Clinical Trial of Two Study Drinks in Detoxification of Environmental Toxicants and Carcinogens
Phase: PHASE2
Conditions: Healthy
Sponsor: Masonic Cancer Center, University of Minnesota

PROTOCOL TITLE: Watercress in Detoxification of Environmental Toxicants and 
Carcinogens  
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PROTOCOL COVER PAGE 
 
 
Protocol Title 
Clinical Trial of Watercress in Detoxification of Environmental 
Toxicants and Carcinogens 
Principal 
Investigator/Faculty 
Advisor 
Name: Dorothy Hatsukami, PhD. & Stephen Hecht, PhD. 
Department: Masonic Cancer Center 
Telephone Number: 612-626-2121 
Email Address:   hatsu001@umn.edu 
Student 
Investigator 
Name: NA 
Current Academic Status (Student, Fellow, Resident): 
  
Department: 
Telephone Number: 
Institutional Email Address: 
Scientific 
Assessment 
Nationally-based, federal funding organizations 
IND/IDE # (if 
applicable) 
NA 
IND/IDE Holder 
NA 
Investigational Drug 
Services # (if 
applicable) 
NA 
Version 
Number/Date: 
Version 3 July 23, 2020 
 

PROTOCOL TITLE: Watercress in Detoxification of Environmental Toxicants and 
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REVISION HISTORY 
 
Revision # 
Version Date 
Summary of Changes 
Consent Change? 
1 
12/3/19 
Made changes to study product 
formulation; Revised the protocol to 
allow for guidelines set forth by the 
Center for Disease Control and the 
University of Minnesota to move any 
study procedures that can be to a virtual 
status. 
Y 
2 
7/23/20 
Changed day 2 to day 4 in schedule 
of events 
Y 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
 
1.0 
Objectives ................................................................................................................ 5 
2.0 
Background .............................................................................................................. 5 
3.0 
Study Endpoints/Events/Outcomes ........................................................................ 12 
4.0 
Study Intervention(s)/Investigational Agent(s)...................................................... 12 
5.0 
Procedures Involved............................................................................................... 13 
6.0 
Data and Specimen Banking .................................................................................. 18 
7.0 
Sharing of Results with Participants ...................................................................... 19 
8.0 
Study Population .................................................................................................... 19 
9.0 
Vulnerable Populations .......................................................................................... 20 
10.0 Local Number of Participants ................................................................................ 20 
11.0 Local Recruitment Methods ................................................................................... 20 
12.0 Withdrawal of Participants .................................................................................... 20 
13.0 Risks to Participants ............................................................................................... 22 
14.0 Potential Benefits to Participants ........................................................................... 22 
15.0 Statistical Considerations ....................................................................................... 22 
16.0 Confidentiality ....................................................................................................... 25 
17.0 Provisions to Monitor the Data to Ensure the Safety of Participants .................... 25 
18.0 Provisions to Protect the Privacy Interests of Participants .................................... 28 
19.0 Compensation for Research-Related Injury ........................................................... 29 
20.0 Consent Process ..................................................................................................... 29 
21.0 Setting .................................................................................................................... 29 
22.0 Multi-Site Research ............................................................................................... 29 
23.0 Resources Available............................................................................................... 30 
24.0 References .............................................................................................................. 30 
 

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ABBREVIATIONS/DEFINITIONS 
 
AE: Adverse Event 
BP: Blood Pressure 
BPM: Beats per Minute 
CVD: Cardiovascular Disease 
DAST: Drug Abuse Screening Test 
DSMB: Data Safety Monitoring Board 
DVT: Deep Vein Thrombosis 
GSTM1: Glutathione-S-transferase M1 
GSTT1: Gluthathione-S-transferase T1 
HMPMA: Abbreviation of mercapturic acid metabolite 
HPMA: Abbreviation of mercapturic acid metabolite 
HR: Heart Rate 
IRB: Institutional Review Board 
IUD: Intrauterine Device 
MAR: Missingness at Random 
MI: Myocardial Infarction (heart attack) 
NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-buatonone 
O2: Oxygen  
PE: Pulmonary Embolism 
PEITC: 2 PHENENTHYL ISOTHIOCYANATE 
PTCA: Angioplasty/stenting 
SAE: Serious Adverse Event 
SMAST: Michigan Alcohol Screening Test Short 
SPMA: Abbreviation of mercapturic acid metabolite 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1.0 
Objectives 
The goal of this proposal is to test the hypothesis that consumption of watercress, a rich 
source of the cancer chemopreventive agent 2-phenethyl isothiocyanate (PEITC), will 
enhance the detoxification of multiple environmental toxicants and carcinogens, 
particularly in subjects who are null for glutathione-S-transferase M1 (GSTM1), 
glutathione-S-transferase T1 (GSTT1), or both. If our hypothesis is supported by the 
results of the trial, watercress could emerge as an inexpensive and plentiful dietary 
constituent which could promote good health by decreasing the effects of 
environmental stressors. 
We and others have carried out extensive studies on the efficacy and mechanisms of 
PEITC in the context of cancer chemoprevention [1-21] Our studies focused on the 
potential use of PEITC in prevention of lung cancer in smokers, as we have 
demonstrated that PEITC is a powerful inhibitor of lung carcinogenesis in rats and mice 
treated with the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-
1-butanone (NNK) [2,20]. These studies led to a clinical trial of PEITC with the goal of 
determining whether it could inhibit the metabolic activation of NNK, as observed in 
rats and mice [11]. The results of that trial demonstrated a modest but significant effect 
of PEITC in decreasing the metabolic activation of NNK in cigarette smokers [11]. 
Unexpectedly however, PEITC showed far greater effects on the detoxification of 
benzene, acrolein, and crotonaldehyde in subjects who were null for GSTM1, GSTT1, 
or both [21] (see Preliminary Results). We now wish to extend these exciting results 
and assess the effects of watercress, as an abundant source of PEITC as well as other 
isothiocyanates, on the detoxification of environmental toxicants and carcinogens in a 
trial that will recruit both non-smokers and smokers in proportion to their prevalence in 
the Minnesota population. All humans are exposed to benzene, acrolein, 
crotonaldehyde and other related toxicants and carcinogens through their diet, polluted 
air, and/or endogenous metabolic sources.  
 
2.0 
Background 
Volatile organic carcinogens and toxicants such as those summarized in Table 1 are 
ubiquitous environmental and endogenous compounds to which virtually all humans are 
exposed. All of these compounds are detoxified by metabolic processes that ultimately 
result in conjugation with glutathione and excretion of mercapturic acids in urine [22]. 
Glutathione conjugation can be upregulated by isothiocyanates through the Nrf2 
pathway and related routes of metabolism [23]. The hypothesis underlying this proposal 
is that watercress consumption, resulting in exposure to milligram amounts of PEITC 
per day, will enhance the detoxification of these and related compounds. As 
summarized in Table 1, three of these compounds – benzene, ethylene oxide, and 1,3- 
butadiene – are classified as “carcinogenic to humans” by the International Agency for 
Research on Cancer. 
Benzene causes acute myeloid leukemia/acute non-lymphocytic leukemia in humans, 
and a positive association has been observed between benzene exposure and acute 
lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, and non-

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Hodgkin lymphoma. Ethylene oxide is believed to be a cause of lymphatic and 
hematopoietic cancers as well as breast cancer, and there is strong evidence that it 
operates by a genotoxic mechanism in humans. 1,3-Butadiene causes cancer of the 
hematolymphatic organs, and there is also strong evidence that it operates by a 
genotoxic mechanism in humans, via formation of epoxides. Glutathione conjugation 
interferes with these genotoxicity pathways. Acrolein and crotonaldehyde are intense 
eye and respiratory tract irritants which cause a variety of toxic effects including 
irritation, inflammation, and cell proliferation [24]. Both are DNA damaging 
compounds and, although lacking strong carcinogenic activity, acrolein-DNA binding 
has been implicated in lung cancer etiology because its pattern of DNA damage in the 
p53 gene is similar to that observed in lung cancer [25]. Propylene oxide has been 
classified as “possibly carcinogenic to humans” [26]. Acrylonitrile causes tumors of the 
central nervous system in rats and has also been evaluated as ‘possibly carcinogenic to 
humans” [27]. Acrylamide has attracted considerable attention because of its presence 
in certain cooked foods. Although it is genotoxic, there is currently uncertainty 
concerning its contribution to the occurrence of human cancers [28]. The structures of 
the mercapturic acids which are formed from these compounds and which will be 
analyzed in this project are shown in Table 1. We will also perform a more extensive 
analysis of urinary mercapturic acids in our study subjects. Significant increases in the 
detoxification of these ubiquitous volatile environmental toxicants and carcinogens, as 
indicated by urinary mercapturic acid levels, would indicate protection against their 
toxic and carcinogenic effects, which could be important in prevention of cancer and 
other diseases. 
 
