Microbial Mechanisms of Methylmercury Metabolism in Humans
Phase: NA
Conditions: Mercury--Toxicology
Sponsor: University of Rochester

 
NCT04060212
Microbial mechanisms of methylmercury metabolism in humans. 
Matthew D. Rand, Ph.D. 
Department of Environmental Medicine 
URMC, Box EHSC, RM 4-6818 
1. Background and Purpose of study.
1 A. Application Background. This submission seeks approval of a new study that 
follows essentially the same protocol of two prior approved studies we have conducted, 
with a modification that includes an additional intervention of administration of a prebiotic 
to influence change in the gut microbiota. The prior studies were 
#RSRB00050613 entitled Methylmercury metabolism and elimination in humans and# 
RSRB00057934 entitled Determination of methylmercury metabolism and elimination 
status in humans. 
1 B. Purpose of the study. 
The overall objective of this study is to investigate the role of gut microbes in 
mediating how humans metabolize and excrete the environmental neurotoxicant 
methylmercury (MeHq). Our secondary objectives are: 1) to derive non-invasive tools to 
evaluate an individual's MeHg metabolizing potential, 2) to identify dietary supplement 
approaches to enhance an individual's capacity to metabolize and excrete toxic MeHg, 
3) to discover symbiotic bacterial strains with probiotic potential. Successful completion 
of the study Aims will eventually lead to advanced methodologies to better assess the 
risks of Me Hg on human health and identify factors that mitigate this risk. 
1 C. Scientific Background. 
MeHg impact on human health. Exposure to MeHg through consumption of fish 
continues to pose a health risk for many populations globally. The developing nervous 
system is a preferred target for MeHg toxicity, making the fetus and young children most 
vulnerable. Conflicting results from large epidemiological studies has lead to uncertainty 
in advising the public on mercury risks associated with fish consumption [1-3]. At odds 
are the potential harmful effects of Me Hg and the beneficial effects of dietary nutrients 
and antioxidants in fish. A great deal of uncertainty for this conflict stems from the fact that 
MeHg metabolism and elimination rate is known to vary widely from individual to 
individual. This translates into the possibility that two individuals consuming the same 
amount of fish with the same frequency could, unknowingly, experience as much as 
4-fold difference in MeHg in their bodies. Thus, there is a need for greater understanding 
of the mechanisms of MeHg metabolism and elimination, as well as for development of 
tools to assess these characteristics in people.
MeHg toxicokinetics: the importance of elimination rate. Orally ingested MeHg 
is absorbed very efficiently in the intestine with 95% of the dose typically being taken up 
and distributed to the bloodstream [4]. MeHg undergoes rapid transport and equilibration 
between blood and target organs [4]. Subsequent to an initial "spike" in concentration, 
mercury levels in blood resolve to a slow steady rate of decline that is dictated by the 
overall elimination rate of Me Hg from the body [4-6]. Elimination rates, 
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expressed as half time (t1/2), range from <30 to >120 days, demonstrating the existence 
of “fast” and “slow” MeHg metabolizers [4-11]. The elimination rate of MeHg is a major 
determinant for accumulation of MeHg to the elevated steady state levels that occur in 
people who consume fish on a regular (e.g. weekly or daily) basis. The ability to 
determine the MeHg metabolism and elimination status of an individual is therefore a 
means of prospectively identifying “fast” versus “slow” metabolizers.
Mechanisms of MeHg metabolism and elimination: the critical role of de-
methylation.   Approximately 90% of the MeHg ingested and absorbed in the body is 
ultimately excreted via the feces [5, 7]. The current dogma holds that MeHg excretion 
occurs via the liver, where glutathione-MeHg conjugates are transported via bile to the 
small intestine [12]. However, a “vicious cycle” exists in that MeHg can be rapidly re-
absorbed in the intestine and returned to circulation [13]. This cycle can be broken by 
MeHg de-methylation, which yields Hg++, a poorly absorbed inorganic (iHg) form of 
mercury [14].  This mechanism is consistent with observations that Hg stemming from a 
MeHg dose is typically found as 90-100% iHg in the feces [15]. Rodent studies, using 
variations in diet and antibiotics, reinforce two principles regarding MeHg de-
methylation: 1) it is a rate-limiting step in elimination of MeHg and, 2) it is supported by 
the microbiota of the gut [16] [17]. The mechanism by which human gut bacteria might 
de-methylate MeHg remains unclear.  Furthermore, distinct species of bacteria with 
MeHg degrading activity within the human gut have yet to be characterized.  
Nonetheless, MeHg degrading bacteria in the environment, common to sediments and 
soils, have been well characterized. Such MeHg degrading strains carry a mercury 
(Mer) locus, which harbors genes encoding alkylmercury lyase (MerB) and mercury 
reductase (MerA) that encode enzymes capable of de-methylating MeHg and reducing 
Hg, respectively [18]. There is a possibility that related strains of microbes carry these 
genes in the human gut. 
A new method to measure MeHg metabolism in people.  We have recently 
overcome a significant barrier to implementing kinetic studies of MeHg in people by 
developing a simple, non-invasive protocol for sampling MeHg metabolism and 
excretion over time.  We have published this in two papers, both stemming from prior 
IRB-approved human studies.  In short, we accomplish this by assaying two samples of 
biological media: human hair and feces.  Through application of current technology and 
instrumentation (i.e., laser ablation-ICP-mass spectrometry (LA-ICP-MS)) we have 
established a protocol to quantify MeHg elimination rate (t1/2) and correlate it with 
demethylation (fecal iHg:MeHg ratio) in individuals who have previously eaten just three 
fish meals.  We are now in a position to capitalize on this methodology to extend our 
investigations of the microbiota of the human gut to identify the mechanisms and the 
bacterial species responsible for MeHg metabolism and excretion.  
1D. Specific Aims. 
It should be noted that the human subject portion of this proposed study is part of 
consortium among the P.I. (M. Rand, U of R) and co-investigators at Montana State 
University (MSU), Drs. Seth Walk and Eric Boyd. This study will be supported by NIH 
ViCTER mechanism specifically designed to support such “Virtual Consortium” with 
three investigators. As such, all aspects of human subject recruitment and bio-
sample collection and sample coding will be performed in Aim 1.  Aims 2 and 3 will 

