
7.1 Results of the study  
No individual data will be shared with the participants.  

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 17 of 25 
 
8.0 
Study Population 
8.1 Inclusion Criteria:  
a) Japanese American – one, but preferably 2 biological parents of Japanese 
descent; 
b) Male or female 21 years or older (adolescents/young adults under the age of 21 
will be excluded); 
c) Daily smoker > 5 cigarettes for at least 3 months; 
d) Eligible urinary ratios of total 3-HCOT to cotinine:  
o “Little or no-CYP2A6 activity”  defined as a ratio of <0.6 or;  
o "Relatively high" CYP2A6 activity defined as a ratio of >3.0.  
e) Stable and good physical health; 
f) Stable and good mental health (e.g., not experiencing unstable or untreated 
psychiatric diagnosis, including substance abuse, within the past 3 months); 
g) Provided written informed consent to participate in the study. 
8.2 Exclusion Criteria:  
a) Unwilling to avoid other nicotine containing products during the study and no 
use of any nicotine-containing products except cigarettes for 1 week prior to 
their study visits); 
b) 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); 
c) Active infection (e.g., influenza, cold, respiratory infection, sinus infection) at 
the time of enrollment); 
d) Experiencing medical conditions that might affect biomarkers of exposure and 
effect; 
e) Pregnant or nursing or planning on becoming pregnant during the study; 
f) Unable to read and understand English; 
g) Unable to identify ancestry. 
 
 
8.3 Screening:  
Participants are initially screened in Study 1: Observational Study where urine and blood 
samples were collected and analyzed to determine rate of nicotine metabolism.  During 
Study 1, Japanese American participants were informed of the opportunity to 
participate in Study 2 if they meet the eligibility criteria.  When eligible participants are 
identified, they will be provided with detailed description of the study and attend a 
Study 2 Screening visit where the study will be described in detail, and informed consent 
will be obtained prior to any study procedures.  Participants have completed several 
forms as part of Study 1 and this information will be shared with Study 2.  As part of the 
Study 2 Screening visit participants will complete: 
a) Brief Medical History 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 18 of 25 
 
b) Concomitant medications 
c) Tobacco Use Exposure and History Update 
d) NIAAA Alcohol Use Questionnaire-short form 
e) Fagerstrom Nicotine Dependence Questionnaire 
Medical history will be reviewed by the study medical professional to determine 
eligibility.  
 
9.0 
Local Number of Participants 
9.1 Local Number of Participants to be Consented:  
Up to 50 participants will be consented in order to complete 20 participants. All 
participants will be recruited at the University of Hawai’i Cancer Center. No 
recruitment will occur at the University of Minnesota. 
10.0 Recruitment Methods 
10.1 Recruitment Process:   
Participants are recruited from Study 1: Observation Study part of "Mechanisms of 
Ethnic/Racial Differences in Lung Cancer Due to Cigarette Smoking"  
IRB ID: Study 00000335; University of Hawai’i IRB ID: 17649. 
10.2 Identification of Potential Participants:  
University of Hawai’i research staff will inform potential participants by phone that 
they are eligible to enroll in Study 2. These participants have already agreed to be 
contacted regarding the opportunity to participate in Study 2. Upon contact they 
can agree or decline further information and screening. 
10.3 Recruitment Materials:  
No additional recruitment materials will be used in this study beyond the Study 1 
recruitment. 
10.4 Payment:   
Participants will receive $40 for time and $10 for travel for each of the 6 clinic 
visits ($300); $50 for each of the four 24 hour urine collections ($200); $10 for 
each of the 8 days of cigarette butt collections ($80) and a bonus of $250 for 
completing all study procedures. Payment is through the University of Hawai’i 
Cancer Center compensation method (gift card) to compensate for effort, time, 
and transportation for a total of $750. 
11.0 Withdrawal of Participants 
11.1 
Withdrawal Circumstances:  
If a participant experiences an adverse event or no longer meet eligibility criteria (e.g., 
quit smoking) they will be withdrawn. 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 19 of 25 
 
11.2 
Withdrawal Procedures: If a participant is no longer eligible, they will be 
informed at the visit and data collection will stop. If withdrawal is due to an adverse 
event, appropriate follow-up will be completed. 
11.3 
Termination Procedures: No additional procedures will be completed however, 
data already collected may be used. 
12.0 Risks to Participants 
12.1 Foreseeable Risks:  
Potential risks: The potential risks for participants are minimal.  
 
In this Clinical Study, the risks in this study include:  
1) Loss of privacy due to a breach in confidentiality; 
2) Risk of pain, bruising or infection at the site of the phlebotomy; and  
3) Use of study cigarettes.   
Protection against breach of confidentiality:  
Participants 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 Hawaii’s institutional oversight. No identifying information concerning 
the data and results will be made known. Participants will have written assurance that 
while de-identified individual participant 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 secure place and accessible only to relevant study staff. 
 