As described under “Preliminary Studies”, we have observed a significant enhancing 
effect of PEITC consumption on the detoxification of benzene and acrolein, as 
determined by analysis of their urinary mercapturic acids SPMA and 3-HPMA [21]. 
The observed effects were particularly strong in subjects lacking the GSTM1 or 
GSTT1 genes, or both, and were also observed for detoxification of crotonaldehyde 

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(as determined by HMPMA in urine) in these individuals. Similar results were 
obtained in a clinical trial of a “broccoli sprouts tea” beverage, rich in the 
isothiocyanate sulforaphane, carried out in China by the Kensler group [33]. In that 
study, in which the urinary mercapturic acid analyses were performed by our 
laboratory, rapid and sustained statistically significant increases in the levels of 
excretion of SPMA from benzene (61% increase) and 3-HPMA from acrolein (23% 
increase) were observed, although there was no statistical effect of GSTM1 and 
GSTT1 status on the results. Collectively, these results clearly demonstrate that 
isothiocyanates, whether administered in pure form, as in the PEITC clinical trial, or 
in the form of a vegetable beverage, as in the broccoli sprouts clinical trial, can 
enhance detoxification of environmental toxicants and carcinogens. We also note that 
in the latter trial, exposure to benzene and acrolein was remarkably higher than in the 
U.S. because of significant levels of air pollution in the Shanghai area where the trial 
was performed. Collectively, these results strongly support our hypothesis that 
watercress consumption can enhance the detoxification of volatile organic 
carcinogens and toxicants, thus enhancing health by providing protection against the 
effects of environmental toxicants and carcinogens. Our results could significantly 
expand the armamentarium of cruciferous vegetables with demonstrated protective 
effects against carcinogens and toxicants. 
Epidemiologic studies provide some evidence for the role of cruciferous vegetables 
and isothiocyanates in protection against several cancers [34]. A meta-analysis of 
greater than 8000 lung cancer cases and 684,000 non-cancer subjects showed an 
overall 20% lower risk of lung cancer associated with cruciferous vegetable intake, 
with the strongest risk reduction in subjects who were GSTM1 or GSTT1 null, and 
particularly in the double nulls (odds ratio = 0.4, 95% CI, 0.20-0.68) [35]. The 
protective effect of cruciferous vegetable consumption against lung cancer was 
observed in both smokers and non-smokers. The greater risk reduction in the 
GSTM1 and GSTT1 nulls has been attributed to inhibition of the metabolism and 
excretion of isothiocyanates, but our clinical trial data as well as other published 
data do not support this hypothesis as GSTM1 and GSTT1 genotypes had no 
significant effect on urinary levels of the mercapturic acid PEITC-NAC, the 
principle metabolite of PEITC [21,36]. Thus, the mechanistic basis for the greater 
protective effect of isothiocyanates in GSTM1 and GSTT1 null individuals remains 
unclear, although we hypothesize that it could be due to induction of GSTP1, which 
we propose to test in later years of this program. 
In this proposal we focus on watercress as the source of PEITC rather than PEITC 
itself, which was used in our completed trial. The study of pure PEITC as a 
chemopreventive agent against lung cancer has been an instructive experience for our 
research team. Our initial studies showing that PEITC inhibited the metabolic 
activation of certain carcinogens were published over 30 years ago [37]. There are 
many challenging steps required to bridge the gap between efficacy studies in 
laboratory animals and human clinical trials. We have overcome numerous 
difficulties, both technical and financial, in bringing PEITC to a clinical trial and 
completing that trial [11]. One of the most vexing issues was formulation of PEITC, 
an oil, into softgels. The expense of doing this under the required GMP conditions 

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and on the scale needed for the clinical trial was prohibitive for an academic 
laboratory. The overall experience with PEITC has convinced our team that using a 
common vegetable as a source of the same agent is more economical and practical. 
Furthermore, we strongly support the thesis put forward by Fahey et al concerning 
“green chemoprevention as frugal medicine [38].” The use of specific foods rather 
than pure compounds derived from these foods for health maintenance and cancer 
prevention is not only credible scientifically and based on substantial evidence from 
published studies, but also more likely to have wide applicability in a global setting 
[38]. 
Watercress is a uniquely abundant source of gluconasturtiin, the glucosinolate 
precursor to PEITC [39]. The structure of gluconasturtiin (β-phenethyl glucosinolate) 
is shown in Figure 1. Glucosinolates are characteristic components of cruciferous 
vegetables. They are plant defense compounds. When cells of the plant are broken, as 
in chewing of the vegetable, the enzyme myrosinase which is normally cellularly 
separated from the glucosinolates, is released and mixes with the glucosinolate 
resulting in conversion of the glucosinolate to an isothiocyanate, as shown for PEITC 
in Figure 1. Multiple studies including our own have shown that gluconasturtiin is a 
major glucosinolate in watercress, generally exceeding all other glucosinolates and 
that watercress is the main natural source of this compound [34,39-41]. Thus, 
consumption of about 10g of watercress (wet weight) will result in uptake of about 2 
mg of PEITC, according to our current preliminary data. Watercress also contains 
other bioactive glucosinolates such as 7-methylsulfinylheptyl-, 8-methylsufinyloctyl-, 
and glucobrassican (the precursor to indole-3-carbinol), although these are generally 
found in considerably lower quantities than gluconasturtiin [34,39-41]. 
 
Several small trials have examined the effects of watercress consumption on 
endpoints associated with cancer prevention. We showed that consumption of 
watercress enhanced the excretion of the NNK metabolite NNAL in smokers, 
suggesting inhibition of its metabolic activation, as observed in studies of PEITC and 
NNK in laboratory animals [42]. Gill et al demonstrated that consumption of 
watercress (85 g daily for 8 weeks) was associated with reductions in DNA damage, 
with a greater affect in smokers than non-smokers [43]. Syed Alwi et al found that 
watercress consumption reduced 4E-BP1 phosphorylation leading potentially to 
inhibition of hypoxia-inducible factor, a key transcription factor involved in cellular 
responses to O2 [44]. Other studies have obtained various results in the examination 
of watercress extracts in cell culture systems [41,44-46]. 
Since the initial studies by Chung et al showing that PEITC inhibited the metabolic 
activation of NNK [37], multiple investigations have confirmed and extended these 
results to demonstrate favorable properties of non- toxic doses of PEITC in a variety 

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of cancer prevention settings, initially focused on mechanisms associated with 
inhibition of carcinogen metabolic activation and tumor induction, but then extended 
by numerous investigators to include multiple effects on important components of 
the tumor induction process [6,9,12,16,19,34,47-54]. These studies leave little doubt 
about the cancer chemoprevention properties of PEITC. Thus, using watercress as a 
source of PEITC and other bioactive glucosinolates, as proposed here, is a practical, 
promising, and potentially significant approach to enhancing detoxification of 
environmental carcinogens and toxicants. 
 
Relatively few clinical studies have examined the efficacy of food-based preparations 
in cancer prevention. Some notable examples have been summarized recently [38] and 
include studies on powdered freeze-dried berries [55], pomegranate [56], tomato [57], 
broccoli sprouts [33], tea [58], garlic [59], and ginger root [60]. There is one clinical 
trial of watercress supplementation for modulation of cancer progression and disease 
recurrence listed on clinical trials.gov. This is quite different from our proposed trial 
which focuses on prevention. Thus, our proposed clinical trial of watercress is 
innovative, especially when combined with our analytical chemistry and mass 
spectrometric expertise which will allow the quantitation of effects on a wide range of 
potentially carcinogenic and toxic environmental chemicals. The great potential of 
watercress and its constituent isothiocyanates to enhance the detoxification of 
environmental toxicants and carcinogens has not been sufficiently investigated in 
previous studies. 
 