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involve studies in mice and with defined bacterial cultures and will be conducted with 
de-identified samples of feces or fecal DNA at Montana State University.   
Our scientific premise is that symbiotic microbes in the human gut are required 
for the efficient biotransformation and excretion of toxic MeHg. There is substantial 
evidence, including our own, that the rate of MeHg elimination in the human body relies 
on gut microbiota.  Importantly, we discovered that MeHg elimination kinetics in an 
individual can vary significantly over time and, furthermore, is significantly slowed when 
taking antibiotics [10, 11].  Our initial findings with microbiome characterization yielded 
logical candidate bacterial taxa that now require validation to test their role in MeHg 
elimination [19]. With this evidence, we hypothesize that a susceptibility for reaching 
harmful levels of MeHg in the body is dictated by select members of the human 
gut microbiome that: a) carry out MeHg demethylation to enhance excretion, b) 
are variably present or variably abundant between and within individuals over 
time and c) are metabolically responsive to diet supplementation.   Our Specific 
Aims are:
Aim 1. Establish gut microbiome samples that exhibit “fast” and “slow” MeHg 
kinetics in humans. 
Aim 2. Validate the microbiome’s role in MeHg kinetics using germ-
free/gnotobiotic mouse modeling.
Aim 3. Identify and isolate microbial species responsible for MeHg demethylation 
in the human gut.
2. STUDY DESIGN
2A. Overview
Study site: This study will involve human subjects recruited from the greater 
Rochester area and will be conducted in the URMC Department of Environmental 
Medicine.  Hg analyses and DNA extraction of fecal samples will be performed in the U 
of R Environmental Health Science Center (EHSC) Elemental Analysis Facility. Fecal 
samples will also be evaluated for MeHg demethylation activity by assaying bacterial 
enzyme activity in Rochester. Hg analyses of hair samples by LA-ICP-MS will be done 
at the Trace Element Analysis lab at Dartmouth College, New Hampshire.  Additional 
studies, exempt from IRB approval, will be done at Montana State University with de-
identified samples of human feces and fecal DNA that are shipped to co-investigators 
Walk and Boyd at MSU. 
Protocol: Subjects will follow a protocol of eating three fish meals within a two-week 
period to incur exposure to naturally occurring levels of MeHg in the fish. Fish meals will 
consist of 6-12 ounces of commercially available tuna steaks.  This will be followed by 
an elimination period of 60 days where no fish or seafood is to be consumed. Single 
hairs collected at 60 days after the last of the three fish meals will be used to determine 
a MeHg elimination rate using LA-ICP-MS to perform longitudinal Hg elemental analysis 
as we have previously reported [10].  De-methylation of MeHg coming from the fish will 
be determined by direct analysis of iHg and MeHg in fecal samples collected 

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approximately four weeks following the final fish meal.  Fecal samples will also be used 
for culture assays and for direct iHg/MeHg analysis to determine of MeHg demethylation 
rates in vitro and in vivo, respectively (Biotransformation, Bin vitro  and Bin vivo). Fecal DNA 
will be used for metagenomic analyses to profile type and abundance of all bacterial 
species present.  Statistical correlations between elimination rate, demethylation rate 
and abundance of bacteria will be used to deduce strains of bacteria that contribute to 
demethylation and overall metabolism and elimination of MeHg.  The above protocol will 
be performed in two separate “trials” (which are denoted as “Study Periods” to the 
participants in the consent form) within the course of 
approximately 10 months and separated by an 
intermediate “washout” period of 4 months (Figure 1). 
Subject will not be required to refrain from fish or 
seafood during this washout period. Prior to the second 
trial subjects will receive a commercially available over 
the counter prebiotic (Prebiotin
TM) to be orally self-
administered on a regular schedule for the entire trial 2 
period. This prebiotic is not the subject of this 
investigation in this study, and is simply selected for its 
known effects of shifting gut microbial composition to 
enrich for healthier strains of bacteria (e.g. 
Bifidobacteria). Fecal sampling prior to and subsequent 
to prebiotic will be used to document changes in the 
microbiota for each individual. The prediction is that 
prebiotic induced change in the gut microbiota will result 
in a change in MeHg metabolism kinetics in each 
individual.
2B. Rationale for Study Design
The main rationale for this study design is based on our evidence that de-
methylation of MeHg in the human gut is the driving force for faster elimination MeHg 
that comes with eating fish. We therefore predict that with the methods we now have 
available to measure MeHg kinetics, coupled that with high-resolution analyses of gut 
microbiome characteristics in individual people who have undergone a controlled 
exposure to MeHg, we can resolve a microbial basis for MeHg metabolism in humans. 
This is an ambitious undertaking, and hence, is designed as a consortium project to be 
executed by three investigators with respective expertise in 1) human MeHg kinetics, 2) 
mouse microbiome manipulations and 3) microbial metagenomics and culturing 
methods. By design, the human subject component of the study is to be carried out at 
URMC.  The objective is to capture “slow” and “fast” MeHg metabolizing human 
microbiome samples, that can be handed off to colleagues at MSU for specialized 
analyses that do not involve the human subjects, simply their de-identified samples.   
3. CHARACTERISTICS OF THE RESEARCH POPULATION
(Many of the characteristics below are reiterated from our previously approved project: 
RSRB00050613 entitled Methylmercury metabolism and elimination in humans and 
RSRB00057934 entitled Determination of methylmercury metabolism and elimination 
Figure 1. Protocol Overview.  
Elimination rate determination, k
el; 
Demethylation (Biotransformation) 
analysis , Bin vitro  and Bin vivo.