Risk with phlebotomy: 
Blood samples will be obtained by a trained phlebotomist with single-use, sterile blood 
collection supplies to minimize the risk associated with a blood draw. 
 
Smoking Study Cigarettes:  
Clinical Study participants will be smoking cigarettes containing deuterium labeled NNK, 
which has not led to any adverse events in the studies that we have completed 
(NCT01158456, NCT00691132). To minimize risk, participants will have a review of their 
medical history prior to entry into this study to screen for any potentially compromising 
medical condition. Participants will be under medical supervision throughout their 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 20 of 25 
 
participation in the clinical study and adverse symptoms will be recorded at each clinic 
visit and monitored by the study’s health professional.  
 
The study cigarettes pose the same risks of conventional cigarettes. Because smoking 
causes a multitude of diseases, if at any time the smoker wants to quit use of all tobacco 
products, this decision will be encouraged and supported. At the end of study, 
participants will be strongly encouraged to stop use of all tobacco products and to set a 
quit date, and will be provided with a treatment resources and referral to local 
treatment options.    
 
Participants will be informed the study cigarettes contain the same risks as smoking any 
combustible tobacco product. Precautions would be similar to other tobacco products 
including smoking related health risks, secondhand smoke exposure and fire danger.  
Participants will be advised to keep out of reach of children or pets. 
 
Throughout their participation in the study, adverse symptoms will be recorded at each 
clinic visit and monitored by the PIs and study medical personnel. Participants who 
experience any significant adverse side effects will be examined by a physician, sent or 
accompanied to the emergency clinic or referred to their physician, depending on the 
nature and severity of the side effect or adverse event. 
12.2 Reproduction Risks:  No additional risk to the fetus than the risk associated with 
conventional cigarettes.  We will conduct a pregnancy screen of child-bearing 
women prior to providing study cigarettes to verify status.   
12.3 Risks to Others: No additional risk of study cigarettes; all cigarettes pose a risk of 
exposure to second-hand smoke. 
13.0 Potential Benefits to Participants 
13.1 Potential Benefits: None 
14.0 Statistical Considerations 
14.1 Data Analysis Plan:  
In subjects with little CYP2A6 activity we expect the metabolism through 
NNAL to be significantly higher and metabolism by α-hydroxylation to be 
much lower than in smokers with "normal" CYP2A6 activity. The extent of 
NNK α-hydroxylation in the two groups will be described by a ratio of NNK 
metabolites, D4- α-hydroxymethyl NNK Gluc (or other products of α-
hydroxylation) to D4-NNAL.  The ratio is used as a measure of the pathway 
as a percent of dose. The values are predicted to be lower in the smokers 
with little or no CYP2A6-activity.  
Statistical considerations: 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 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 22 of 25 
 
A Data and Safety Monitoring Board will be convened and will be comprised of 
three experts in the areas of tobacco toxicants, clinical studies. The 
Board/Committee will include individuals with an MD, a PhD, and a statistician. 
The Data and Safety Monitoring Board will begin by reviewing the protocol and 
establishing guidelines for data and safety monitoring. This will include 
developing standard procedures for day-to-day monitoring by the internal 
monitors, investigators and study staff. This Board will meet at regular intervals 
(at least once a year) to evaluate the progress of the study, review data quality, 
patient recruitment, study retention, and examine other factors that may affect 
study outcome. They will also review the participant’s ability to achieve the 
study requirements and the rates of adverse events to determine whether there 
has been any change in participant risk. Their review will ensure that participant 
risk does not outweigh the study benefits. A brief report will be generated from 
each of these meetings for the study record and forwarded to each of the study 
site’s Institutional Review Boards (IRB). The DSMB will be available to convene 
outside of their regular meetings, if necessary, if concerns should arise regarding 
a particular participant, or any troublesome trends are observed in the 
participant experiences. They will make appropriate recommendations for 
changes in protocol, if needed. The UHCC also has a Cancer Protocol Review 
Committee that meets to review all cancer related protocols and this study will 
also be subjected to review by this committee on a yearly basis.  
All study cigarette-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 will be reported by telephone to the IRB 
within 3 days of our receipt of information regarding the event and written 
reports will be submitted within 10 days. The Data and Safety Monitoring Board 
will review all serious or unexpected adverse events and provide 
recommendations. 
NIH will be informed of any significant action taken as a result of the Data and 
Monitoring Board’s findings. We will inform the participants of any changes in 
risk. 
At the time of IRB annual review, a copy of the submission will be provided to the UHCC 
and UMN Cancer Center Protocol Review Committee. 
16.0 Provisions to Protect the Privacy Interests of Participants 
16.1 Protecting Privacy:  
For this study, most physiological and subjective measures will be noninvasive and 
should present no psychological or medical risk to the participant. Some 
questionnaires may be of a sensitive nature assessing participants’ alcohol or drug 
use. Participants will be told that they may refuse to answer, however, refusal may 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 23 of 25 
 