One group of people routinely exposed to high levels of volatile toxic combustion 
products is firefighters. We will work with firefighters to increase awareness of our 
approach and have contacted Mr. Ken Bence in this regard. Watercress consumption 
has the potential to ameliorate the toxic effects of volatile combustion products such as 
acrolein and benzene to which firefighters are exposed. 
 
Preliminary Studies 
Effects of PEITC on detoxification of benzene, acrolein, and crotonaldehyde 
The results of the clinical trial of PEITC that form the basis of this proposal have 
been described [21]. The study was a randomized, placebo-controlled, double-blind 
phase II clinical trial with a crossover design. Current smokers of 10-45 cigarettes per 
day who were 21 years or older and in good health were enrolled. For reasons 
unrelated to this proposal, the subjects smoked commercial cigarettes with added 
[pyridine-D4]NNK during the study. After 2 weeks of adaptation to smoking these 
cigarettes, the smokers were randomly assigned to either the PEITC then placebo arm 
or the placebo then PEITC arm of the trial. During the treatment period, each subject 
was asked to take PEITC (10 mg in 1 ml olive oil) 4 times/day, once every 4 h, for 
five days (week 3 or 5), or the placebo agent (olive oil) on the same schedule (week 3 
or 5). There was a one week washout period between the PEITC and placebo 
treatments (week 4). Twenty-four h urine samples were collected at the end of the 
smoking adaptation period (week 2), on 3 days (3rd, 4th, and 5th day) of each of the 
two treatment periods (weeks 3 and 5), and at the end of the washout period (week 
4). Blood and buccal cell samples were collected collected at baseline, and at the end 

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of the smoking adaptation period, each of the two treatment periods, and the washout 
period. Forty-one subjects in the PEITC-placebo group and 41 subjects in the 
placebo-PEITC group completed the study. Their urine samples were analyzed for 
SPMA, 3-HPMA, and HMPMA, mercapturic acids derived from benzene, acrolein, 
and crotonaldehyde, respectively, using validated methods [61,62]. 
 
As shown in Table 2, GSTT1 and GSTM1 status had a strong effect on levels of 
SPMA from benzene, but not on levels of 3-HPMA or HMPMA from acrolein and 
crotonaldehyde, respectively. This is consistent with previous results [62-64]. 
Among all subjects, intake of PEITC significantly increased the urinary excretion of 
SPMA by 24.6% (P=0.002) and 3-HPMA by 15.1% (P = 0.005), but did not have a 
significant effect on levels of HMPMA. But as shown in Table 3, larger effects were 
observed when the subjects were classified by GST genotype.  

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Thus, for SPMA, PEITC caused significant 43.1%, 74.2%, and 95.4% increases in 
SPMA in the subjects who were GSTM1 null, GSTT1 null, and GSTM1 plus GSTT1 
null, respectively. For the acrolein metabolite 3-HPMA, the corresponding figures 
were 24.6%, 26.6%, and 32.7%, all statistically significant, while for the 
crotonaldehyde metabolite HMPMA statistically significant increases of 15.4%, 
22.0%, and 29.8% were seen in the GSTM1 null, GSTT1 null, and GSTM1 plus 
GSTT1 nulls, respectively. Thus, GST status had a remarkable effect on the ability of 
PEITC to enhance detoxification of benzene, acrolein, and crotonaldehyde. This was 
particularly evident for SPMA in the nulls, because these individuals have relatively 
low basal levels of SPMA, although the increases still did not raise their levels above 
those of non-nulls. We hypothesize that the significant increases in mercapturic acids 
observed upon treatment with PEITC is due to induction by PEITC of GSTP1, which 
is an established catalyst of these reactions, but further studies are required, as 
induction of GSTP1 by isothiocyanates appears to be complex [65,66]. These studies 
are beyond the scope of the present proposal, but we will examine this hypothesis in 
the future, pending the results of the clinical trial proposed here. 
Analysis of Watercress for Gluconasturtiin and PEITC 
We purchased watercress from a local market, divided it into portions, and freeze 
dried it. We analyzed each for gluconasturtiin using a published HPLC method which 
we described previously [40]. The average level of gluconasturtiin was 2 µmol per 
gram wet weight watercress. In an important new experiment, we determined that 
when this freeze-dried watercress was placed in H2O for 10 min, PEITC (2 µmol/g 
wet weight watercress) was released. The gluconasturtiin in this freeze-dried 
watercress was stable in a -20 °C freezer for at least 9 months. This experiment 
provides a clear pathway for dose formulation in the clinical trial. Watercress can be 
purchased and freeze-dried, conditions under which there is no loss of gluconasturtiin 
because of its relatively high molecular weight (423 g/mol). This freeze-dried 
watercress is stable at -20 °C. When this freeze-dried watercress is added to H2O, an 
equivalent amount of PEITC is released within 10 min because the cells in the 
watercress have been broken during freeze-drying which allows myrosinase to mix 
with gluconasturtiin when the freeze-dried powder is placed in H2O, thus releasing 
an equivalent amount of PEITC. 
3.0 
Study Endpoints/Events/Outcomes 
 
The primary endpoint is to conduct a clinical trial with 350 subjects (both non-smokers 
and smokers) to determine the effects of the watercress preparation on detoxification of 
environmental toxicants and carcinogens. 
 
4.0 
Study Intervention(s)/Investigational Agent(s) 
 
We will freeze-dry approximately 2000 lbs of watercress to produce a powder (about 
100 lbs, or  45.4 kg). Freeze-drying on the large scale required for this study will be 
performed by Van Drunen Farms. The resulting powder will  contain the 
gluconasturtiin precursor to about 3 mg PEITC per gram (which will be released upon 
addition to H2O.)  Subjects will come to the clinic and receive a pre-filled sealed jar of 

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freeze-dried watercress powder and a pre-packaged flavoring product, which they will 
add to water and stir. Each jar of freeze-dried watercress (FDWC) will contain 125 g, 
two jars will be given to each participant for a total of 250 g. Dosing calculations will 
vary depending on the potency of PEITC for each batch of FDWC (a small sample will 
be taken from each batch and potency will be measured). An example calculation is as 
follows: a total of forty-two 4.4-gram portions will be taken over the 2-week period; 
each 4.4 gram portion will release about 13.2 mg of PEITC. The 2-week supply will 
provide for an extra 14 doses for a total of 56 doses (4.4 x 56 = 246). For the placebo, 
we plan to use maltodextrin, which is a polysaccharide and does not contain 
glucosinolates, and the same flavoring product used to prepare the watercress drink. 
Subjects will prepare the placebo drink in the same way as the watercress drink. Each 
jar of maltodextrin will contain 250 g, one jar will be given to each participant. A total 
of forty-two 4.4-gram portions will be taken over the 2-week period and each container 
will hold an extra 14 doses for a total of 56 doses (4.4 x 56 = 246).  
 
Flavoring Product 
We will use a commercially available flavoring product such as Crystal Light or 
SweetLeaf Water Drops™.  Participants will mix a pre-determined amount of flavoring 
product with the FDWC or placebo.  
 
Drink Preparation 
Freeze-dried Watercress. Participants will be instructed to mix one scoop (73 mL) of 
FDWC with 16 oz of water and stir for 30 seconds, then let the mixture rest for 2 
minutes. Next, they will add in the flavoring product and stir for 10 seconds. 
Participants will be asked to finish the drink within 10 minutes of mixing .  
 
Maltodextrin. Participants will be instructed to mix one scoop (11 mL) of maltodextrin 
with 16 oz of water and stir for 30 seconds, then let the mixture rest for 2 minutes. 
Next, they will add in the flavoring product and stir for 10 seconds. Participants will be 
asked to finish the drink within 10 minutes of mixing. 
 
5.0 
Procedures Involved 
The study will be a randomized, placebo-controlled, single-blind, phase II clinical trial 
with a crossover study design. Participants will be assigned to active or placebo study 
product for 14 days, then undergo a 4 week wash-out period, and will then be crossed 
over to the other product for another 14 days (see Figure 2). During the treatment 
phase, subjects will consume the watercress beverage or placebo, 3 times per day. The 
target dose will be 40 mg/day of PEITC, as in our previous study. Urine, oral swabs, 
saliva, and blood will be collected.  