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status in humans.)
3A. Subject Characteristics
Number of Subjects: We aim to obtain data on 40 adults, 20 males and 20 
females, for this study. We will therefore target consent of 46 participants (23 women, 
23 men) to account for potential drop out.  We expect the recruitment will be achieved 
locally in the Rochester area including the URMC community, but will not exclude 
interested people from outside this region. With the limited exclusion criteria below, we 
anticipate completing recruitment within the first several months of the study. 
Gender and Age of Subjects:  Study subjects will aim to be comprised of an equal 
number of men and women with no restriction on age ranging from 18 to 80. 
Racial and Ethnic Origin: There will be no restrictions on racial or ethnic origin in 
subject recruitment. It is anticipated the racial and ethnic diversity of the study group will 
reflect the various ethnicities that comprise the general population of the greater 
Rochester metropolitan area.
Dental amalgams:  Dental amalgams are a source of inorganic mercury. This study 
is not directed at inorganic mercury and prevailing literature indicates that inorganic 
mercury stemming from amalgams will not affect the biological and metabolic processes 
of MeHg under investigation here. Nonetheless, we will identify presence or absence of 
amalgams in subjects so that we can account for the contribution of this inorganic 
source of Hg to the Hg content measured in feces.  This will be critical for evaluating the 
extent of MeHg demethylation that has occurred in each subject.
Vulnerable Subjects:  Vulnerable subjects potentially include students and staff of 
the University of Rochester.  Advertisements will be placed in areas where they may be 
seen by students, employees and the general public.  For students and staff of the 
University of Rochester consent forms will clearly delineate that refusal to participate will 
have no consequence on job status, work place relationships, grades, or any other 
benefits to which the student/staff will otherwise be entitled. 
3B. Inclusion and Exclusion Criteria
Inclusion Criteria: 
Subjects must be 18-80 years in age.
Subjects must be in good general health based on self-reported health status, 
with the exception of self-reported conditions listed in the exclusion criteria.
Subject must be willing to comply with study procedures.
Exclusion Criteria: 
Known allergy to fish.
Pregnant or lactating women.
Use of hair dyes or chemical treatments within the prior month and/or intent to 
use such treatments during the two trial periods. (Hair treatment during the 
washout period up to one month prior to the second trial is acceptable).
Known gastrointestinal or renal disorders.
Subjects with diminished mental capacity.
Use of antibiotics within two months leading up to the study.
. 
3C. Discussion of Subject Population

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The inclusion criteria are very broad and it is anticipated a large diversity in age and 
ethnicity will result.  This is acceptable as age and ethnicity, although not primary study 
objectives in this pilot, are likely to produce variation in MeHg metabolism and 
elimination status, the determination of which is an objective of the study.  Exclusion 
criteria are defined based on factors that are likely to result in either harm to the subject 
or to substantially alter the metabolic processes or analytical procedures of MeHg and 
thus obscure results. Of particular concern is exposure to the fetus or infant to MeHg, 
which is known to target the developing nervous system.  This concern warrants 
exclusion of pregnant or breast-feeding women. 
4. SUBJECT IDENTIFICATION, RECRUITMENT, CONSENT AND COMMUNICATION
4A. Method of Subject Identification and Recruitment
Subjects will be recruited through local advertising by posting RSRB-approved 
flyers around the University of Rochester Medical Center. Response via phone and 
email to these postings will be anticipated. The P.I. and Lab personnel will also inform 
friends and acquaintances about the study.  Lab personnel will be prepared to tell 
interested persons of the existence of the study and provide interested persons with 
contact information for the P.I. for follow up. The Lab personnel will not inquire about 
health status or other personal information required of interested persons for consent. 
For phone inquiries the P.I. will perform an initial screening. Potential subjects will be 
asked if any of the following apply to them:
Do you have any known allergy to fish?
Are you pregnant or currently breastfeeding?
Have you used hair dyes or treatments within the last month?
Do you have any gastrointestinal or renal (kidney) disorders?
Have you used antibiotics within the last month?
Subjects answering “no” to all of the above will be asked to follow up with the initial 
office visit.
Process of Consent
Subjects with Capacity: Only subjects with capacity will be recruited into the 
study. Subjects with capacity will be consented by the P.I. or study coordinator (TBD) 
listed on the protocol.  The consent form will be reviewed with the subject directly, and 
the subject will also be offered the opportunity to review the consent form on their own. 
They will be encouraged to ask any questions and will be free to choose whether or not 
to participate. 
Subject Comprehension: All subjects will be asked, in a private setting, if they 
understand all aspects of the consent form and expectations of participation in the 
protocol.  They will be provided with contact information for the investigator and 
encouraged to ask for clarification at any time.  Reading skills will be assessed by 
asking all participants, in private, if they would like any assistance in reading forms.
Documentation of Consent.  All subjects will receive a signed copy of the 
consent form, and all consent forms will be kept on file in a locked office SMD Room 4-
6818.

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Costs to Subjects.  There will be no costs incurred by subjects participating in 
the study.  
Payment for Participation.  Subjects will be reimbursed with “rewards cards” 
from Wegman’s Supermarket. Renumeration will be prorated such that participants will 
receive $25 upon completion of trial 1 and an additional $50 ($75 total renumeration) 
upon completion of trial 2. 
Use of E-mail for Communication in Research 
E-mail communication will be used in recruitment and in scheduling visits and 
follow ups. E-mail will not be used in screening or consenting of subjects.  E-mail will 
also be a means for subjects to contact Dr. Rand with any study concerns. When using 
e-mail to communicate with subjects in this study, the researcher cannot guarantee, but 
will use reasonable means to maintain security and confidentiality of e-mail information 
sent and received.  Minimizing the risks for this is described below (9A, B). 
5. METHODS AND STUDY PROCEDURES
5A. Overview:  
The study will follow the protocol outlined below under “specific methods”.  A 
timeline of study activities can be seen in Figure 1 and Appendix 1. The study will 
engage human subjects in six activities to execute this protocol:  1) recruitment, 2) 
collection of body metrics (weight, height) and oral observation for presence/absence of 
amalgam fillings, 3) fish meal consumption 4) fecal sample collection and submission 
and 5) hair sample collection, 6) prebiotic self-administration (see below). A total of four 
visits will be mandatory, one at the beginning and one at the end of each trial.  Fecal 
sample submission will be done with a shipping protocol, or scheduled pick up by lab 
staff, and will not require a visit. Steps 2-4 above are anticipated to occur over a period 
of approximately 74 days (see Figure 1 and Appendix 1). Step 5 will commence one 
week prior to the first fish meal in the second trial (Figure 1).  Subjects will be asked to 
keep a food diary in a provided notebook and include notes on their general health, and 
particularly any illness or unusual diet habits, over the study period. The entire study, 
including pre-study preparations, subject enrollment, two trials of fish meal 
administration and hair and feces sampling, Hg measurements, microbiome analysis 
and statistical analysis of the data is anticipated to take place over a period of three 
years. 
5B. Approach. 
Our approach is summarized schematically in Figure 1.  Subjects will consume 
three meals of tuna steaks, each meal separated by seven days (on days 0, 7 and 14”).  
This will be followed by an elimination period of 60 days where subjects will resume 
normal daily diets and activities.  Hair will be sampled on or near the 60th day after the 
last fish meal.  Fecal samples will be taken at three time points: one each within 30 days 
following the last fish meal for each trial and one prior to, or upon the day of initiating the 
prebiotic administration in the second trial.  Longitudinal Hg analysis of single hair 
strands will be performed with LA-ICP-MS.  iHg and MeHg levels will be determined in 
fecal samples using tetra-ethylborate (NaBEt
4) derivatization and GC-ICP-MS methods.   