effect continued participation in the study. Participants will be told that their data 
will be kept confidential.  
16.2 Access to Participants:  
Participants will be referred from Study 1: Observation Study and may also be 
recruited locally through advertisements   
17.0 Consent Process 
Consent Process (when consent will be obtained): The consenting process will take place 
at the University of Hawai’i Cancer Center. The Japanese American participants will have 
been initially been briefed regarding the potential to be part of Study 2. They are told 
that after the analysis of the biomarker samples we will determine if they meet the 
eligibility criteria for Study 2 and if so, we would contact them by phone with 
information about the study. They can determine if they are interested in being 
screened for the study at that time. Only participants who agreed to be contacted 
regarding the second study will be recruited.  
At the screening visit, the Research Assistant will present a PowerPoint that describes 
the study in detail. The participant will have ample time to read the consent form, ask 
questions and receive answers. The participant will respond to several questions 
regarding the purpose, procedures and risks of the study prior to signing the consent 
forms. Afterwards, the screening procedures can commence. 
18.0 Setting 
All recruitment activities will take place at the University of Hawai’i Cancer Center, 701 
Ilalo Street, Honolulu, HI, 96813.  
 
Biomarker specimens will be sent to the Sharon Murphy lab at the University of 
Minnesota, Masonic Cancer Center, 2231 6th Avenue, Room 2-127, Minneapolis, MN 
55455. 
 
19.0 Multi-Site Research 
There is only one recruitment site, the University of Hawai’i Cancer Center. Dr. 
Hatsukami at the University of Minnesota is the PI for this study. 
 
 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 24 of 25 
 
 
20.0 References 
 
(1) 
Hecht, S. S. (2012) Lung carcinogenesis by tobacco smoke. Int J Cancer 131, 2724-2732. 
(2) 
Stram, D. O., Park, S. L., Haiman, C. A., Murphy, S. E., Patel, Y., Hecht, S. S., and Le 
Marchand, L. (2019) Racial/Ethnic Differences in Lung Cancer Incidence in the 
Multiethnic Cohort Study: An Update. J Natl Cancer Inst. 
(3) 
Haiman, C. A., Stram, D. O., Wilkens, L. R., Pike, M. C., Kolonel, L. N., Henderson, B. E., 
and Le Marchand, L. (2006) Ethnic and racial differences in the smoking-related risk of 
lung cancer. N. Engl. J. Med 354, 333-342. 
(4) 
Nakajima, M., Fukami, T., Yamanaka, H., Higashi, E., Sakai, H., Yoshida, R., Kwon, J. T., 
McLeod, H. L., and Yokoi, T. (2006) Comprehensive evaluation of variability in nicotine 
metabolism and CYP2A6 polymorphic alleles in four ethnic populations. Clin. Pharmacol. 
Ther 80, 282-297. 
(5) 
Park, S. L., Tiirikainen, M. I., Patel, Y. M., Wilkens, L. R., Stram, D. O., Le Marchand, L., 
and Murphy, S. E. (2016) Genetic determinants of CYP2A6 activity across racial/ethnic 
groups with different risks of lung cancer and effect on their smoking intensity. 
Carcinogenesis 37, 269-279. 
(6) 
Hukkanen, J., Jacob, P., III, and Benowitz, N. L. (2005) Metabolism and disposition 
kinetics of nicotine. Pharmacol. Rev 57, 79-115. 
(7) 
Jalas, J. R., Hecht, S. S., and Murphy, S. E. (2005) Cytochrome P450 enzymes as catalysts 
of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-
specific carcinogen. Chemical Research in Toxicology 18, 95-110. 
(8) 
Dicke, K., Skrlin, S., and Murphy, S. E. (2005) Nicotine and 4-(methylnitrosamino)-1-(3-
pyridyl)-butanone (NNK) metabolism by P450 2B6. Drug Metab. Dispos 33, 1760-1764. 
(9) 
Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl. Cancer Inst 91, 
1194-1210. 
(10) 
Hecht, S. S. (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-
nitrosamines. Chem. Res. Toxicol 11, 559-603. 
(11) 
Cancer, I. A. f. R. o. (2007) Smokeless tobacco and some tobacco-specific nitrosamines, 
In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans,  , IARC, Lyon, 
FR. 
(12) 
Yuan, J. M., Koh, W. P., Murphy, S. E., Fan, Y., Wang, R., Carmella, S. G., Han, S., 
Wickham, K., Gao, Y. T., Yu, M. C., and Hecht, S. S. (2009) Urinary levels of tobacco-
specific nitrosamine metabolites in relation to lung cancer development in two 
prospective cohorts of cigarette smokers. Cancer Res 69, 2990-2995. 
(13) 
Yuan, J. M., Gao, Y. T., Murphy, S. E., Carmella, S. G., Wang, R., Zhong, Y., Moy, K. A., 
Davis, A. B., Tao, L., Chen, M., Han, S., Nelson, H. H., Yu, M. C., and Hecht, S. S. (2011) 
Urinary levels of cigarette smoke constituent metabolites are prospectively associated 
with lung cancer development in smokers. Cancer Res 71, 6749-6757. 
(14) 
Park, S. L., Carmella, S. G., Ming, X., Vielguth, E., Stram, D. O., Le Marchand, L., and 
Hecht, S. S. (2015) Variation in levels of the lung carcinogen NNAL and its glucuronides 
in the urine of cigarette smokers from five ethnic groups with differing risks for lung 
cancer. Cancer Epidemiol Biomarkers Prev 24, 561-569. 