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Screening Visit  
This study will be conducted at two Minnesota sites: University of Minnesota Twin 
Cities and Hormel  Institute, which is a part of the University of Minnesota. Subjects 
will first undergo a prescreening visit, which will be conducted via phone or online 
REDCap survey. Those who meet the eligibility criteria will be invited to complete a 
virtual screening visit (completed via secured video-conferencing link). In order to 
follow guidelines set forth by the Centers for Disease Control and the University of 
Minnesota, a significant portion of the study procedures will be completed remotely.  
 
During the virtual screening visit, we will explain, that in the trial, subjects will be asked 
to use a drink prepared from watercress or an inert substance (placebo) that will be 
provided by us. The order in which they receive the watercress or placebo product will 
be randomized. Each product will be used for a period of 14 consecutive days with a 4-
week interval separating the two periods. An electronic informed consent (e-consent) 
will then be obtained. Following consent, study participants will be asked to complete 
questionnaires to assess demographics, medical and medication history, psychiatric 
status and substance abuse history (see Table 4). Our Licensed Medical Professional, 
will review these health questionnaires for consistency and eligibility for the study. 
Tobacco users will be asked to complete a tobacco use history form that assesses amount 
and frequency of tobacco use. Eligible participants will then be scheduled for a clinic 
visit and asked to refrain from eating any cruciferous vegetables during the course of the 
study. (Cruciferous vegetables are the only other major source of isothiocyanates that 
could affect the study results.) A list and pictures of cruciferous vegetables will be 
provided to each participant. Participants will be provided a urine cup (via mail) and 
asked to collect their first morning urine on the day of their next clinic visit. A reminder 
call or text will be sent to the participant prior to this visit. 
 
 
 
Experimental Period 

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Study design. Participants will be asked to attend clinic visits on days 0 (the day of 
product distribution), 4 (±1 day), 7 (±1 day) and 14 (±1 day) for each of the periods they 
are receiving product and weekly during the 4 week washout between periods (see 
Figure 2). We use a 4-week washout period because the 1-week washout in our 
preliminary studies may not have been sufficient [11]. Each visit is composed of three 
parts and will be completed in a single day. Part 1: Prior to the clinic visit the participant 
will collect their first morning void urine. Part 2: In a virtual visit, participants will be 
asked to complete multiple online surveys (see Table 4).  Part 3: Participants will drop 
off their first morning void urine (also used to test for pregnancy) at the research clinic 
(University of Minnesota – Tobacco Research Programs, 717 Delaware St. SE). Blood 
(once allowed), blood pressure, saliva, oral rinse, and buccal cells will be collected. 
After this collection, participants will be provided with either the watercress powder or 
the placebo and the flavoring product. Subjects will be given written instructions on how 
to add the watercress/placebo powder and flavor product to water and will also be 
provided a link to a video with instructions. They will be provided three different flavors 
from which they can sample. At the Day 4 visit, they will be asked to choose one of the 
flavors to be used during the rest of this phase. They will be asked to drink the 
formulation at breakfast, lunch and dinner.  Participants will receive one jar of the 
FDWC on day 0 and the second jar on day 7. Participants will receive one jar of 
maltodextrin on day 0, which will last until the end of the study. A log will be kept of 
the products that are dispensed and any amounts unused. At the end of each two-week 
period (or end of study participation, whichever comes first), we will ask all participants 
to return any unused product. Compliance of use of the study products will be 
determined by the product accountability log and biochemical verification (e.g., urinary 
PEITC-NAC and total isothiocyanates). Participants will be provided standardized 
counseling about the importance of using the assigned products as directed and if 
procedural compliance is an issue, the research assistant will discuss the obstacles and 
develop a plan to overcome these obstacles. Participants will also be provided a urine 
collection cup, dietary intake log and dosing log at each visit. 
 
Biospecimen collection. All collection containers will be labeled with study number, 
subject number, collection date and type of biological sample (urine, blood, saliva, 
buccal cells). At each clinic visit, first morning void urine samples will be collected and 
participants will be asked to submit saliva and oral cells. Participants will be asked to 
brush their teeth in the bathroom 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 5 min prior to obtaining oral cells. Saliva samples 
will be collected by asking the participant to spit in a sterilized 10 ml tube. These 
samples will be frozen at -20 °C for possible future oral microbiome studies. Oral cells 
will be collected by first, doing an oral rinse. Participants will squirt 15 mL of saline 
solution into their mouths and swish for 45 seconds. The oral rinse sample will be 
expectorated back into a 50 mL tube. After completing the rinse, participants will scrape 
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 saline, swirled for a 

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few seconds, and tapped against the walls of the tube to ensure the cells are transferred 
from the brush to the saline. Oral cells will be stored at -20°C for DNA isolation and 
research in future years of this study, which may include analysis for protein expression, 
including GSTP1. The use of oral cells for protein expression studies has been described 
[70]. 
 
Blood collection. Blood will not be collected until allowed per University policy. Once 
allowed by the University, blood collection will occur in two phases. Phase 1: One tube 
of blood will be collected (8 mL purple top). Depending on when blood collection is 
allowable, it will be collected on day 0 or if the participant has already completed the 
study, we will contact them again to have them come to the clinic for a one time blood 
draw. Phase 2: Blood will be collected on days 0, 7 and 14. Two tubes of blood (8 mL 
each, purple top) will be drawn by a trained technician. Blood samples will be used for 
DNA and protein isolation. The DNA will be used for genotyping (described under 
Specific Aim 3) and the protein reserved for possible future studies of expression of 
GSTP1 and other proteins. Protein will be isolated from peripheral blood mononuclear 
cells as described [69].  
 
Sample preparation Blood collected into purple top tubes will be processed within 12 h 
of phlebotomy. Samples should remain on ice until processing. The tubes will be 
centrifuged at 2500 × g for 15 min 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. For 
genetic analysis, genomic DNA is purified from buffy coats using a PureGene Blood Kit 
(Gentra Systems, Minneapolis, MN) according to the instructions provided by the 
manufacturer. DNA samples are aliquoted into 96-well plates using robotic equipment to 
minimize human error. The extracted DNA samples will be used for the determination 
of GST genotypes. The urine samples will be aliquoted into cryotubes and frozen at -20 
°C. Tubes with buccal cell samples will be centrifuged at 2500 × g for 15 min to pellet 
the cells and the supernatant will be discarded. The cell pellets will be washed by adding 
1 mL of pH 7.4 buffer, swirling the tube gently, repeating the centrifugation, and 
discarding the buffer. The washed buccal cell pellet will be re-suspended in 100 µL cold 
buffer, transferred into a pre-labeled cryovial, and stored at -20 °C. Throughout the 
study, biological samples (blood, buccal cells, urine, saliva) will be collected, labeled 
and registered on our Biomarker Website. The Hormel  Institute will send batches to the 
Masonic Cancer Center, University of Minnesota, to be stored in the Biorepository with 
de-identified information. The Hormel  Institute and Masonic Cancer Center 
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 will be presented 
in the informed consent for this study. A Data and Biospecimen Sharing Plan is also 
provided below. 
 
Other measures Health changes, medication use, and adverse events since the last visit 
will be determined, vital signs measured and ingestion of any cruciferous vegetables 

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assessed. Among tobacco users, specific type of tobacco product use and amounts of use 
will be determined using the timeline follow-back method. 
 