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Fecal DNA will be extracted and stored frozen. Hair Hg analyses will give Hg levels over 
time and will be used to determine a MeHg elimination rate (t
1/2).  Fecal Hg analyses will 
be used to determine iHg/MeHg ratio as a proxy of MeHg de-methylation, and adjusted 
based on presence of dental amalgam. Fecal samples and isolated DNA will be shipped 
to MSU for germ-free mouse microbiome experiments and metagenomic sequencing 
and bacterial culturing and abundance analyses.  Statistical analyses of correlations of 
elimination rate, MeHg demethylation and bacterial taxa abundance will be made.  
5C. Specific methods.
(Many of the specific methods below are reiterated from our previously approved 
project: RSRB00050613 entitled Methylmercury metabolism and elimination in humans 
and RSRB00057934 entitled Determination of methylmercury metabolism and 
elimination status in humans.)
Subject recruitment and subject information.  Potential subjects responding 
to flyer announcements of the study will be scheduled for a confidential visit in the office 
of the Principal Investigator (P.I.) who will serve as the study leader. The study methods 
and objectives and subject responsibilities, risks and benefits will be explained to 
prospective participants. Willing participants will sign a consent form provided by the P.I. 
at the time of the first visit. Alternatively, potential subjects will be given the opportunity 
to take home a written description of the study for further consideration before 
consenting. The study will target 40 participants, 20 women and 20 men. To achieve 
this subject recruitment will target 46 consenting participants (23 women, 23 men) to 
account for attrition due to drop out. When 46 participants have signed a consent form  
recruitment will cease. Once consent is obtained the subject will be assigned an alpha-
numerical identifier that will be used to label all associated samples, data and record 
keeping throughout the study.  This identifier number will be recorded on the consent 
form and kept in a locked and secure file in the P.I.’s office.
Information collected from the subjects at the initial study visit will be: age, sex, body 
weight, presence/absence of dental amalgams and medications currently taken. This 
will be collected without the need to access medical records. Subjects will be provided 
with a notebook carrying templates to fill in for a food diary.  Alternatively, subjects who 
currently use a personal food tracking application (e.g. “My Fitness Pal” or “Healthwatch 
360”) can substitute a print out from this application of their food consumption record for 
period spanning the trial.   
Fish meal administration. Just prior to each trial, individually weighed, wrapped 
and frozen portions of tuna steaks in 6-8 oz (170-227gm) portions will be prepared by 
and purchased from a local market (Wegman’s). Portions will be kept frozen in a 
dedicated freezer in the P.I.’s lab until the time of distribution to the subjects. Frozen fish 
portions will keep over the time of distribution to enrolling participants (~ 2-3 months, 
https://stilltasty.com/fooditems/index/18559). Tuna portions will be prepared from one 
large fish fillet, so subjects will be receiving portions from the same fish within each 
Trial. A sample of the fish tissue will be set aside at the time of purchase to determine 
mercury content in the fish. Mercury measurements will be performed at the URMC 
EHSC Elemental Analysis Facility.  Fish mercury values will be used to calculate 
predicted exposures to subjects based on individual body weights (see 9B, Protection 

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against risks, below). Where needed, instruction for adjustment of the portion size will 
be made to assure that MeHg exposure levels will not exceed federal guidelines for 
each subject.  Each subject will be provided with the three frozen portions of tuna in a 
Thermosafe styrofoam shipping container affixed with prepaid return postage for later 
return (see below). Two polar-pack freezer blocks will be included to maintain the frozen 
steaks during transport home.  For safety, subjects will be instructed on the steps 
necessary to keep the fish frozen until preparation for eating.  Since cooking does not 
affect MeHg content in fish, fish meals can be prepared to the subject’s own liking and 
eaten at home.  Suggestions for preparation of the fish will be provided to the subjects. 
Fish meals will be eaten on Days 0, 7 and 14. Subjects will record the date and time of 
consumption of each meal. Subjects will be instructed not to eat any fish or seafood 
during the 60 days following the final tuna fish meal. Subjects will be asked to keep a 
record of all types of food and drink consumed at each meal during the 60-day 
elimination period using a provided template or their personal food tracking application
Prebiotic administration.  Prebiotin™ (oligosaccharide enriched inulin), a 
commercially available prebiotic supplement from Jackson GI Medical 
(https://www.prebiotin.com/about-us/), will be distributed to the subjects for self-
administration subsequent to the first trial and washout period and prior to the second 
trial (Figure 1). An intended use for Prebiotin™ is not the subject of investigation in this 
study. Prebiotic supplementation with this lawfully marketed over-the-counter dietary 
supplement is being used for its known properties of changing gut microbiome 
composition. Regarding FDA approval, use of Prebiotin™ in this study will be IND 
exempt in that all six (6) of the conditions under section 7.1 of Policy 605 of the Office of 
Human Subject Protection at the Univ. of Rochester are met. Briefly, with respect to 
Prebiotin™, this study: is not intended to support a new indication for use (7.1.1); is not 
intended to support a significant change in the advertising of Prebiotin™ (7.1.2); does 
not involve a change in route of administration, dosage level, or population at risk 
(7.1.3); will be conducted in compliance with the IRB review (7.1.4); will be conducted in 
compliance with requirements concerning the promotion and sale of Prebiotin™ (7.1.5); 
and, does not intend to request exception from informed consent requirements for 
emergency use (7.1.6).  Individually packaged portions (4 grams) of the Prebiotin™ 
Prebiotic Fiber Stick Pac will be provided to subjects 
(https://prebiotin.net/collections/our-products/products/prebiotin-prebiotic-fiber-stick-pac-
4g). Prebiotin™ is the prebiotic of choice in several published studies on gut microbiota 
control of obesity [20-22] and has been used in an NIH study on reducing inflammation 
in kidney patients [23]. For this study, we are targeting a dose of 8 grams/day. This 
dose amount is based on published effects of inulin on gut microbiota in the range of 4 
to 16grams/day dosages [20, 24].  We have selected 8 grams/day based on the finding 
of Holscher et al [24], who demonstrated clear effects on shifting the microbiota 
composition and activity at the 7.5 gram/day dose level in healthy adults in a controlled 
double-blinded study. Prebiotin™ self-administration will be initiated one week prior to 
fish consumption in the second trial and extend through the entirety of the second trial 
(i.e. 11 weeks, Figure 1). Subjects will be given a 11-week supply of Prebiotin™ Stick 
Pacs and instructed on how to self-administer on either a once or twice daily dosing 
schedule. For the first week, subjects will be instructed to begin with a daily supplement 
of one Pac (4grams) a day, taken with meals. Starting the second week, and additional 