PROTOCOL TITLE: Study 2: Metabolism of NNK Among JA 
VERSION DATE: October 11, 2019 
 
Page 25 of 25 
 
(15) 
Park, S. L., Carmella, S. G., Chen, M., Patel, Y., Stram, D. O., Haiman, C. A., Le Marchand, 
L., and Hecht, S. S. (2015) Mercapturic Acids Derived from the Toxicants Acrolein and 
Crotonaldehyde in the Urine of Cigarette Smokers from Five Ethnic Groups with 
Differing Risks for Lung Cancer. PLoS One 10, e0124841. 
(16) 
Stepanov, I., Upadhyaya, P., Carmella, S. G., Feuer, R., Jensen, J., Hatsukami, D. K., and 
Hecht, S. S. (2008) Extensive metabolic activation of the tobacco-specific carcinogen 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone in smokers. Cancer Epidemiol Biomarkers 
Prev 17, 1764-1773. 
(17) 
Liu, Y. L., Xu, Y., Li, F., Chen, H., and Guo, S. L. (2013) CYP2A6 deletion polymorphism is 
associated with decreased susceptibility of lung cancer in Asian smokers: a meta-
analysis. Tumour. Biol 34, 2651-2657. 
(18) 
Liu, T., Xie, C. B., Ma, W. J., and Chen, W. Q. (2013) Association between CYP2A6 genetic 
polymorphisms and lung cancer: a meta-analysis of case-control studies. Environ. Mol. 
Mutagen 54, 133-140. 
(19) 
Park, S. L., Murphy, S. E., Wilkens, L. R., Stram, D. O., Hecht, S. S., and Le Marchand, L. 
(2017) Association of CYP2A6 activity with lung cancer incidence in smokers: The 
multiethnic cohort study. PLoS One 12, e0178435. 
(20) 
Patel, Y. M., Park, S. L., Han, Y., Wilkens, L. R., Bickeboller, H., Rosenberger, A., Caporaso, 
N., Landi, M. T., Bruske, I., Risch, A., Wei, Y., Christiani, D. C., Brennan, P., Houlston, R. S., 
McKay, J., McLaughlin, J., Hung, R. J., Murphy, S. E., Stram, D. O., Amos, C. I., and Le 
Marchand, L. (2016) Novel Association of Genetic Markers Affecting CYP2A6 activity and 
Lung Cancer Risk. Cancer Res 76, 5768-5776. 
(21) 
Scherer, G., Engl, J., Urban, M., Gilch, G., Janket, D., and Riedel, K. (2007) Relationship 
between machine-derived smoke yields and biomarkers in cigarette smokers in 
Germany. Regul. Toxicol. Pharmacol 47, 171-183. 
(22) 
Murphy, S. E., Park, S. S., Thompson, E. F., Wilkens, L. R., Patel, Y., Stram, D. O., and Le 
Marchand, L. (2014) Nicotine N-glucuronidation relative to N-oxidation and C-oxidation 
and UGT2B10 genotype in five ethnic/racial groups. Carcinogenesis 35, 2526-2533. 
(23) 
Upadhyaya, P., Kenney, P. M. J., Hochalter, J. B., Wang, M., and Hecht, S. S. (1999) 
Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol 
(NNAL) enantiomers and metabolites in the A/J mouse. Carcinogenesis 20, 1577-1582. 
(24) 
Dator, R., von Weymarn, L. B., Villalta, P. W., Hooyman, C. J., Maertens, L. A., 
Upadhyaya, P., Murphy, S. E., and Balbo, S. (2018) In Vivo Stable-Isotope Labeling and 
Mass-Spectrometry-Based Metabolic Profiling of a Potent Tobacco-Specific Carcinogen 
in Rats. Anal Chem 90, 11863-11872.