Table 4. Procedures for each visit 
 
Pre-Screening 
Screening 
Clinic visits during  
the  
14 days of study 
product use (days 
0,4,7,14) 
Clinic visits (weekly) 
during  
one-month wash-out 
period 
Telephone screening 
(remote) 
X 
 
 
 
Consent form (remote) 
 
X 
 
 
Screening 
questionnaires 
(remote)a,b 
 
X 
 
 
Body Mass Index 
(remote) 
 
X 
 
 
First urine void 
 
 
X 
X 
Urine Pregnancy Test 
(in-person clinic) 
 
X 
 
 
Blood draw (in-person 
clinic)c 
 
 
X 
X 
Oral cell collection (in-
person clinic) 
 
 
X 
X 
Saliva collection (in-
person clinic) 
 
 
X 
X 
Vital signs (in-person 
clinic)  
 
X 
X 
X 
Health changes 
(remote) 
 
 
X 
X 
Adverse effects 
(remote) 
 
 
X 
X 
Medications 
questionnaire (remote) 
 
 
X 
X 
Affect liking scale 
(remote)d 
 
 
X 
 
Dietary intake Log 
(remote) 
 
 
X 
X 
Study product dose log 
(remote) 
 
 
X 
 
Dispense study 
products (in-person 
clinic) 
 
 
X 
 
a Demographics, Medical History; Medications Questionnaire, PRIME MD(b), Michigan Alcohol Screening Test(b), Drug Abuse 
Screening Test(b), and Tobacco Use History for those who are tobacco users. 
b Questionnaires will only be administered if the participant responds ‘yes’ to related items in the Medical History Questionnaire 
c Blood will be only be drawn once allowable by the University.  
d Affect liking scale will be done on Day 14 for each study product use period 
 
 
 

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Description of Measurements at the Research Clinic 
Screening measures. 1) Demographics such as age and gender, race, ethnicity, income, 
current occupation and usual occupation; 2) Medical history and current health status for 
current diagnoses, symptoms and past health problems; 3) Medications Questionnaire to 
rule out medications that affect the metabolism of targeted constituents; 4) Prime MD for 
depression and anxiety symptoms [71]; 5) Michigan Alcohol Screening Test Short form 
(SMAST) [73]; and 6) Drug Abuse Screening Test (DAST) [74]. For those who are 
tobacco users, we will administer a Tobacco Use History that assesses amount and 
frequency as well as duration of tobacco use [11]. The following measures comprise the 
Biomarker modifier questionnaires (measures of factors that may moderate biomarkers at 
each clinic visit): 1) Dietary Intake assesses consumption of cruciferous vegetables since 
the prior visit; 2) Medications Questionnaire to assess any changes in medications taken 
since last visit and to determine use of any drugs that may affect the Nrf2 pathway; 3); 
Safety measures: Health Changes and Adverse Events Scale to assess the nature, severity, 
duration, action taken, and outcome of any adverse events. Vitals: Blood pressure and 
heart rate will be measured.  
Information about each subject will be entered into a database by the Study Coordinator.  
Each subject will be coded with a unique number, and only these coded ID’s will be 
entered into the database.  All raw data will be kept in locked file cabinets.  Only the 
Project Manager, Study Coordinator and Principal Investigators will have access to 
individually identifiable private information about participants.  Coded ID’s will be used 
throughout the study by all the researchers involved. While all the samples and 
information will be collected specifically to achieve the goals of this proposal, de-
identified individual subject data and back-up samples may be available to other 
researchers for research purposes after our study is complete.   Permission will be 
obtained from participants to allow de-identified biosamples to be stored in a 
biorepository for future analyses of biomarkers or genotyping. Standard operating 
procedures will be developed, as in our previous studies. A tracking database will be 
developed to follow each participant from enrollment to study completion. A detailed 
study manual will be developed with step-by-step instructions for interviewers and 
phlebotomists. All staff will be trained in the procedures and visits will be made to the 
Hormel Institute site to monitor the case report books and ensure that the protocol is 
strictly followed. 
 
6.0 
Data and Specimen Banking 
Biomarker specimens will be collected and stored at the study sites (University of 
Minnesota - Tobacco Research Programs, 717 Delaware St. SE and Hormel  Institute – 
801 16th Ave NE, Austin, MN) until delivery to the Masonic Cancer Center’s Dr. Stephen 
Hecht’s laboratory for storage and analysis. 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 analyses and destroyed if it is determined they are no longer needed. The 
samples, which may also include DNA or RNA, may be stored up to a maximum of 10 
years from the study’s end. A subject has the right to withdraw consent at any time by 
informing the Principal Investigator by following the instructions provided in the consent 

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and HIPAA documents. If this occurs, any remaining identifiable research sample(s) will 
be destroyed. 
Data will be stored in a secure database (RedCap) at the study site at the 
University of Minnesota, Masonic Cancer Center. Only study personnel directly 
involved with the study will have access to the linked records.  The samples that 
will be transferred to the laboratory or stored for future analyses will be de-
identified.  We will not be transferring data that has any identifying information 
over the Internet.     
Biomarker samples (blood, urine, saliva and buccal/oral cells) that are banked after the 
completion of the primary analyses will be stored at the Masonic Cancer Center Hecht 
laboratory located at the Cancer and Cardiovascular Research Building for future use. 
No identifying information will be shared with outside investigators. If used in any 
collaborative efforts beyond the scope of this study, any shared data will be de-identified. 
However, records for the study may be reviewed by departments at the University with 
appropriate regulatory oversight.  The records may also be reviewed by a representative 
of the funding agency, National Institutes of Health, and the Food and Drug 
Administration.  
 
7.0 
Sharing of Results with Participants 
Information will not be shared with participants.  
 
8.0 
Study Population 
Inclusion Criteria:  
1. Male or female age 18 years or older. Participants can be smokers or non-
smokers; 
a. Smokers must report daily use (at least one cigarette per day) 
2. In good physical health with no unstable or serious medical conditions as 
determined by the licensed medical professional; 
3. In stable and good mental health (i.e. not currently, within the past 6 months, 
experiencing unstable or untreated psychiatric diagnosis, including substance 
abuse) as determined by the licensed medical professional; 
4.  Not using any medications that may affect the Nrf2 pathway; 
5. Women who are not pregnant or nursing or planning to become pregnant; 
6. Participants have provided written informed consent to participate in the study. 
Exclusion Criteria:  
1. Significant immune system disorders, respiratory diseases, kidney or liver 
diseases or any other medical disorders that may affect biomarker data as 
determined by the licensed medical professional; 
2. History of Type I or II diabetes; 
3. Vital signs outside of the following range (participants failing for vital signs will 
be allowed to re-screen once): 

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a. Systolic BP < 90 or ≥ 160 mmHg 
b. Diastolic BP < 50 or ≥ 100 mmHg 
c. Heart rate < 45 or  ≥ 105 bpm 
4. Not willing to abstain from eating cruciferous vegetables during the course of the 
study; 
5. Known allergy or contraindication to watercress or any of the study product 
components; 
6. PKU disease (as some of the flavoring packets contain phenylalanine). 
 
Participants who agree to go through the telephone screening process will be assigned a 
screening number and taken through a screening questionnaire.  Participants who meet 
the eligibility criteria described above will be invited to participate in a virtual screening 
visit, where the entire study will be explained in detail, informed consent will be obtained 
and the screening measures will be completed. 
9.0 
Vulnerable Populations 
No vulnerable populations will be used. 
10.0 Local Number of Participants 
Three hundred and fifty participants are needed for the study. Anticipating a 15% attrition 
rate, a total of 400 of participants will be enrolled into the study. We expect about 300 
non-smokers and about 100 smokers.  
11.0 Local Recruitment Methods 
We will recruit smokers and non-smokers using various media sources (flyers, newspaper 
ads, Craigslist, Facebook, etc.). We will also recruit participants who have previously 
participated in other research studies through the Tobacco Research Programs and agreed 
to be contacted for future studies.  
For each clinic visit, the participant will be paid $25 for the screening visit. Eligible 
participants will be paid $10 for transportation costs and $25 for time spent at each visit 
(12 visits × $35 = $420). A $245 bonus will be provided for completion and compliance 
to the study procedures (including biochemical validation of watercress consumption by 
measurement of PEITC-NAC in urine) for a total of $690. 
12.0 Withdrawal of Participants 
A study participant may be discontinued from the study if investigators determine that 
this is the best decision in order to protect his/her safety. In the event that a participant 
either withdraws from the study or the investigators decide to discontinue a participant 
due to an adverse event (AE) or serious adverse event (SAE), the participant will have 
appropriate follow-up assessments and if necessary, referrals will be made for medical 
care. The participant experiencing an AE/SAE will be followed until the problem 
resolves, stabilizes, or is clearly unrelated to the study product. Any AE that remains 
open will be reviewed and closed at the last study visit.  