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Pac (2 Pacs total) will be consumed to achieve 8 grams/day. Both Pacs can be 
consumed at breakfast, or alternatively, divided between meals, e.g. breakfast and 
dinner. Prebiotin™ has a mild natural sweetness and will dissolve readily in any liquid. It 
can also be added to any food such as cereal, soups, salads, or sauces. If disruptive 
bloating or gas occurs with the 8gram dose, then the amount can be reduced to one 
pouch (4grams/day) until bloating and/or gas is manageable. While Prebiotin is safe to 
use in very high quantities, subjects will be advised on what to expect with prebiotic 
ingestion, including the anticipated digestive health benefits as well as possible feelings 
of bloating and excess flatulence that are harmless yet may produce some annoyance.  
Where bloating or gas is not manageable/tolerable and is disruptive to daily life, 
participants will be advised to contact study staff for further recommendations on 
reducing supplementation amounts to 2 grams/day or pursuing the option to withdraw 
from the study. 
Fecal sample collection, DNA isolation and shipping.  Feces will be used to 
capture the profile of the gut microbiota for each subject.  Collection methods will 
therefore be aimed at capturing and preserving anaerobic species. At the initial visit, 
subjects will also be provided with instructions on the stool sampling protocol and 
provided with an easy to use commercially available collection container (Fisherbrand 
Commode Specimen Collection System) and an Omnigene-Gut collection tube for DNA 
subsampling (below). Stools will be sampled at three time points as indicated in Figure 
1: approximately four weeks after the 3rd fish meal (for each trial) and at approximately 
one week prior to the 2nd Trial, preceding or contemporaneously with prebiotic 
supplementation (e.g. day 44 of the Trial 1 and day -7 and 44 of Trial 2). Collection of 
stools within 2-3 days centered on these target collection times will be acceptable. For 
all fecal samplings, the whole stool will be collected and frozen within 30 minutes to 
preserve microbe viability [25]. To stabilize a sample of the fecal DNA at the time of 
stool collection, a sub-sampling of feces with the Omnigene-Gut collection kit (DNA 
Genotek, Ottawa, ON, Canada) will be done by the subject according to the provided 
instructions. Subjects will be instructed to triple contain the stool collection with freezer-
grade zip-lock bags provided.  Subjects will ship the stool sample and the DNA 
stabilized sample to the P.I.’s lab using express shipping (FedEx) as soon as possible 
using the freezer packs and Thermosafe styrofoam packaging provided (see above). 
Alternatively, sample pick up by lab staff will be arranged. Fecal samples will be logged-
in and coded for identity protection in the P.I.’s laboratory and kept frozen until analysis 
and DNA isolation.  Fecal DNA will be isolated with the ZR fecal prep kit (Zymo 
Research) in the Rand lab and will be distributed to the Boyd lab at MSU. Samples of 
feces will also be distributed to the Walk lab at MSU. Human feces falls under the 
“exempt human/animal specimens” category for shipping.  A portion of frozen stool for 
each subject will be prepared in appropriate triple-layer packaging and containment 
(http://www.fedex.com/us/packaging/guides/Clinical_fxcom.pdf) and marked 
appropriately with “exempt human specimens” for shipping to MSU.  
Hair sample collection and submission.  Hair samples will be collected during 
a visit of the subject to the P.I.’s laboratory on approximately day 74 (60 days after the 
last fish meal). Sampling within a five-day window of this target time will be acceptable. 
Hair will be sampled manually by direct withdrawal from the scalp.  Sampling from the 
posterior region of the head will be done for all the subjects. Direct withdrawal of three 

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to five strands at once is found to elicit little discomfort and yield root material that can 
be used as a reference landmark for the start of the LA-ICP-MS analysis. A total of 20-
25 hair strands will be collected for each subject and stored in a zip-lock bag. Hairs will 
then be mounted on double stick tape on a microscope slide and stored at room 
temperature until LA-ICP-MS analyses (see below). Hair samples will also be logged-in 
and coded for identity protection in the P.I.’s laboratory.
Food and health diary.  Subjects will be requested to keep a food diary for the 
periods spanning each of the 75 days of Trial 1 and 2.  The diary is to include a list of 
types of food eaten and drinks consumed at each meal.  Careful accounting of amounts 
of food or caloric intake will not be requested.  In addition, subjects will be asked to 
record incidents of illness (e.g. cold or flu) or stresses (e.g. work or personal) that are 
likely to contribute to unusual eating patterns, digestive distress or metabolic imbalance.  
A food diary will not be requested for the intervening 4-month washout period. However, 
participants, at the visit prior to Trial 2, will be asked to comment on fish/seafood eating 
frequency and if illness or undue stress was experienced during the washout period.  
Analytical procedures. 
LA-ICP-MS of hair samples.  Hair analyses will be done on individual strands of 
hair using LA-ICP-MS at the Dartmouth College Trace Element Analysis (TEA) Lab at 
Dartmouth College (http://www.dartmouth.edu/~toxmetal/program-resources/trace-
element-analysis/). These measurements will be done by the TEA lab staff on a fee for 
service basis.   Laser ablation will be carried out with a UP213 laser (ESI, Bozeman, 
MT) equipped with a programmable X-Y stage for predetermining coordinates for 
ablations of a series of spots on the hair shaft.   Parameters of laser power, gas flow 
rates, dwell time and acquisition time will be optimized with Dr. Jackson’s assistance.  
Methods of Hg determination will follow previously described protocols [10, 11].  Briefly, 
spots of 50µm will be ablated at 333µm intervals along a 3.0cm length of the initial 
segment of hair closest to the scalp.  Hair growth occurs at approximately 1.1 cm per 
month. Therefore, 3.3cm of the initial segment of hair will be analyzed to cover the 75 
days previous in which MeHg administration and elimination have occurred.  The 202Hg 
isotope will be detected and normalized to the amount of hair sampled in the ablation 
event by detecting 34S in parallel and determining the Hg/S ratio.  Quantification of Hg 
will be done by comparing Hg/S ratios to a matrix match hair sample [11].  
Determination of MeHg elimination rate.  Elimination rate will be determined 
from the normalized relative Hg concentrations in an individual hair strand over time, 
which is determined from the length of hair that grew over the interval from the last fish 
meal to the 60 day collection point.  Growth rate of each individual hair will be calibrated 
by measuring the distance between the “peaks” of Hg observed in the hair resulting 
from the first and last fish meal, a period representing 14 days. Elimination rate will be 
determined by linear regression analysis of Hg concentration over time in a semi-log 
plot and expressed as t1/2 in units of days. 
Hg determinations in fecal samples. iHg and MeHg analysis and quantitation 
will be done in the URMC EHSC Elemental Analysis Lab.  Analyses will employ a 
standard protocol of acid digestion followed by tetra-ethylborate (NaBEt4) derivatization 
and separation on GC-ICP-MS.
Data & Specimen Banking for Future Research Use.  Samples (hair, feces) 

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will be stored in the P.I.’s laboratory until the analytical steps are completed.  With 
subject consent, samples will be stored for future research purposes. Data emerging 
from the study will remain on the P.I.’s password protected personal computer. 
Genetic/Genomic Research Activities
Genomic analysis will be carried out only on microbial samples from each subject.  
These studies are distinct from genomic testing of the subject’s own DNA.  Microbial 
genomic analyses will be done with a sequencing-based metagenomic method of 
identifying the presence and abundance of specific strains of bacteria.  The relevance of 
the composition of the gut microbiota to MeHg toxicity will be explained to the subject.  
Recent focus on the gut microbiome has revealed several important roles for gut 
bacteria in health and disease.  The subject will be made aware of the potential health 
benefits of management of gut microbiota composition and will have the opportunity to 
engage the P.I. in answering questions related to micriobiome research. 
Return of Individual Research Results
A summary of the study results will be shared with the participants upon request in a 
one-page electronic document. Participants will also be alerted to any public forum 
where study results will be presented and they are welcome to attend.   
6. CONCOMITANT AND DISALLOWED MEDICATIONS
Administration of antibiotic medication within two months prior to the study is an 
exclusion criterion. Antibiotic administration during the elimination period of the 75 day 
of protocol will not be disallowed, however, but must be reported to the P.I. No 
additional treatments or alteration of concomitant medication schedules will be 
necessary.  
7. SUBJECT WITHDRAWALS
Subjects will be advised orally and in writing in the consent forms that they have the 
right to withdraw from the study at any time without prejudice.  Subjects will be 
withdrawn from the research without their consent in instances of non-compliance or 
termination of funding for the study.  Subjects withdrawn from the study will be replaced 
with a follow up recruitment.
8. SAFETY AND REPORTABLE EVENTS
At the first visit, subjects will be alerted to self-monitor for any adverse event and to 
contact study staff by phone or email with any concerns. To monitor subject adverse 
events, study staff will also contact subjects with a phone call or email within the week 
subsequent to the last fish meal in each Trial (e.g. between days 15-21 of the Trial). An 
additional call will be made on day “0” of Trial 2 to monitor effects of prebiotic self-
administration. Study staff will not call subjects to monitor adverse events during the 
intermediate 4-month washout period. The most likely causes for an adverse event 
would be a reaction to consumption of tuna or the prebiotic administered in the second 
trial.  Subjects will be advised to contact the P.I. by phone or email in the instance that 
an adverse symptom, sign, illness or experience is encountered.  In the instance that 
this occurs before all tuna meals or prebiotic are consumed, the subject will be advised 

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to refrain from further fish or prebiotic consumption.  Adverse events or problems are 
not expected but will be reported to the RSRB according to OHSP Policy 801 
guidelines.
 
9. RISK/BENEFIT ASSESSMENT
9A. Potential Risks
The risk of MeHg toxicity from fish consumption remains a topic of considerable 
debate. MeHg poses the greatest risk for neural development in the fetus and young 
children, hence, the exclusion of pregnant and breast-feeding women and of children 
(17yrs and younger) in this study.  The risk to adults (18yrs and older) is substantially 
less.  Fish consumption implemented in this protocol will be carefully calculated so that 
blood MeHg levels incurred will not exceed that of the EPA reference dose (RfD) levels.  
The EPA reference dose includes a safety factor of 10. Thus, steps to avoid any 
potential risk due to MeHg have been averted in this study design. 
There is a minimal risk of physical discomfort associated with hair collection and with 
examination for presence of dental amalgam surfaces. There is minimal risk for 
gastrointestinal discomfort with consumption of prebiotic.
There is a potential risk of infringement on privacy or confidentiality. 
Risks of e-mail use:
Sending the subject’s information by e-mail has a number of risks that the subject 
should consider. These include, but are not limited to, the following:
a) E-mail can be circulated, forwarded, stored electronically and on paper, and 
broadcast to unintended recipients.
b) E-mail senders can easily misaddress an e-mail.
c) Backup copies of e-mail may exist even after the sender or the recipient has deleted 
his or her copy.
d) Employers and on-line services have a right to inspect e-mail transmitted through 
their systems.
e) E-mail can be intercepted, altered, forwarded, or used without authorization or 
detection.
f) E-mail can be used to introduce viruses into computer systems.
9B. Protection Against Risks
Protection against MeHg toxicity will be implemented by careful calculations that 
consider: 1) the amounts of MeHg administration that will occur in the three fish meals, 
2) known kinetic parameters of MeHg accumulation and elimination rates in the blood 
compartment and 3) each subject’s individual body weight and 4) the EPA reference 
dose (RfD).   These calculations are summarized below:
The EPA RfD is derived from a benchmark dose (BMD) analysis using a value of 
5.8µg/L in blood, which includes a safety factor of 10 
(http://www.epa.gov/iris/subst/0073.htm).  
A study by Kershaw, et al. [4] concludes that 5.9% of a MeHg dose is deposited in 
the blood compartment after complete tissue distribution.