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For the participant’s protection, they will be withdrawn immediately from the study 
if any of the following occur: 
1) Cardiovascular disease (CVD) event: Typically includes MI (heart attack), PTCA 
(angioplasty/stenting), bypass surgery, stroke, or peripheral vascular disease.  
Less common CVD problems would be new cardiac arrhythmias (e.g., new atrial 
fibrillation) or new valvular disease (e.g., mitral or aortic regurgitation). 
2) DVT/PE (deep vein thrombosis/pulmonary embolism, i.e., blood clots in the 
venous system). 
3) Suicide Attempt:  A participant will be withdrawn if he/she attempts suicide at 
any time during participation in the study. 
4) Psychiatric Hospitalization: A participant will be withdrawn if he/she is 
hospitalized for psychiatric reasons at any time during participation in the study. 
5) Pregnancy: If a participant becomes pregnant, she be immediately withdrawn 
from the study.  In addition, the licensed medical professional will follow-up after 
delivery to ask questions about the health of the baby. 
The following will be monitored and can lead to the participant being withdrawn by 
the Principal Investigators or Medical Monitor: 
1) Blood pressure (BP) or heart rate (HR) changes: If any of the following occur post-
enrollment:  
1) BP is at or above 160 systolic/100 diastolic; 2) BP is below 90 systolic/50 
diastolic and the participant is experiencing symptoms listed on the ‘Blood Pressure 
and Heart Rate Symptom Checklist; 3) HR is at or above 105 bpm; 4) or below 45 
bpm and the participant is experiencing symptoms listed on the ‘Blood Pressure and 
Heart Rate Symptom Checklist.   
2) Medication changes: If a participant begins taking any of the exclusionary 
medications or other medications that could potentially have an interaction post- 
enrollment, the licensed medical professional will determine how best to monitor 
and minimize potential risks (including withdrawal if warranted).   
3) Any hospitalization or debilitation in which participation in the study could be 
detrimental to the recovery process.  This will be self-reported by the participant 
and reviewed by the investigators and medical professional to determine if 
continued participation in the study is appropriate.   
4) If a participant is behaving in an inappropriate or threatening manner, admits to 
lying about eligibility criteria, including omitting previous medical diagnoses and 
medications, is participating in other tobacco research studies that could affect the 
primary outcome measures, does not follow study instructions, etc., then he/she can 
be withdrawn at the investigators’ discretion. 
 
Participants will be informed about the withdrawal at the visit and data collection will 
stop. No additional procedures will be conducted if participants are withdrawn. We will 
ask that all unused product be returned along with completed dosing diaries. 
 
 
 
 

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13.0 Risks to Participants 
The potential risks for participants recruited for this study are minimal. Physiological and 
subjective measures will be noninvasive and should present no psychological or medical 
risk to the subject.  Blood and oral samples will be obtained by trained study personnel. 
Watercress in doses that are administered has the potential to lead to gastrointestinal 
distress (e.g. flatulence, bloating, nausea). Participants will be under medical supervision 
throughout their study participation and adverse symptoms will be recorded at each clinic 
visit and monitored by the project Co-PIs Dr. Hecht and Dr. Hatsukami along with the 
study’s licensed medical professional.  
Pregnant women will not be recruited.  If participants choose to be sexually active, they 
should use an appropriate “double barrier” method of birth control (such as female use of 
a diaphragm, intrauterine device (IUD), or contraceptive sponge, in addition to male use 
of a condom) or the female should be using prescribed “birth control” pills, injections, or 
implants. Female participants with child-bearing potential will be tested for pregnancy at 
the first  clinic visit using the first morning void urine sample. If a participant becomes 
pregnant during the study, she will be withdrawn from the study. Approximately 30 days 
after being withdrawn or having a positive pregnancy test, the research staff will call the 
participant to confirm her due date. The licensed medical professional will follow-up 
with the participant after delivery to ask questions about the baby’s health. 
 
14.0 Potential Benefits to Participants 
While there might not be any direct benefit to the participant, if proven effective, 
watercress can become part of a health maintenance diet to possibly reduce risks for 
certain cancers by enhancing the detoxification of toxicants and carcinogens. 
 
15.0 Statistical Considerations 
Descriptive statistics will be used to summarize subject characteristics, for example 
subject demographics (age and gender, race, ethnicity, income, current occupation and 
usual occupation), medical history and current health status for current diagnoses, 
symptoms and past health problems. Categorical variables will be described by their 
frequencies and the continuous measurements will be described by their mean, median, 
range, and standard deviations. We will use paired t-test to compare distributions of 
continuous variables (e.g., age, number of cigarettes per day, number of pack-years of 
cigarettes smoked over lifetime) between the two treatment groups (watercress and 
placebo arms). The McNemar’s Chi-squared test will be used to compare categorical 
variables, such as sex, GSTM1 and GSTT1 genotypes between the two treatment groups.  
 
The statistical analysis section particularly focuses on Specific Aim 3, which investigates 
the hypothesis that detoxification of these noxious agents by conjugation with 
glutathione, as indicated by mercapturic acid levels in urine, will be significantly elevated 
compared to placebo in subjects who consumed the  watercress preparation and are null 
for GSTM1, GSTT1, or both. We can test for a Period x Treatment interaction such as 
testing if the effect of the watercress preparation varies depending on whether it is given 
in the first period compared to the second period. 

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We will have baseline measurements on each subject. Participants will be asked to attend 
clinic visits on days 0 (baseline), 2, 7 and 14 for each of the periods they are receiving 
product (watercress or placebo). We will compare the baseline measurements on the 
outcome variables and some important covariates between the two treatment groups 
through the two-sample t test to see if there is any significant difference between the two 
groups in order to ensure homogeneous treatment groups. We will also perform repeated 
data analysis using the measurements on each subject and consider different baseline 
parameters for the watercress and the placebo group in our multivariate regression 
analysis described later. 
 
Measurements: For the comparison between the two treatment groups, we will consider 
measurements excluding the baseline observation. Measurements will be taken from each 
subject in the two supplementation sequences as seen in the following diagram: 
Group 1: Period #1 (watercress; A1) – washout – Period #2 (placebo; B2) 
Group 2: Period #1 (placebo; B1) – washout – Period #2 (watercress A2) 
The letter is used to denote supplementation (A for watercress and B for placebo) and the 
number, 1 or 2, denotes the period. We will assume, in the formulation of statistical 
models, that the measurements A1, A2, B1, and B2 are “normally distributed” in log-
scale. These letters can represent one of our outcome variables: urinary mercapturic 
acid levels of specific toxicants (e.g. benzene, acrolein, crotonaldehyde, propylene oxide, 
etc.) or for mercapturic acids generally, or DNA adducts of acrolein. 
Main Outcome Variables: Our data analysis will be based on the following “outcome 
variables”: 
X1 = B2 – A1; and X2 = B1- A2. 
This subtraction will cancel the “within-sequence” effects of all subject-specific factors, 
leaving only two to be modeled and analyzed: the Treatment (or Supplementation) effect 
and the Period (or Order) effect. The measurements will result in two independent 
samples. Each will have the same sample size if there are no dropouts or missing data. 
Otherwise the number of subjects for each group of measurements (i.e., X1 and X2) will 
differ if there are dropouts and missing data. 
 
We would obtain values of X1 or X2, which is a log of "ratio" for each subject in each of 
the two groups with different supplementation order, it’s (watercress/placebo) for group 1 
and (placebo/watercress) for group 2. We'll then compute the averages of these log ratios, 
i.e x̅̅̅ 1 and x̅̅ 2.  We will do this calculation separately for the double null group (α̂nn), and 
single null group (α̂np). We will compare this average ratio with the average ratio of the 
double positive group (α̂pp). With the double nulls being about 15% of the population, 
GSTM1 single nulls (45%) and GSTT1 single nulls (23%), we expect to see 60 double 
nulls, 180 GSTM1 single nulls, 92 GSTT1 single nulls, and 68 double positive 
individuals. If there is no influence of GSTM1 or GSTT1, the effect of watercress 
supplementation would be the same between the double null and the double positive 
group or between the single null and the double positive group. We will test for the 
significance of the genotypic effect by using a two-sample t test.  