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The blood compartment is ~7.1% of the body mass, equating to a range of 4.2 to 6.5 
liters in 130 to 200 pound individuals, respectively. 
Therefore, a blood level of 5.8µg/L equates to a single dose of:
5.8µg/L X (4.2-6.5L) X 1/0.059 = 413-639µg MeHg for individuals ranging 
from 130-200 pounds, respectively. 
Fresh tuna steaks have an average mercury content of 0.39ppm* (µg/g wet weight)
One 8oz (227gm) portion of tuna contains 88.5µg MeHg. Therefore, three 8oz 
portions of tuna will give a cumulative dose of 265.5µg MeHg, well below that 
needed to achieve blood levels equal to the EPA RfD.
*A recent in-house analysis of yellowfin tuna tissue obtained from the designated 
vendor (Wegman’s) revealed a mercury content of 0.23-1.35ppm.  For this highest level 
of mercury, three 8oz meals in 14 days would yield MeHg in blood exceeding to the RfD 
level for a 130-pound person.  For this reason, the tuna used in this study will be 
sampled and analyzed for Hg content prior to distribution to the subjects.  Subjects will 
be allocated tuna steak portion sizes according to their body weight and above 
calculations to ensure that EPA RfD levels are not exceeded. 
Risks of physical discomfort of hair sampling will be managed by limiting sampling to 4-
8 strands with each extraction. Subjects will be informed of what to expect with prebiotic 
administration, which could include feelings of bloating and excess flatulence, which 
have no adverse health effects beyond being an annoyance. 
Privacy will be maintained as best possible by coding samples and storing documents 
under locked files in the P.I.’s office.
Risk of email breach will be minimized by utilizing teh U of R SecureMail Encryption 
process following the steps outlined here: 
https://tech.rochester.edu/tutorials/sending-securemail-messages/
 
9C. Potential Benefits to Subjects
There will be no direct benefit to individual subjects in this study.  
10. RESEARCH INFORMATION IN MEDICAL RECORDS
No information from this research will be included in the participants’ medical records.
11. DATA ANALYSIS AND MONITORING
Sample Size Determination
We will target 40 participants (20 women, 20 men)for the reason that some 
statistical power is expected while we also are aiming to keep the study size appropriate 
for the logistics of recruiting and retention of subjects for both trials. We aim to recruit 
and consent 46 participant (23 women, 23 men) in anticipation of some drop out. A 

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complete cohort of 20 women and 20 menwill afford the opportunity to assess sex-
based differences.  We have performed power analysis on the pairwise correlations 
between kel and MeHg demethylation rate and/or bacterial taxa abundance.  With 
grouped sexes, 40 subjects will have at least 80% power to detect a linear relationship 
where 19% or more of the variance in elimination rate is explained by de-methylation 
status or particular bacterial species abundance.
Planned Statistical Analysis
Relationships of taxa abundance with kinetic parameters of MeHg elimination rate 
(kel), biotransformation in vivo (Bvivo, %iHg in feces) and in vitro (Bvitro, %MeHg 
biotransformation in cultures) will be evaluated.  A total of 117 numerical values for kel 
and Bvivo, Bvitro will be available (37 from our prior study and 40 subjects X 2 trials in the 
present study). Evaluation of slopes and R2 values for elimination rate calculations in 
the prior study demonstrates that a difference of two days in MeHg half-life between two 
subjects can be determined with significance.  Pairwise t-tests will be used to assess 
significance among pre- vs. post-prebiotic elimination rates (Aim1).  The correlation 
between the (kel) and biotransformation (Bvivo and Bvitro) will evaluated for the 40 X 2 = 
80 new human subject determinations in Aim 1. Graphical and numerical diagnostics 
will be used to test the linearity of the relationships and the homogeneity of variance. If 
these conditions are met, linear regression will be used to estimate functions for 
predicting kel from Bvivo or Bvitro.  The R system for statistical computing will be used 
throughout [26].  
Data and Safety Monitoring.
All records with information identifying a study subject will be kept in a secure, 
locked office or on a secure computer at the U of R site.  Only the P.I. will have access 
to these records. All samples will be coded so as to remove any subject specific 
information. The laboratory staff will only have access to subject samples and various 
non-identifying analytical parameters. Samples shared with co-investigators at Montana 
State University (MSU) will be de-identified.  In addition, the PI will consult with the MSU 
Co-Is to advise on practices of avoiding any possible disclosure of subject’s personal 
information.  In accordance with regulations, we may share a copy of the consent form 
and records that identify individual subjects with the U of R, MSU Department of Health 
and Human Services and National Institutes of Health.
12. OVERSIGHT PLAN. As PI, Dr. Rand will provide administrative and scientific 
oversight for all activities carried out by co-investigators Walk and Boyd at Montana 
State University. The PI will be responsible for conducting monthly meetings via Zoom 
or telephone to review study activities. Ongoing training of staff will be implemented to 
ensure protocol compliance. Data analysis will be reviewed on a monthly basis. In 
instances where there is a breach of privacy/confidentiality it will be promptly reported to 
the PI. Additional oversight and review of data analysis will occur at an annual meeting 
where PI, co-I’s and students and staff members of the study will physically convene in 
either Rochester or Bozeman Montana.
13. FOLLOW UP COMMUNICATIONS.  The Study PI will not initiate unsolicited contact 
with subjects but subjects may contact the Study PI to inquire about study status and 