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In order to estimate the power for that comparison, we need information on the variability 
of mercapturic acid levels within and between subjects. We will compute the variances of 
each of these genotypic groups separately and then obtain a pooled variance estimate to 
compute our pooled t test statistic. Based on our previous work, we used an intra-subject 
CV of 0.4 and an inter-subject CV of 0.6, which translates to a pooled standard deviation 
of 0.657. According to Table 3, the percentage difference between the Placebo and 
PEITC groups with the double null genotypes varied from 29.8% to 95.4%, whereas the 
percentage difference for the double positive genotypes varied from 4% to 7%. In 
general, we will have 80% power to detect a difference of 35% between the double null 
and double positive group and given the range of difference between these two groups in 
Table 3, we expect to have good power to study this interaction between  watercress and 
GSTM1 or GSTT1 genotypes.  
 
We will also perform the above analysis separately for smokers and non-smokers. We 
expect to see about 25% smokers (~ 100 smokers) in our sample. We might have limited 
power to detect the impact of watercress for the double-nulls among the smokers, 
however this subgroup analysis would reveal if there are suggestive differences in the 
association of urinary mercapturic acid levels, watercress and GSTM1, GSTT1 between 
the smokers and the non-smokers. 
 
Multivariate Regression Analysis: We will use multivariate regression analysis to 
supplement the t-test by pooling the data together and using each of the primary outcome 
variables as dependent variable, separately. In addition to the variables representing the 
supplementation, treatment order, and status of GST (M1, T1) genotypes, the list of 
independent variables will include socio-demographic characteristics such as gender, age, 
smoker vs non-smoker, baseline smoking level (cigarettes per day), and intake of dietary 
nutrients. We will fit a linear mixed-effect model where the dependency in the repeated 
measurements on each individual will be captured through a random effect. The status of 
GST (M1, T1) genotypes will be modeled as fixed-effects and we will test for the null 
hypothesis of no association between the genotypes and each outcome. Our primary 
parameter of interest will be the interaction between the treatment order (pre vs post) and 
the genotype groups (double null, GSTM1 single null, GSTT1 single null, and double 
positives). We will perform a t-test to assess the significance of the interaction parameter. 
The residual covariance structure should be selected among several candidates, i.e. 
autoregressive, compound symmetry, unstructured, etc., to estimate the fixed and random 
effects. We do not anticipate much missing data on the participants. In general, we will 
assume missingness at random (MAR) and the likelihood-based linear mixed effect 
model will provide consistent estimates of our parameters of interest in presence of 
MAR. The models will be assessed through their Akaike’s Information Criterion and 
Bayesian’s Information Criterion. The normality of the outcome variables will be 
assessed and in presence of non-normality, we will also implement Generalized 
Estimating Equations to study the impact of watercress and genotypes on each outcome 
variable. We will use the statistical software R packages nlm and gee to perform the 
above analysis. These packages will also provide the flexibility to fit non-linear mixed 
effect models to assess the impact of watercress and genotype groups. 

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A variety of measures will be taken to ensure data accuracy and completeness. The 
regular research team meetings will include discussions of proper methods for data 
collection, transmission, and storage, limiting data collection to those in protocol required 
to answer a research question, de-identifying data, and encryption methods. 
A comprehensive data dictionary will be created to specify definitions and value codes 
for all variables that will be entered into REDCap and other study databases. Electronic 
forms for the collection of subjective measures via REDCap will include programming 
features to ensure valid data (i.e., input masks, validation criteria, skipout logic) and will 
be stored on the University of Minnesota, HIPAA compliant, computing system.  Double 
entry will be used for all other de-identified data entered into REDCap.  Biological 
specimens will be labeled with barcode labels that incorporate the participant ID. The 
secured biospecimen website will identify the location of each sample at the clinic (prior 
to submission to the laboratory) and in the Hecht laboratory.   
Oversight of the randomization and product distribution will be conducted by the Project 
Manager in collaboration with the co-Principal Investigators. The randomization 
schedules and the link between the alphabetic code and treatment assignment will be 
maintained securely by the Project Manager.  An Online Randomization and Product 
Tracking database will be created in collaboration with the study biostatistician. This 
database will be used to track product purchases, product inventory, and assignment of 
study product to participants based on the randomization schedule. 
Quality control procedures will be conducted for all data collected, including analysis of 
missing data, and logic checks for out of range and other anomalous values. Queries will 
be made regarding such data issues, with documentation of any changes made in the data. 
16.0 Confidentiality 
All investigators and staff associated with this project have been trained, and new hires 
will be trained, on human research ethics and Good Clinical Practice in accordance with 
the requirements of the University of Minnesota. 
Only the immediate study team (Project Manager, Study Coordinator and Principal 
Investigators) will have access to individually identifiable private information about 
participants.  Coded ID’s will be used throughout the study by all the researchers 
involved. Original signed consent forms and other identifiable information will be kept 
separate from the research information in a secure and locked space/database. 
 
17.0 Provisions to Monitor the Data to Ensure the Safety of 
Participants 
Oversight for quality control and adherence to protocol procedures will be conducted by 
the Project Manager in collaboration with the co-Principal Investigators. A start-up 
meeting with the whole research team will take place prior to participant enrollment. 
During this meeting, there will be training on the study protocol, standard operating 
procedures, equipment and data collection platforms. The co-Principal Investigator, 
Dorothy Hatsukami, will closely monitor the research staff on the procedures to be used 
in this study.  Such monitoring will consist of frequent in-person discussion of study 
visits and other procedures to make sure that all protocol procedures are followed, and 

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regular research team meetings to provide updates on study progress and review the data 
collection process, the results from data monitoring and other issues of concern. 
Standard operating procedures will be developed for consistent implementation of the 
protocol. The Study Coordinator will be administering all measures during clinic visits 
and entering the information about each subject into a database. Each visit will have a 
checklist of all measures that need to be obtained and the order by which they will be 
administered. The Project Manager will be directly supervising the Study Coordinator 
and will periodically review protocol compliance and implementation, and adherence to 
good clinical practice procedures. 
The Study Coordinator will go over the questionnaire instructions and will be available to 
the participant to answer any questions he/she may have.  Questionnaires will be 
reviewed for completeness, however, they may choose not to answer questions.   
Once a participant has completed all study procedures and all open events have been 
closed, the PI will review the participant’s binder and sign a form indicating study 
completion for that participant. 
This is a minimal risk, non-therapeutic study. While participating in the trial, AEs and 
concomitant medications will be assessed at every study visit and vital signs will be 
obtained periodically. The Co-Principal Investigator, Dorothy Hatsukami will meet 
regularly with the study staff to review recruitment progress and any adverse events.  
Entrance criteria will be reviewed following screening.  Study participants will be under 
medical supervision while in the study and our research staff will make appropriate 
referrals to the physician should any adverse events occur.  The Data Safety Monitoring 
Board (DSMB) and other regulatory bodies will be informed of any adverse events either 
at the regularly convened meetings or in the annual report, or if necessary, immediately.  
The DSMB, potentially comprised of a biostatistician, clinical research scientist (MD) 
and medicinal chemist, 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 trial, 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 subject 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 the Institutional Review Boards (IRB). 
 
The DSMB will be available to convene outside of the regular meetings, if necessary, if 
concerns should arise regarding a particular subject, or any troublesome trends in the 
subject experiences. They will make appropriate recommendations for changes in 
protocol, if needed. 
AEs will typically be identified during the administration of the Health Changes 
Questionnaire. Other events may be identified from physiological study measures or by 
spontaneous reports during assessments. 

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Assessment of Questionnaire Items  
• Health Changes Questionnaire: If the participant answers ‘YES’ to Questions 1, 2, 
or 3, the interviewer will assess for an ‘Adverse Event.’  
1) Have you had any negative changes in your health since your last visit?  
2) Have you had any changes in medication since your last visit? 
3) Since your last visit, have you received any form of medical care? 
Assessment of Physiological Data 
• Blood Pressure:  
o The ‘Adverse Event Log’ and ‘Blood Pressure and Heart Rate Symptom 
Checklist’ will be completed if an automatic and subsequent manual blood 
pressure measurement during the same visit is at or above 160 systolic or 
100 diastolic. 
o The ‘Adverse Event Log’ and ‘Blood Pressure and Heart Rate Symptom 
Checklist’ will be completed if an automatic and subsequent manual blood 
pressure measurement during the same visit is below 90 systolic or 50 
diastolic and the participant is experiencing symptoms listed on the ‘Blood 
Pressure and Heart Rate Symptom Checklist.’ 
• Heart Rate:  
o The ‘Adverse Event Log’ and ‘Blood Pressure and Heart Rate Symptom 
Checklist’ will be completed if an automatic and subsequent manual heart 
rate measurement during the same visit is at or above 105 bpm. 
o The ‘Adverse Event Log’ and ‘Blood Pressure and Heart Rate Symptom 
Checklist’ will be completed if an automatic and subsequent manual heart 
rate measurement during the same visit is below 45 bpm and the 
participant is experiencing symptoms listed on the ‘Blood Pressure and 
Heart Rate Symptom Checklist.’ 
Adverse Events Communicated by Participants 
The occurrence of AEs will be sought by non-directive questioning of the participant at 
each visit during the study. AEs also may be detected when the participant volunteers 
them during or between visits or through physical examination, laboratory test, or other 
assessments. 
Review and Reporting of Adverse Events and Serious Adverse Events 
Co-Principal Investigators with oversight from Naomi Fujioka, M.D. (Medical Monitor) 
and the clinic’s licensed medical professional will review all AEs and assess whether 
they are related to the study product. 
An AE is defined as the appearance or worsening of any undesirable sign, symptom, or 
medical condition occurring after starting the study procedures even if the event is not 
considered to be related to the study product. Medical conditions/diseases present before 
starting the study are only considered adverse events if they worsen after starting the 
study product. Abnormal laboratory values or test results constitute adverse events only if 
they induce clinical signs or symptoms, are considered clinically significant, or require 
therapy; there are no plans for active monitoring of laboratory tests as part of this project.   

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To the extent possible, each adverse event will be evaluated to determine: 
1. the severity grade (mild, moderate, severe) 
2. its relationship to the study product used (suspected/not suspected) 
3. its duration (start and end dates or if continuing at final exam) 
4. outcome (resolved/improved/unchanged/worsened; study product 
temporarily interrupted or permanently discontinued due to this adverse 
event; concomitant medication taken; non-drug therapy administered) 
5. whether it constitutes a SAE 
Information about all SAEs will be collected and recorded on the project’s Serious 
Adverse Event Report Form. A SAE is defined an undesirable sign, symptom or medical 
condition which: 
1. is fatal or life-threatening; 
2. requires or prolongs hospitalization; 
3. results in persistent or significant disability/incapacity; 
4. constitutes a congenital anomaly or a birth defect; 
5. is medically significant, in that it may jeopardize the subject and may 
require medical or surgical intervention to prevent one of the outcomes 
listed above. 
Events not considered to be SAEs are hospitalizations that are: 
1. elective or pre-planned, for a pre-existing condition that is unrelated to the 
products under study and did not worsen; 
2. for general care, and/or overnight observation; 
3. treatment on an emergency, outpatient basis for an event not fulfilling any 
of the definitions of serious given above and not resulting in hospital 
admission. 
Safety evaluation will be performed on all accrued participants for whom study products 
were dispensed. The assessment of safety will be based on the frequency of AEs and 
severity grade of AEs. Other safety data (e.g. vital signs) will be considered as 
appropriate. 
 
Any SAE occurring after the participant has signed the consent form and until the last 
encounter with the participant will be reported. All AEs will be summarized by 
presenting, for each treatment group, the number and percentage of participants who 
experienced any AE, the number reporting AEs in each body system and the number of 
AEs by type. Any other information collected (e.g., severity or relatedness to study 
product) will be listed as appropriate. A summary of clinically relevant toxic events, such 
as AEs leading to death or rated as SAEs, those with a suspected relationship to study 
product, or AEs requiring further medication or non-drug therapies will be provided. 
Reports will be reviewed regularly by the study investigators. 
 
18.0 Provisions to Protect the Privacy Interests of Participants 
It will be made clear to participants that all information obtained during assessments is 
confidential and that no information will be shared with the participants’ clinicians unless 
the participant requests this in writing.  

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While all the samples and information will be collected specifically to achieve the goals 
of this proposal, de-identified individual subject data and back-up samples may be made 
available to other researchers for research purposes after our study is complete.  We will 
obtain permission from participants to allow de-identified biosamples to be stored in a 
biorepository for future analyses of biomarkers or genotyping. 
There will be no access to medical records or any other sources of private information 
about the participating participants. 
19.0 Compensation for Research-Related Injury 
The study poses minimal risk to participants. 
20.0 Consent Process 
The consenting process will take place virtually via a secured video-conferencing 
meeting invitation. Potential participants will be told the nature of the research 
over the phone during pre-screening and then at the screening visit. They will be 
told they may discontinue participation at any time and will not be discriminated 
against if they choose to do so. Interested subjects will be provided considerable 
time to review the consent form, consider whether or not to participate, and have 
any questions answered by the coordinator. Participants will be required to 
demonstrate an understanding of the study purpose and procedures prior to 
signing the consent form. Assessment of the subject’s understanding will be 
completed via questions by a slideshow presentation. The consent form must be 
signed before the research is started. Immediately after signing, the participant 
will received an email with a signed copy of the consent form.  
The electronic consent forms will be stored in a REDCap database with restricted 
access for essential study personnel only. The electronic informed consent (eIC) 
will be built us the ‘UMN e-Consent HRP-592-TEMPLATE-Biomedical’. The 
template will be customized to match the written informed consent form exactly. 
The electronic signatures obtain in the outline above are intended to be the 
equivalent of handwritten signatures. Therefore, the electronic signatures will 
occur in accordance with the predicated rule (e.g. approved, reviewed and 
verified) as outlined in the Food and Drug Adminstrations’s CFR part 11. 
21.0 Setting 
The study will be conducted at the University of Minnesota, Twin Cities and The Hormel 
Institute 
• Subject recruitment and sample collection will take place at Tobacco Research 
Programs (717 Delaware St. SE, Minneapolis, MN 55414) and The Hormel 
Institute 801 16th Ave NE, Austin, MN 55912. 
• Biochemical analysis will be carried out in the Masonic Cancer Center (2231 6th 
St. SE, Minneapolis, MN 55455). 
 
22.0 Multi-Site Research 

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A total of 400 of participants will be enrolled into the study. Each site will recruit 
smokers and non-smokers using various media sources (flyers, newspaper ads, Craigslist, 
Facebook, etc.).  
Regular teleconferences will be scheduled between the two sites to discuss the study 
procedures, recruitment, and any safety concerns.  
 
23.0 Resources Available 
The lead site for this study will be the University of Minnesota’s Tobacco Research 
Programs housed at the Delaware Clinical Research Unit at 717 Delaware St. SE 
Minneapolis, MN. Dr. Dorothy Hatsukami serves as the Director for this Program. We 
have a Research Project Manager who oversees all research and is responsible for 
logistics of implementing the protocols and standard operating procedures. She is also 
responsible for the quality control of the projects by ensuring that all studies follow 
ethical scientific standards and that procedures meet GCP standards, that all regulatory 
forms are completed including Institutional Review Board applications, and that the 
DSMB process is in place. We also have an Administrator  who ensures the smooth 
operation of the daily activities of the Program.  In addition, the Program has a registered 
nurse practitioner and many research project coordinators. The shared space at the 
Delaware Clinical Research Units includes a shared waiting room with a receptionist, 7 
physical exam rooms (two dedicated to the Tobacco Research Programs), 1 phlebotomy 
room, 5 interview rooms, 2 day hospital rooms, an infusion room, 1 smoking laboratory 
with one way observation room, laboratory space for processing blood, urine processing 
laboratory, a locked medication supply room, locked protocol room for subject files, 
cubicles for data entry, management and analyses, locked supply storage and access to 
three conference rooms. Two restrooms are in the clinical space for urine collections. We 
have dedicated space for our biorepository with key card access containing ten -20 
freezers. We also have access to all of the resources of the University of Minnesota for 
our use, as needed. 
24.0 References 
Reference List 
 
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