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results subsequent to completing their study activities.  It is anticipated subjects will 
contact the Study PI via email, at which point the Study PI will call the subject by phone 
to review an information sheet delineating the risks and conditions of communication via 
email, and proceed to obtain verbal consent for using email. The timeframe within which 
the Study PI will receive and respond to email inquiries will be through August 31
st of 
2023, when the project funding is anticipated to expire, and study results will be 
complete.  Information about the study that is anticipated to be shared with subjects 
includes aggregate results and individual results specific to the subject only. Prior to 
sharing any data, the PI will confirm the subject data set matches the subject identity via 
a cross reference to the assigned alpha-numeric identifier assigned and recorded on the 
consent form as detailed in section 5C above.  In addition, the Study PI, when 
requested by the subject, will inform the subject of published results and, where allowed 
by the publisher guidelines, share such document(s) via an electronic file (e.g. PDF file), 
or refer the subject to a publicly available site to access the publication. 
REFERENCES
1.  
Clarkson, T.W., L. Magos, and G.J. Myers, Human exposure to Mercury: Three Modern 
Dilemmeas. J. Trace Elements in Exp. Med., 2003. 16: p. 321-343.
2.  
Hibbeln, J.R., et al., Maternal seafood consumption in pregnancy and 
neurodevelopmental outcomes in childhood (ALSPAC study): an observational cohort 
study. Lancet, 2007. 369(9561): p. 578-85.
3.  
Budtz-Jorgensen, E., P. Grandjean, and P. Weihe, Separation of Risks and Benefits of 
Seafood Intake. Env. Health Persp., 2007. 115: p. 323-327.
4.  
Kershaw, T.G., T.W. Clarkson, and P.H. Dhahir, The relationship between blood levels 
and dose of methylmercury in man. Arch Environ Health, 1980. 35(1): p. 28-36.
5.  
Aberg, B., et al., Metabolism of methyl mercury (203Hg) compounds in man. Arch 
Environ Health, 1969. 19(4): p. 478-84.
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Miettinen, J.K., et al., Elimination of 203Hg-methylmercury in man. Annals of clinical 
research, 1971. 3(2): p. 116-22.
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Toxicol Appl Pharmacol, 1994. 128(2): p. 251-6.
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Al-Shahristani, H. and K.M. Shibab, Variation of biological half-life f methylmercury in 
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Yaginuma-Sakurai, K., et al., Hair-to-blood ratio and biological half-life of mercury: 
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Journal of toxicological sciences, 2012. 37(1): p. 123-30.
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Rand, M.D., et al., Methods for Individualized Determination of Methylmercury 
Elimination Rate and De-Methylation Status in Humans Following Fish Consumption. 
Toxicol Sci, 2016. 149(2): p. 385-95.
11. 
Caito, S.W., et al., Variation in Methylmercury Metabolism and Elimination Status in 
Humans Following Fish Consumption. Toxicol Sci, 2018. 161: p. 443-53.
12. 
Ballatori, N. and T.W. Clarkson, Biliary transport of glutathione and methylmercury. Am 
J Physiol, 1983. 244(4): p. G435-41.

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13. 
Norseth, T., Biliary excretion and intestinal reabsorption of mercury in the rat after 
injection of methyl mercuric cloride. Acta pharmacologica et toxicologica, 1973. 33(4): 
p. 280-8.
14. 
Norseth, T. and T.W. Clarkson, Biotransformation of methylmercury salts in the rat 
studied by specific determination of inorganic mercury. Biochem Pharmacol, 1970. 
19(10): p. 2775-83.
15. 
Ishihara, N., Excretion of methyl mercury in human feces. Arch Environ Health, 2000. 
55(1): p. 44-7.
16. 
Rowland, I.R., M.J. Davies, and J.G. Evans, The effect of the gastrointestinal flora on 
tissue content of mercury and organomercurial neurotoxicity in rats given 
methylmercuric chloride. Developments in Toxicology and Environmental Science, 
1980. 8: p. 79-82.
17. 
Landry, T.D., R.A. Doherty, and A.H. Gates, Effects of three diets on mercury excretion 
after methylmercury administration. Bulletin of environmental contamination and 
toxicology, 1979. 22(1-2): p. 151-8.
18. 
Miller, S.M., Bacterial detoxification of Hg(II) and organomercurials. Essays in 
biochemistry, 1999. 34: p. 17-30.
19. 
Caito SW, G.A., Vorojeikina D, Scrimale T, Gill A,  Gill SR,  van Wijngaarden E, Rand 
MD, Characteristics of gut microbiota associated with variation in methylmercury 
metabolism and elimination in humans. Toxicological Sciences, 2019. in revision.
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Salazar, N., et al., Inulin-type fructans modulate intestinal Bifidobacterium species 
populations and decrease fecal short-chain fatty acids in obese women. Clin Nutr, 2015. 
34(3): p. 501-7.
21. 
Nicolucci, A.C., et al., Prebiotics Reduce Body Fat and Alter Intestinal Microbiota in 
Children Who Are Overweight or With Obesity. Gastroenterology, 2017. 153(3): p. 711-
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Hume, M.P., A.C. Nicolucci, and R.A. Reimer, Prebiotic supplementation improves 
appetite control in children with overweight and obesity: a randomized controlled trial. 
Am J Clin Nutr, 2017. 105(4): p. 790-799.
23. 
Prebiotic, P. Successful Use of Prebiotin® Prebiotic Fiber in NIH/NIDDK Study to 
Reduce Inflammation in Kidney Patients. 2018  Nov. 2018]; Available from: 
https://www.prebiotin.com/successful-use-prebiotin-prebiotic-fiber-nihniddk-study-
reduce-inflammation-kidney-patients/.
24. 
Holscher, H.D., et al., Agave Inulin Supplementation Affects the Fecal Microbiota of 
Healthy Adults Participating in a Randomized, Double-Blind, Placebo-Controlled, 
Crossover Trial. J Nutr, 2015. 145(9): p. 2025-32.
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Marcobal, A., et al., A metabolomic view of how the human gut microbiota impacts the 
host metabolome using humanized and gnotobiotic mice. ISME J, 2013. 7(10): p. 1933-
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Appendix 1

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Schedule of Activities.
*Call will be made between day 15-21.
# Fecal sample can be collected within 2-3 day period centered on this day.
$Hair sample can be collected within 5-day period centered on this day.
&Prebiotic is to be consumed daily from days -7 to 74 of Trial 2.
@ Food diary is to be filled in every day during Trial 1 and 2, but not during washout.